http://2012.igem.org/wiki/index.php?title=Special:Contributions/Lisa.O&feed=atom&limit=50&target=Lisa.O&year=&month=2012.igem.org - User contributions [en]2024-03-28T19:12:18ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/File:22497_470225256351975_566801605_n.jpgFile:22497 470225256351975 566801605 n.jpg2012-10-27T04:04:51Z<p>Lisa.O: </p>
<hr />
<div></div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-27T03:56:54Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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<ul><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Post-Regionals">Post-Regionals</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
</html>|<br />
<br />
TITLE=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/3/3d/UCalgary2012_FRED_and_OSCAR_Achievements.png" style="width: 280px; float: right; padding: 10px;"></img><br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<li><p><a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Returned to industry experts</b></a> to see if we accomplished our goals and what the <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy"><b>next steps</b></a> should be.</li></p><br />
<br />
<li><p><a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation#Prha_results"><b>Characterized</b></a> the functionality of our previously submitted <a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902066"><b><i>Prha</i> (BBa_K902066)</b></a> promoter using GFP fluorescence.</li></p><br />
<br />
<br />
<li><p>Tested an additional <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation"><b>complete kill switch device</b></a> using the <a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084"><b><i>Prha</i> (BBa_K902084)</b></a> promoter with our S7 kill gene.</li></p><br />
<br />
<li><p><a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation"><b>Further characterized</b></a> our previously validated <b>magnesium riboswitch kill gene construct</b><a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018"> <b>(BBa_K902018)</b></a>.</li></p><br />
<br />
<li><p>Tested a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy"><b>glycine auxotroph killswitch</b></a>.</li></p></ul><br />
<br />
<br><br />
<p><b>In terms of <FONT COLOR=#159900>FRED</FONT>, we...</b></p><br />
<ul><br />
<br />
<li><p><a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting"><b>Electrochemically characterized</b></a> an inducible <a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902090"><b><i>lacz</i> (BBa_K902090)</b></a> circuit, replacing a faulty existing circuit in the registry containing a frameshift.</p></li><br />
<br />
<li><p><a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting"><b>Electrochemically characterized</b></a> a constitutive <a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902005"><b><i>bglX</i> (BBa_K902005)</b></a> <b>generator</b> in terms of its electrochemical activity.</p></li><br />
<br />
<li><p><a class="green" href="https://2012.igem.org/Team:Calgary/Project/Synergy"><b>Tested one of our transposon library hits</b></a> with our electrochemical reporter, showing that we can use our system to <b>detect toxins electrochemically</b>.</p></li><br />
<br />
<li><p><a class="green" href="https://2012.igem.org/Team:Calgary/Project/Synergy"><b>Validated our system with tailings</b></a> using our transposon system and electrochemical reporter to be able to selectively detect toxins on <b>real oil sands samples</b>.</p></li><br />
</ul><br />
<br />
<br><br />
<br />
<p><b>In terms of <FONT COLOR=#1088CC>OSCAR</FONT>, we...</b></p><br />
<ul><br />
<li><p>Obtained <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>additional characterization data</b></a> in validation of our flux-variability analysis model.</p></li><br />
<br />
<li><p>Characterized the ability of our <b>novel</b> <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041"><b><i>amdA</i> (BBa_K902041)</b></a> <b>BioBrick</b> to selectively remove primary amides from ring structures with remarkable efficiency, turning them into a substrate that the <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025"><b>Petrobrick (BBa_K590025)</b></a> can likely convert into hydrocarbons.</p></li><br />
<br />
<br />
<li><p><a class="blue" href="https://2012.igem.org/Team:Calgary/Project/Synergy"><b>Validated a scale up model</b></a> for using our bioreactor/belt skimmer system in producing and extracting hydrocarbons.</p></li><br />
</ul><br />
<br />
<h2> <br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-27T03:56:30Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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<ul><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Post-Regionals">Post-Regionals</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
</html>|<br />
<br />
TITLE=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/3/3d/UCalgary2012_FRED_and_OSCAR_Achievements.png" style="width: 280px; float: right; padding: 10px;"></img><br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<li><p><a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Returned to industry experts</b></a> to see if we accomplished our goals and what the <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy"><b>next steps</b></a> should be.</li></p><br />
<br />
<li><p><a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation#Prha_results"><b>Characterized</b></a> the functionality of our previously submitted <a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902066"><b><i>Prha</i> (BBa_K902066)</b></a> promoter using GFP fluorescence.</li></p><br />
<br />
<br />
<li><p>Tested an additional <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation"><b>complete kill switch device</b></a> using the <a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084"><b><i>Prha</i> (BBa_K902084)</b></a> promoter with our S7 kill gene.</li></p><br />
<br />
<li><p><a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation"><b>Further characterized</b></a> our previously validated <b>magnesium riboswitch kill gene construct</b><a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018"> <b>(BBa_K902018)</b></a>.</li></p><br />
<br />
<li><p>Tested a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">glycine auxotroph killswitch</a>.</li></p></ul><br />
<br />
<br><br />
<p><b>In terms of <FONT COLOR=#159900>FRED</FONT>, we...</b></p><br />
<ul><br />
<br />
<li><p><a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting"><b>Electrochemically characterized</b></a> an inducible <a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902090"><b><i>lacz</i> (BBa_K902090)</b></a> circuit, replacing a faulty existing circuit in the registry containing a frameshift.</p></li><br />
<br />
<li><p><a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting"><b>Electrochemically characterized</b></a> a constitutive <a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902005"><b><i>bglX</i> (BBa_K902005)</b></a> <b>generator</b> in terms of its electrochemical activity.</p></li><br />
<br />
<li><p><a class="green" href="https://2012.igem.org/Team:Calgary/Project/Synergy"><b>Tested one of our transposon library hits</b></a> with our electrochemical reporter, showing that we can use our system to <b>detect toxins electrochemically</b>.</p></li><br />
<br />
<li><p><a class="green" href="https://2012.igem.org/Team:Calgary/Project/Synergy"><b>Validated our system with tailings</b></a> using our transposon system and electrochemical reporter to be able to selectively detect toxins on <b>real oil sands samples</b>.</p></li><br />
</ul><br />
<br />
<br><br />
<br />
<p><b>In terms of <FONT COLOR=#1088CC>OSCAR</FONT>, we...</b></p><br />
<ul><br />
<li><p>Obtained <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>additional characterization data</b></a> in validation of our flux-variability analysis model.</p></li><br />
<br />
<li><p>Characterized the ability of our <b>novel</b> <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041"><b><i>amdA</i> (BBa_K902041)</b></a> <b>BioBrick</b> to selectively remove primary amides from ring structures with remarkable efficiency, turning them into a substrate that the <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025"><b>Petrobrick (BBa_K590025)</b></a> can likely convert into hydrocarbons.</p></li><br />
<br />
<br />
<li><p><a class="blue" href="https://2012.igem.org/Team:Calgary/Project/Synergy"><b>Validated a scale up model</b></a> for using our bioreactor/belt skimmer system in producing and extracting hydrocarbons.</p></li><br />
</ul><br />
<br />
<h2> <br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/FRED/DetectingTeam:Calgary/Project/FRED/Detecting2012-10-27T02:14:51Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Transposon-Mediated Mutant Library for Toxin Detection|<br />
<br />
CONTENT=<br />
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<html><br />
<img src="https://static.igem.org/mediawiki/2012/5/52/UCalgary2012_FRED_Detecting.png" style="float: right; padding: 10px; height: 280px;"></img><br />
<p align="justify"><br />
This year, our team wanted to identify a novel responsive element capable of detecting and quantifying different tailings ponds toxins (e.g. naphthenic acids, NAs) in solution. While numerous studies have begun to identify species of bacteria capable of surviving and sensing a variety of toxic compounds (e.g. NAs), the degradation pathways have not yet been fully characterized. Therefore, we needed to design and implement novel approaches to efficiently isolate the genetic elements that detect and potentially lead to the breakdown of these toxins.<br />
</p><br />
<h2>Transposons: What, How, Why?</h2><br />
<p align="justify"><br />
The transposable element (TE), Tn5, is a conservative transposon that can insert a segment of genes bordered by specific 19bp insertion sequences from one part of the genome (e.g. plasmid vector) randomly to another location like a chromosome (Reznikoff, 2008). The transposition event is catalyzed by a transposase enzyme encoded by <i>tnp</i> gene included in the TE. Using the appropriate selective pressure, the insertion can be maintained permanently in the genome.</p><br />
<br />
</html>[[File:Transposon.jpg|thumb|700px|center|Figure 1: "Transposition reaction from plasmid entry into the recipient cell to integration of the transposon into the genome. Modified from Transposons: Shifting Segments of the Genome" by McGraw Hill]]<html><br />
<br />
<br />
<p align="justify">By inserting a vector construct containing the TE with selectable markers (such as tetracyclin resistance and lacZ) into an organism with a desirable phenotype, we can find out what genetic elements (e.g. genes and promoters) are responsible for that particular function. This can happen via a random insertion of a TE containing a promoterless reporter gene downstream of promoter elements that creates a transcriptional fusion, providing activity in response to specific environmental stimuli. Using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">bipartite-mating (conjugation) method</a> to transfer the TE vector into the organism of choice is an efficient method for creating the massive number of mutants required.</p><br />
<p align="justify"><br />
Due to the complexity of biological systems, our team focused our efforts on utilizing a system for identification of promoter elements that respond specifically in the presence of environmental stimuli. Our hypothesis requires that the organisms we use respond specifically to particular toxins and result in upregulation of metabolic genes with little background effect in the cell. We recognize that any number of biological molecules may play a role in toxin sensing, such as enzymes, transcription factors, and even RNA elements (e.g. riboswitches). However, the identification of a promoter sequence takes us further in that we can better understand the degradation mechanism by elucidating the genes involved.<br />
</p><br />
<br />
<br />
<br><br />
<h2>Toxin-Degrading Organism Used</h2><br />
<p align="justify"><br />
<i>Pseudomonas spp. </i>have been isolated from oil sands tailings ponds and shown to biodegrade model and tailings-associated NAs and nitrogen- and sulfur-containing heterocyclic aromatic compounds (Ramos-Padrón <i>et al</i>. 2010; Herman <i>et al</i>., 1994; Del Rio <i>et al</i>., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins.</p><p> We wanted to use a commercially available strain of <i>Pseudomonas fluorescens</i> characterized for a response to toxins found in tailings pond water (TPW). The <i>P. fluorescens </i>PF-5 strain (Paulsen <i>et al</i>., 2005) is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, the genome sequence is available for this strain with annotations (Pseudomonas Genome Database V2, http://pseudomonas.com/). This allows us to use sequencing data from the mutants and identify where in the genome the TE insertion occurred, and what genes (if present) are located downstream of it.<br />
</p><br />
<br />
<br><br />
<br />
<br />
<a name="library"></a><h2>Method Design</h2><br />
<h3>Mutant Library Generation</h3><br />
<p align="justify"><br />
To construct the promoter library, a pOT182 vector construct (containing a IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR transposable element) is introduced into commercially purchased <i>E. coli SM10</i> donor strain.</p><br />
<br />
<p align="justify"><br />
</html>[[File:Transposonproject Tn5OT182constructucalgary.png|thumb|750px|center|Figure 2: The transposable Tn5 element used in the pOT182 plasmid, containing a lacZ reporter gene, ampicillin and tetracycline resistance, an<br />
<i> E. coli</i> origin of replication for use during downstream sequencing protocols, and transposase. The genes are flanked by the transposon insertion elements]]<html><br />
</p><br />
<br />
<p align="justify">The plasmid contains a RP4 mob conjugation region and a p15A origin of replication (ori), and is engineered to only replicate in <i>E. coli</i>. The TE construct is transferred from the <i>E. coli</i> donor strain to the recipient <i>P. fluorescens </i> PF-5 using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in <i>P. fluorescens</i>, and selected by isolating the recipients that have a genomic TE insertion on Pseudomonas Isolation Agar/PIA with tetracycline. If a promoter element is fused upstream of the TE construct, then promoter activation will turn on the expression of lacZ, which can be detected by the degradation of a colorless compound, X-Gal, to an insoluble blue pigment product (an indoxyl compound) (Juers <i>et al</i>., 2012). If the fused promoter is activated in response to a stimulus, then the lacZ enzyme will be produced in response. Mutant strains sensitive to the particular toxic stimulus will appear as blue colonies on the selective plate.</p><br />
<br />
<h3>Mutant Strain Characterization</h3><br />
<p align="justify">Mutants generated are characterized for their roles in the response to toxins with dose response experiments, and compared to general stress-inducing agents (e.g. H<font style="text-transform: lowercase;">2</font>O<font style="text-transform: lowercase;">2</font>) and compounds such as fatty acids to ensure the specificity of the response. These measurements help to determine thresholds of detection, robustness of the signal, and specificity of response. The dose response curves will also assess the usefulness of correlating the concentration of NA to the level of response, and the possibility of measuring NA concentrations in a sample, rather than simply by presence/absence.</p><br />
</p><br />
<h3>Self-Cloning and Sequencing</h3><br />
<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman and Lamont, 1993). The TE used is a self-cloning construct because it contains all the elements required for plasmid replication (i.e. origin of replication) and selection (Tet resistance). Genomic DNA from a desirable mutant is isolated, and restriction digested with BglII (a restriction enzyme that does not cut within the TE but numerous times within the genome). The resulting fragments may contain the TE construct with flanking sequences. The genomic fragments are circularized by self-ligation and transformed into <i>E. coli</i>. Plasmids from the transformed cells contain the TE construct with the upstream and downstream flanking sequencing connected by the BglII restriction site. Sequencing primers designed against the 19 bp recognition sequence in the TE to sequence the isolated plasmids.</p><br />
<br />
<p align="justify">For a detailed protocol, please consult our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">methods section</a>.</p><br />
<br />
<h2>Results</h2><br />
<h3>Detection by Mutant <i>Pseudomonas fluorescens</i> PF-5</h3><br />
<br />
<br />
<p align="justify">After mating experiments and plating on selective media (Pseudomonas isolation agar, with tetracycline and naphthenic acids), 24 responsive (blue) colonies were found. Screens were conducted on these blue colonies found on selective plates comparing a response in LB and LB with 100mg/L naphthenic acids (both with X-Gal). When results were observed it was found that 4 mutant strains are differentially regulated in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. These colonies were further screened to test the specificity of their responses.</p><br />
<br />
<p align="justify"></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 3: Transposons: Shifting Segments of the Genome: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16hr. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><p align="justify"><br />
<br />
<br />
Screens involving the use of different toxins at environmentally relevant concentrations were performed to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used as one testing condition to determine if the response is simply stress-induced.<br />
</p><br />
<p align="justify"></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 4: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:170-1data.png|thumb|650px|center|Figure 5: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 6: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 7: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<p align="justify"><br />
From these screens, it was seen that both colony 66-1 and colony 170-1 appear to respond to toxins when compared to a response in LB media. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure 8: Stress response screen on <i>P. fluorescens</i> Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions (shown are '''A:''' 66-1 undiluted, '''B:''' 66-1 at 1/10 dilution, '''C:''' 170-1 undiluted, '''D:''' 170-1 at 1/10 dilution) into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 uL of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.]]<html><p><br />
<p><br />
These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.</p><br />
<br />
<h2>Promoter Constructs Isolated</h2><br />
<p align="justify">To determine the location of the transposon insertion, we utilized the self-cloning properties of the transposon. By digesting the genome, religating, and transforming the ligated genomic fragments into <i>E. coli</i>, plasmids containing the transposon and flanking gene sequences were isolated. These plasmids have been isolated and sent for sequencing. However, we are having difficulty with getting sequencing reactions to produce a read. The results so far are a promising step towards finding a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds. </p><p> <br />
Our next steps were to test these strains in conjunction with our electrochemical detector as well as see if they could detect tailings toxins. <b>The results of this can be seen on the x page</b>.</p><br />
<br><br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
</html><br />
<br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/FRED/DetectingTeam:Calgary/Project/FRED/Detecting2012-10-27T02:14:26Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Transposon-Mediated Mutant Library for Toxin Detection|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/5/52/UCalgary2012_FRED_Detecting.png" style="float: right; padding: 10px; height: 280px;"></img><br />
<p align="justify"><br />
This year, our team wanted to identify a novel responsive element capable of detecting and quantifying different tailings ponds toxins (e.g. naphthenic acids, NAs) in solution. While numerous studies have begun to identify species of bacteria capable of surviving and sensing a variety of toxic compounds (e.g. NAs), the degradation pathways have not yet been fully characterized. Therefore, we needed to design and implement novel approaches to efficiently isolate the genetic elements that detect and potentially lead to the breakdown of these toxins.<br />
</p><br />
<h2>Transposons: What, How, Why?</h2><br />
<p align="justify"><br />
The transposable element (TE), Tn5, is a conservative transposon that can insert a segment of genes bordered by specific 19bp insertion sequences from one part of the genome (e.g. plasmid vector) randomly to another location like a chromosome (Reznikoff, 2008). The transposition event is catalyzed by a transposase enzyme encoded by <i>tnp</i> gene included in the TE. Using the appropriate selective pressure, the insertion can be maintained permanently in the genome.</p><br />
<br />
</html>[[File:Transposon.jpg|thumb|700px|center|Figure 1: "Transposition reaction from plasmid entry into the recipient cell to integration of the transposon into the genome. Modified from Transposons: Shifting Segments of the Genome" by McGraw Hill]]<html><br />
<br />
<br />
<p align="justify">By inserting a vector construct containing the TE with selectable markers (such as tetracyclin resistance and lacZ) into an organism with a desirable phenotype, we can find out what genetic elements (e.g. genes and promoters) are responsible for that particular function. This can happen via a random insertion of a TE containing a promoterless reporter gene downstream of promoter elements that creates a transcriptional fusion, providing activity in response to specific environmental stimuli. Using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">bipartite-mating (conjugation) method</a> to transfer the TE vector into the organism of choice is an efficient method for creating the massive number of mutants required.</p><br />
<p align="justify"><br />
Due to the complexity of biological systems, our team focused our efforts on utilizing a system for identification of promoter elements that respond specifically in the presence of environmental stimuli. Our hypothesis requires that the organisms we use respond specifically to particular toxins and result in upregulation of metabolic genes with little background effect in the cell. We recognize that any number of biological molecules may play a role in toxin sensing, such as enzymes, transcription factors, and even RNA elements (e.g. riboswitches). However, the identification of a promoter sequence takes us further in that we can better understand the degradation mechanism by elucidating the genes involved.<br />
</p><br />
<br />
<br />
<br><br />
<h2>Toxin-Degrading Organism Used</h2><br />
<p align="justify"><br />
<i>Pseudomonas spp. </i>have been isolated from oil sands tailings ponds and shown to biodegrade model and tailings-associated NAs and nitrogen- and sulfur-containing heterocyclic aromatic compounds (Ramos-Padrón <i>et al</i>. 2010; Herman <i>et al</i>., 1994; Del Rio <i>et al</i>., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins.</p><p> We wanted to use a commercially available strain of <i>Pseudomonas fluorescens</i> characterized for a response to toxins found in tailings pond water (TPW). The <i>P. fluorescens </i>PF-5 strain (Paulsen <i>et al</i>., 2005) is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, the genome sequence is available for this strain with annotations (Pseudomonas Genome Database V2, http://pseudomonas.com/). This allows us to use sequencing data from the mutants and identify where in the genome the TE insertion occurred, and what genes (if present) are located downstream of it.<br />
</p><br />
<br />
<br><br />
<br />
<br />
<a name="library"></a><h2>Method Design</h2><br />
<h3>Mutant Library Generation</h3><br />
<p align="justify"><br />
To construct the promoter library, a pOT182 vector construct (containing a IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR transposable element) is introduced into commercially purchased <i>E. coli SM10</i> donor strain.</p><br />
<br />
<p align="justify"><br />
</html>[[File:Transposonproject Tn5OT182constructucalgary.png|thumb|750px|center|Figure 2: The transposable Tn5 element used in the pOT182 plasmid, containing a lacZ reporter gene, ampicillin and tetracycline resistance, an<br />
<i> E. coli</i> origin of replication for use during downstream sequencing protocols, and transposase. The genes are flanked by the transposon insertion elements]]<html><br />
</p><br />
<br />
<p align="justify">The plasmid contains a RP4 mob conjugation region and a p15A origin of replication (ori), and is engineered to only replicate in <i>E. coli</i>. The TE construct is transferred from the <i>E. coli</i> donor strain to the recipient <i>P. fluorescens </i> PF-5 using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in <i>P. fluorescens</i>, and selected by isolating the recipients that have a genomic TE insertion on Pseudomonas Isolation Agar/PIA with tetracycline. If a promoter element is fused upstream of the TE construct, then promoter activation will turn on the expression of lacZ, which can be detected by the degradation of a colorless compound, X-Gal, to an insoluble blue pigment product (an indoxyl compound) (Juers <i>et al</i>., 2012). If the fused promoter is activated in response to a stimulus, then the lacZ enzyme will be produced in response. Mutant strains sensitive to the particular toxic stimulus will appear as blue colonies on the selective plate.</p><br />
<br />
<h3>Mutant Strain Characterization</h3><br />
<p align="justify">Mutants generated are characterized for their roles in the response to toxins with dose response experiments, and compared to general stress-inducing agents (e.g. H<font style="text-transform: lowercase;">2</font>O<font style="text-transform: lowercase;">2</font>) and compounds such as fatty acids to ensure the specificity of the response. These measurements help to determine thresholds of detection, robustness of the signal, and specificity of response. The dose response curves will also assess the usefulness of correlating the concentration of NA to the level of response, and the possibility of measuring NA concentrations in a sample, rather than simply by presence/absence.</p><br />
</p><br />
<h3>Self-Cloning and Sequencing</h3><br />
<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman and Lamont, 1993). The TE used is a self-cloning construct because it contains all the elements required for plasmid replication (i.e. origin of replication) and selection (Tet resistance). Genomic DNA from a desirable mutant is isolated, and restriction digested with BglII (a restriction enzyme that does not cut within the TE but numerous times within the genome). The resulting fragments may contain the TE construct with flanking sequences. The genomic fragments are circularized by self-ligation and transformed into <i>E. coli</i>. Plasmids from the transformed cells contain the TE construct with the upstream and downstream flanking sequencing connected by the BglII restriction site. Sequencing primers designed against the 19 bp recognition sequence in the TE to sequence the isolated plasmids.</p><br />
<br />
<p align="justify">For a detailed protocol, please consult our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">methods section</a>.</p><br />
<br />
<h2>Results</h2><br />
<h3>Detection by Mutant <i>Pseudomonas fluorescens</i> PF-5</h3><br />
<br />
<br />
<p align="justify">After mating experiments and plating on selective media (Pseudomonas isolation agar, with tetracycline and naphthenic acids), 24 responsive (blue) colonies were found. Screens were conducted on these blue colonies found on selective plates comparing a response in LB and LB with 100mg/L naphthenic acids (both with X-Gal). When results were observed it was found that 4 mutant strains are differentially regulated in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. These colonies were further screened to test the specificity of their responses.</p><br />
<br />
<p align="justify"></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 3: Transposons: Shifting Segments of the Genome: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16hr. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><p align="justify"><br />
<br />
<br />
Screens involving the use of different toxins at environmentally relevant concentrations were performed to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used as one testing condition to determine if the response is simply stress-induced.<br />
</p><br />
<p align="justify"></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 4: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:170-1data.png|thumb|650px|center|Figure 5: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 6: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 7: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<p align="justify"><br />
From these screens, it was seen that both colony 66-1 and colony 170-1 appear to respond to toxins when compared to a response in LB media. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure 8:Stress response screen on <i>P. fluorescens</i> Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions (shown are '''A:''' 66-1 undiluted, '''B:''' 66-1 at 1/10 dilution, '''C:''' 170-1 undiluted, '''D:''' 170-1 at 1/10 dilution) into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 uL of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.]]<html><p><br />
<p><br />
These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.</p><br />
<br />
<h2>Promoter Constructs Isolated</h2><br />
<p align="justify">To determine the location of the transposon insertion, we utilized the self-cloning properties of the transposon. By digesting the genome, religating, and transforming the ligated genomic fragments into <i>E. coli</i>, plasmids containing the transposon and flanking gene sequences were isolated. These plasmids have been isolated and sent for sequencing. However, we are having difficulty with getting sequencing reactions to produce a read. The results so far are a promising step towards finding a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds. </p><p> <br />
Our next steps were to test these strains in conjunction with our electrochemical detector as well as see if they could detect tailings toxins. <b>The results of this can be seen on the x page</b>.</p><br />
<br><br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
</html><br />
<br />
}}</div>Lisa.Ohttp://2012.igem.org/File:PrhaFinal.pngFile:PrhaFinal.png2012-10-27T02:12:25Z<p>Lisa.O: uploaded a new version of &quot;File:PrhaFinal.png&quot;</p>
<hr />
<div>A finalized version of rhamnose kill switch.</div>Lisa.Ohttp://2012.igem.org/File:NativeRhamnosePromoter_Calgary2012.jpgFile:NativeRhamnosePromoter Calgary2012.jpg2012-10-27T02:11:49Z<p>Lisa.O: uploaded a new version of &quot;File:NativeRhamnosePromoter Calgary2012.jpg&quot;</p>
<hr />
<div>The organization of the rhamnose metabolic genes in E. coli.</div>Lisa.Ohttp://2012.igem.org/File:NativeRhamnosePromoter_Calgary2012.jpgFile:NativeRhamnosePromoter Calgary2012.jpg2012-10-27T02:07:32Z<p>Lisa.O: uploaded a new version of &quot;File:NativeRhamnosePromoter Calgary2012.jpg&quot;</p>
<hr />
<div>The organization of the rhamnose metabolic genes in E. coli.</div>Lisa.Ohttp://2012.igem.org/File:PrhaFinal.pngFile:PrhaFinal.png2012-10-27T01:59:31Z<p>Lisa.O: uploaded a new version of &quot;File:PrhaFinal.png&quot;</p>
<hr />
<div>A finalized version of rhamnose kill switch.</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T01:41:27Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Interviews|<br />
CONTENT=<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team began our project with human practices in mind. While we had established a research objective to produce a biosensor and bioreactor system, we wanted to ensure that our system was relevant to the industry where it would be employed. As well, we wanted to ensure that academic, government, and industry professionals' concerns were taken into consideration during the design process of our system. In order to best accomplish this, we conducted interviews with two leaders in oilsands reclamation. We approached a major oilsands company, Suncor, and talked to Christine Daly, an Ecologist who works in Environmental Cleanup. We then approached Ryan Radke, the president of BioAlberta. BioAlberta focuses on bringing biotechnology to our province and develop these in an industrial setting. His experience allowed us to better predict if our project would have any concerns amongst legislators and industrial leaders. <br />
</p><br />
<br />
<h2> <u>Initial Interviews</u> </h2><br />
<br />
<h3>Talking with Suncor's Christine Daly on Biology in the Oil Sands</h3><br />
<p>We spoke with Christine Daly, an Aquatic Reclamation Research Coordinator at Suncor Energy Inc. Christine expressed an interest in our <a href="https://2011.igem.org/Team:Calgary">project in 2011</a> and was willing to discuss this year’s project design with us. One major point that was brought up early on in our design was that there is an opportunity for engineered organisms to outcompete existing tailings ponds bacteria, and we were pleased to hear that Christine had a similar concern. To address these concerns, we created our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">bioreactor</a> system, which would physically contain our bacteria, and also a <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">genetic killswitch mechanism</a>. Another interesting point brought up in this discussion was how the oil industry is currently looking into biology as one of many potential alternative methods to remediate the toxic components of tailings ponds and the oil sands in general. Research exists with other systems such as algal bioremediation, but practical implementations of biology in the oil sands appear to be rather few and far between. Oil industries do, however, appear to show an increased interest in biology (and in turn, synthetic biology) as a possible solution to various problems, a sentiment reflected in <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">our dialogue with the Oil Sands Leadership Initiative</a>.</p><br />
<p>The full interview can be viewed below.</p><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/GiM6EIC9XBo" frameborder="0" allowfullscreen></iframe><br />
<br />
</div><br />
<br />
<h3>BioAlberta's Ryan Radke on Biology in the Oil Sands</h3><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/86XQ-Kg5fJ4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<a name="postregionals"></a><br />
<h2><u>Follow-Up Interviews</u></h2><br />
<p>Our second iteration of interviews were conducted once we had a more concrete product built. The purpose of these interviews was to see whether we had successfully addressed the concerns of the first iteration interviews. We also wanted to see whether any new issues with the design existed, which would provide us with potential future directions to take FRED and OSCAR. Kelly Roberge, an independent oil consultant, suggested we look into various ways to deal with the clay and silt particles that can enter our bioreactor system, which can be a major problem since mature fine tailings have a thick consistency that could clog the system.</p><br />
<br />
<h3>Kelly Roberge, of K. Roberge Consulting Ltd. Discussing Bioreactor Improvements</h3><br />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our design at this point were issues with scale-up. These were things such as the amount of toxins that would need to be added to the system to provide constant production of our product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do towards this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we will have to look at the limitations in terms of the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to this grime and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that William brought up was that there needs to be extra safety factors put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design"> structural and genetic killswitch devices</a>. In the later part of our project, we have also been trying to work on establishing a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">glycine auxotrophic killswitch</a> to add yet another layer of safety which we feel is necessary. </p><br />
<br />
<p>Similar to Mr. Roberge, another thing Mr. Sawchuk brought up was scale-up. Specifically, he talked about feasibility and cost a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we’ve been experimenting with starting to get our bioreactor working and have performed an initial validation assay that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons, which can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. The next step is to scale-up further! The exact cost is a bit tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<p>Gordon Lambert is the VP Sustainable Development at Suncor Energy Inc. We asked him whether or not the oil sands industry would find technology such as this useful. There was a very positive response. The Oil Sands Leadership Initiative is very keen on searching for any solutions to tackle the tailings ponds, which are considered to be one of the biggest issues in the oil sands currently. OSLI is collaborating with organizations that run competitions globally for oil sands solutions and other bodies such the Canada's Oil Sands Innovation Alliance (COSIA). Similar to Kelly Roberge's comment, mature fine tailings can be dried and solidified, but in turn it liberates water from the clay and sand. This water cannot be used for any industrial purposes until it is detoxified. Ideally, this water can be detoxified sufficiently to be returned as tailings pond surface water and become reusable in the bitumen extraction process.</p><br />
<p>In order to deploy our biosensor and bioreactor system, it was suggested that we look into various regulatory boards within Alberta such as Alberta Environment and the Energy Resources Conservation Board (ERCB) to attempt to obtain permits to begin attempting pilot programs. Scale-up of the bioreactor is also a major consideration in order for us to push it off the bench and into the field.</p><br />
<p>The full interview can be found below. If it does not load, <a href="http://www.youtube.com/watch?v=7KbEjQVUsFA">click here</a>.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
</div><br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T01:37:31Z<p>Lisa.O: </p>
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TITLE=Interviews|<br />
CONTENT=<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team began our project with human practices in mind. While we had established a research objective to produce a biosensor and bioreactor system, we wanted to ensure that our system was relevant to the industry where it would be employed. As well, we wanted to ensure that academic, government, and industry professionals' concerns were taken into consideration during the design process of our system. In order to best accomplish this, we conducted interviews with two leaders in oilsands reclamation. We approached a major oilsands company, Suncor, and talked to Christine Daly, an Ecologist who works in Environmental Cleanup. We then approached Ryan Radke, the president of BioAlberta. BioAlberta focuses on bringing biotechnology to our province and develop these in an industrial setting. His experience allowed us to better predict if our project would have any concerns amongst legislators and industrial leaders. <br />
</p><br />
<br />
<h2> <u>Initial Interviews</u> </h2><br />
<br />
<h3>Talking with Suncor's Christine Daly on Biology in the Oil Sands</h3><br />
<p>We spoke with Christine Daly, an Aquatic Reclamation Research Coordinator at Suncor Energy Inc. Christine expressed an interest in our <a href="https://2011.igem.org/Team:Calgary">project in 2011</a> and was willing to discuss this year’s project design with us. One major point that was brought up early on in our design was that there is an opportunity for engineered organisms to outcompete existing tailings ponds bacteria, and we were pleased to hear that Christine had a similar concern. To address these concerns, we created our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">bioreactor</a> system, which would physically contain our bacteria, and also a <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">genetic killswitch mechanism</a>. Another interesting point brought up in this discussion was how the oil industry is currently looking into biology as one of many potential alternative methods to remediate the toxic components of tailings ponds and the oil sands in general. Research exists with other systems such as algal bioremediation, but practical implementations of biology in the oil sands appear to be rather few and far between. Oil industries do, however, appear to show an increased interest in biology (and in turn, synthetic biology) as a possible solution to various problems, a sentiment reflected in <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">our dialogue with the Oil Sands Leadership Initiative</a>.</p><br />
<p>The full interview can be viewed below.</p><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/GiM6EIC9XBo" frameborder="0" allowfullscreen></iframe><br />
<br />
</div><br />
<br />
<h3>BioAlberta's Ryan Radke on Biology in the Oil Sands</h3><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/86XQ-Kg5fJ4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<a name="postregionals"></a><br />
<h2><u>Follow-Up Interviews</u></h2><br />
<p>Our second iteration of interviews were conducted once we had a more concrete product built. The purpose of these interviews was to see whether we had successfully addressed the concerns of the first iteration interviews. We also wanted to see whether any new issues with the design existed, which would provide us with potential future directions to take FRED and OSCAR. Kelly Roberge, an independent oil consultant, suggested we look into various ways to deal with the clay and silt particles that can enter our bioreactor system, which can be a major problem since mature fine tailings have a thick consistency that could clog the system.</p><br />
<br />
<h3>Kelly Roberge, of K. Roberge Consulting Ltd. Discussing Bioreactor Improvements</h3><br />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our design at this point were issues with scale-up. These were things such as the amount of toxins that would need to be added to the system to provide constant production of our product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do towards this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we will have to look at the limitations in terms of the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to this grime and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that William brought up was that there needs to be extra safety factors put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both structural and genetic killswitch devices. In the later part of our project, we have also been trying to work on establishing a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">glycine auxotrophic killswitch</a> to add yet another layer of safety which we feel is necessary. </p><br />
<br />
<p>Similar to Mr. Roberge, another thing Mr. Sawchuk brought up was scale-up. Specifically, he talked about feasibility and cost a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we’ve been experimenting with starting to get our bioreactor working and have performed an initial validation assay that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons, which can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. The next step is to scale-up further! The exact cost is a bit tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<p>Gordon Lambert is the VP Sustainable Development at Suncor Energy Inc. We asked him whether or not the oil sands industry would find technology such as this useful. There was a very positive response. The Oil Sands Leadership Initiative is very keen on searching for any solutions to tackle the tailings ponds, which are considered to be one of the biggest issues in the oil sands currently. OSLI is collaborating with organizations that run competitions globally for oil sands solutions and other bodies such the Canada's Oil Sands Innovation Alliance (COSIA). Similar to Kelly Roberge's comment, mature fine tailings can be dried and solidified, but in turn it liberates water from the clay and sand. This water cannot be used for any industrial purposes until it is detoxified. Ideally, this water can be detoxified sufficiently to be returned as tailings pond surface water and become reusable in the bitumen extraction process.</p><br />
<p>In order to deploy our biosensor and bioreactor system, it was suggested that we look into various regulatory boards within Alberta such as Alberta Environment and the Energy Resources Conservation Board (ERCB) to attempt to obtain permits to begin attempting pilot programs. Scale-up of the bioreactor is also a major consideration in order for us to push it off the bench and into the field.</p><br />
<p>The full interview can be found below. If it does not load, <a href="http://www.youtube.com/watch?v=7KbEjQVUsFA">click here</a>.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
</div><br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/DesulfurizationTeam:Calgary/Notebook/Desulfurization2012-10-27T01:18:48Z<p>Lisa.O: </p>
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<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Desulfurization Journal|<br />
<br />
CONTENT=<br />
<html><br />
<h2>Week 1 (May 1-4)</h2><br />
<p>During this week, literature search was performed.</p><br />
<br />
<br />
<br />
<br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<br />
<br />
<p>Along with the rest of the team, this week was dedicated to familiarizing ourselves on the protocols that will be utilized during this years project; specifically the polymerase chain reaction, gel verification, preparation of overnight cultures, as well as developing a procedural flowchart to transform competent cells with registry biobricks. With regards to our sub-group specific goals, we reviewed the current available literature around various industrial and laboratory approaches to desulfurization of organic groups, especially in the petroleum industry. This included a comparison of non-biological processes such as conventional hydrodesulfurization, which is currently employed in petroleum product refinery stages, and how a biological approach would supplement and perhaps even offer several advantages over these methods. Current limitations to biological desulfurization, however, include such factors as biocatalyst stability, enzyme specificity and desulfurization rate. We also identified the enzyme desulfinase (DszB) as being one of the bottlenecks in the desulfurization 4S pathway. Overall, our goals moving forward involve determining the specific pathways involved in the desulfurization process as well as the reaction conditions we would want to employ, and identifying specific model compounds in addition to dibenzothiophene (DBT) that we could use to test the effectivity of our biosystem in order to determine its functionality in the conversion of naphthenic acids to economically valuable hydrocarbons.</p><br />
<br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<br />
<br />
<p> Building on the previous week's literature review, the 4S pathway was recognized as the preferred biological mechanism that we would explore in devising a desulfurization biosystem. Of specific interest is the dsz operon consisting of the genes for <i> dszA, dszB, </i>and<i> dszC</i> which selectively and non-destructively remove the sulfur from the hydrocarbon structure, and therefore preserves the carbon skeleton. In addition to these, another dsz gene exists.<i> dszD</i>, which codes for a FMN:NADH reductase, is an essential component of the pathway, but not part of the operon. Instead, it is on the genome. The enzyme produced by this gene is required to regenerate the FMNH2 consumed by the reactions carried out by DszA and DszC. <i>Rhodococcus erythropolis</i> IGTS8 is the most studied model organism in investigations of the 4S pathway, and has been shown in many different research endeavors to be capable of converting DBT to 2-HBP.</p><p><br />
</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|thumb|750px|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD. Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO2) through the addition of oxygen to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the heteroatom, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-hydroxybiphenyl. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.]]<html></p><p><br />
An alternative to the DszD gene is HpaC, an oxidoreductase encoded in the <i>E. coli</i> W genome. This enzyme has been shown to increase the rate of desulfurization. Following this, other protocols added to our growing lab methods 'toolkit' were a restriction digest protocol, PCR purification, and finally, DNA construction digest. Aims moving forward include obtaining strains of the <i> R. erythropolis </i>, while also executing a timeline devised to biobrick, test, and incorporate the genes necessary in the above processes in a biobrick circuit.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<br />
<br />
<p>This week was kicked off with a project development meeting with Emily and David, and we devised a protocol for biobricking the <i> hpaC </i> gene. Additionally, methods to place the genes coding for the 4 enzymes, DszA,B,C and HpaC into a single construct were explored. Within the lab, the PCR performed on the resuspended pUC18-hpaC was not successful initially. <br />
Furthermore, we ordered the substrates/compounds that we intend to use for desulfurization tests. Once the substrates and the <i>Rhodococcus</i> strain arrive we are going to test how effectively the bacteria can desulfurize different sulphur-containing compounds that resemble naphthenic acids. Finally, we came across a paper where a team had developed an improved efficiency DszB through site-directed mutagenesis in 2007. This was through a point mutation to the gene, converting a tyrosine at position 63 to a phenylalanine residue. A member of this team was contacted to request the plasmid that contains the mutated gene. The conversion step carried out by DszB is the major bottleneck in the 4S pathway and if a strain or sample containing this mutation was obtained, it would significantly bolster our later testing efforts on DBT, as well as other compounds such as thiophane.<br />
</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<br />
<br />
<p>Since we wanted to make sure we would not run out of pUC18(plasmid containing the hpaC gene), we transformed some E.coli cells with it. We grew them on plates containing ampicillin (A), kanamycin (K), tetracycline (T) and chloramphenicol (C) antibiotics and they only grew on A. Therefore pUC18 has A resistance. We did a three sets of PCR with primers designed against <i>hpaC</i>, one using 1/10 dilution of pUC18, the other using 1/100 dilution of pUC18 and one with the colonies we had just obtained by transforming the <i>E.coli</i> cells. </p><br />
<p>The PCR worked and we saw bands of the same size for all three sets of PCR. (Unfortunately, the picture we saved is not a good one since some of the bands faded away under UV due to prolonged exposure. Following this, PCR purification was performed to obtain the pure <i>hpaC</i> with biobrick prefix and suffix attatched to gene, which would allow us to insert the sequence into a biobrick standard backbone. 3 sets of digestion, ligation, and transformation (using pairs of X&P enzymes, E&S enzymes and E&P enzymes) were carried out in order to insert the <i>hpaC </i> gene into the <a href=" http://partsregistry.org/Part:pSB1C3">pSB1C3</a><br />
vector. All the sets grew successfully.<br />
Following the above successes with<i> hpaC</i>, the arrival of our <i>Rhodococcus</i> strain afforded us the opportunity to begin investigation of the Dsz operon using the primers currently in our possession. This strain is an environmental isolate that has been shown to be an active desulfurizer. The gram-positive nature of the strain also dictated we explore various lysing strategies before the genes encoding the Dsz enzymes could be amplified for further purification and biobrick construction steps. PCR was carried out using <i>dszA</i> primers on three different treatments {microwave, lysate buffer, and a control} which yielded banding pattern around 1200 base pairs for the lysate treatment (2%SDS and 10% tritonX-100, plus heat for 5mins at 98C).<br />
</p> <br />
<br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<br />
<br />
<p> In order to confirm the <i>hpaC</i> biobrick construction, two sets of colony PCR were performed, choosing white colonies from the 3 plates we grew last week (white colonies indicate a loss of the RFP generator in the <a href=" http://partsregistry.org/Part:pSB1C3">pSB1C3</a><br />
backbone, and therefore allow for weeding out of the colonies which are simply the original plasmid vector). These reactions were carried out both with <i>hpaC</i> primers and with standard biobrick primers designed against the plasmid backbone. After running them on the gel we saw equal bands for the PCR reactions performed using <i>hpaC</i> primers (However, a PCR using biobrick primers was performed later and the same results were obtained). Colonies 1(-) and 5(-) were used to make overnight cultures, which were then miniprepped the following day to obtain the plasmid DNA of the putative <i>hpaC</i> biobrick. Digestions were performed on the miniprep products using EcoRI and PstI to look for part size as further verification for the genes presence in the plasmid. The results were good and two bands were observed on each column (one for vector and the other for <i>hpaC</i>)). <i>hpaC</i> was sent in for sequencing. </p><br />
<p><br />
<br />
<br />
<br />
</html>[[File:UCalgary2012_04.06.2012-desulfurisation_hpacverification.jpg|thumb|700px|center|Figure 2: HpaC verification cPCR. HpaC gene was inserted into the pSB1C33 vector and E. coli cells were transformed. In order to confirm that pSB1C3 contains the hpaC gene, two sets of colony PCR's were conducted. One with biobrick primers, and the other with hpaC primers. Bands indicate successful amplification at the approximate size of the hpaC gene (517 bp)]]<br />
[[File:Ucalgary2012 06.06.2012-digestion of hpaC with E and P.jpg|thumb|500px|center|Figure 3: HpaC confirmation digest. Plasmid was miniprepped and digested for 2h with EcoRI and SpeI before being run on a 1% gel for 1h and 120V. Bands between 500 and 700 bp indicate the hpaC part is present as an insert. Bands of about 2000bp show the size of the psb1c3 vector.]]<html></p><br />
<br />
<br />
<br />
<p> PCR reagents were prepared to re-test/confirm previous results of <i>dszA</i> amplification following two different lysing treatments (microwave + lysate buffer). This time, all three genes were amplified and gel verification showed clear banding patterns around 500bp range for all three genes for the microwave treatment. Remaining PCR products were run on a gel and extracted for further purification steps; however, presence of any genetic material were not confirmed through nanodropping which raised concerns about the composition of the purified products, the success of the initial amplification step, or perhaps even the lysis treatment. Further experimentation will have to be carried out to troubleshoot.</p><br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<br />
<br />
<p> This week, we focused on amplifying <i>dsz</i> genes from our <i>Rhodococcus</i> strain for construction into biobricks. We also wanted to purify the <a href=" http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"> BBa_K902057 <i>hpaC</i></a> and pUC18-<i>hpaC</i> plasmids to replenish our current stocks. For the <i>dsz</i> aspect, we were able to successfully grow extra plates of <i>Rhodococcus</i> strain which was used to inoculate PCR tubes. The PCR did not go well, with significant streaking and false positives with similar banding pattern to previous gels run in the previous week. A final gel verification of a random sample of a tube of PCR products from dszA,B,C respectively and two negative control treatments involving master mix only and the lysed cells only illustrated the lack of discrepancy between the supposed successful amplification and the lysed cells (with lysate buffer) alone. Because of this we decided to take a different approach involving plasmid isolation carried out before PCR, rather than applying the PCR reagents directly to a lysed culture sample.</p><br />
<p> <a href=" http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> verification through sequencing was successful, confirming the construction of our first biobrick. Subsequently, O/N cultures of the plasmid containing cultures were prepared and stored in glycerol at -80C. Furthermore, verification of catalase gene part (<a href=" http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a>), which was sent as a culture stab from the parts registry was initiated. Our newly identified biobricked-hpaC was used as a positive control, but the banding pattern was not very conclusive. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<br />
<br />
<p>PCR was reattempted on <i>Rhodoccocus</i> that was lysed using two different dilutions of the lysate buffer, but the gel verification confirmed the previous failure in using this approach. An alternative that involved preparation of an overnight culture of the <i>Rhodococcus</i> cells followed by a plasmid purification was followed. The plasmid purification eventually yielded plasmid samples with concentrations of 98.6ng/μL to 182.7ng/μL (4 samples obtained overall). Additionally, the catalase biobrick was used to transform some stock competent cells, and samples of some colonies were subsequently PCR'ed. Although, the gel verification showed some potential contamination, and the required banding patterns at around 2200bp was not obtained. </p><br />
<br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<br />
<br />
<p> PCR was attempted to amplify the genes of the <I>dsz</i> operon utilising an adapted PCR protocol with purified <i>Taq</i> polymerase that had been isolated from the host organism. Eventually, some banding pattern was obtained between 1200 and 1500 base pairs when a gradient thermocycler was used with melting temperatures ranging betweeen 55°C to 65°C. This was assumed to be indicative of successful amplification of <i>dszB</i>; however, further purification and gel verification results were inconclusive and no yield was obtained when placed tested using a nanodrop machine. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<br />
<br />
<p>Top 10 E.coli cells were transformed with <a href="http://partsregistry.org/Part:BBa_R0011">BBa_R0011</a><br />
(IPTG inducible promoter in psb1C3 backbone), and resulting colonies were tested using cPCR. <br />
Colony PCR was performed on cells containing the catalase biobrick. Catalase is 2217bp long but since biobrick primers add about 200bp, bands of 2400 bp were expected if the part was present in the biobrick. These bands were observed, indicating that the <a href=" http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a> gene was most likely present.</p><br />
<p></html>[[File:Ucalgary2012 4.7.2012 catalase colony pcr 2.jpg|thumb|500px|center|Figure 4: Colony PCR on potential colonies containing katG-LAA. Biobrick primers were used. Bands at 2200 indicate successful amplification of the catalase part within the biobrick backbone. Smaller bands are indicative of primer degradation and are nonspecific amplification. +C indicates the positive control, and RFP generator, which should amplify at around 1000 bp.]]<html></p> <p><br />
<br />
PCR using Phusion high fidelity polymerase was carried out on <i>dszA</i>, <i>dszB</i>, and <i>dszC</i> in a gradient thermocycler. Amplification of non-specific bands was present for <i>dszA</i> and <i>dszB</i>, however strong banding for the desired size of the gene was observed for both (around 1500 for <i>dszA</i>, 1100 for <i>dszB</i> </p><br />
<br />
<p></html>[[File:Ucalgary2012 6.7.2012.dszABphusionPCR.jpg|thumb|500px|center|Figure 5:DszA and DszB PCR from <i>Rhodococcus</i>. DszA amplicon is around 1300 bp, and is observed to run higher on the gel. DszB amplicon is expected to be 1098 bp, which is observed in addition to multiple nonspecific banding. +C indicates the positive control, and RFP generator, which when PCRed with biobrick primers should be around 1000 bp. As this control is running high as well, it is believed that both amplicons for DszA and DszB have been obtained. No contamination is observed in the NTC (no template control).]]<html><br />
</p> <br />
<p></html>[[File:Ucalgary2012 7.5.2012 dszC.png|thumb|500px|center|Figure 6:DszC PCR from <i>Rhodococcus</i>. The numbers above the figure show the colony number. DszC amplicon is around 1450 bp, and is observed to run at almost the right size on the gel. No contamination is observed in the NTC (no template control).]]<html><br />
<p><br />
Examining the sequences of the <i>dszABC</i> genes led to the discovery that all 4 had multiple illegal enzyme cut-sites in them that we have to eliminate before biobrick composite part construction can occur. <i>dszA</i> has four PstI cut sites, <i>dszB</i> has a PstI and a NotI and <i>dszC</i> has a PstI cut site. The Stratagene QuickChange mutagenesis procedure is going to be used to eliminate illegal cut sites with the only alteration being that Kapa HiFi polymerase would be used during the process. Primers needed for the mutagenesis were designed based on the procedure mentioned above. </p><br />
<br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<br />
<p> Following successful amplification of the <i>dsz</i> operon genes in the previous week, the genes were constructed into the <a href="http://partsregistry.org/Part:pSB1C3"> pSB1C3</a> vector. Colony PCR verifications were observed to be positive. Furthermore, the insertion of part <a href="http://partsregistry.org/Part:BBa_J13002"> BBa_J13002, (P<sub>tetR</sub>-RBS)</a><br />
in front of the previously biobricked <i>hpaC</i> was attempted. Overnight cultures were also prepared using two colonies each for <a href="http://partsregistry.org/Part:BBa_J13002"> <i>P<sub>tetR</sub>-RBS</i></a> and <a href="http://partsregistry.org/Part:BBa_R0011"> <i>P<sub>lacI</sub></i> </a> (an IPTG inducible promoter that we hope to build in front of <ahref="http://partsregistry.org/Part:BBa_B0034">an RBS site, BBa_B0034</a>). These cultures were then miniprepped to yield the respective parts.</p> <br />
<br />
<p>Additionally, <a href="http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a> was built into a <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> backbone. The construction and availability of all these parts will be critical in the construction of our overall circuit for biodesulfurization. Colonies which looked good on cPCR were used to prepare overnight cultures, and were miniprepped and sent in for sequencing verification the following day.<br />
On the side, M9 minimal media was also prepared to carry out growth experimentation and overall desulfurization capability of <i>Rhodococcus</i> when exposed to DBT. The various growth treatments were M9 Media and glucose only, M9+glucose+DBT, M9+glucose+MgSO4+/-DBT, M9+glucose+MgCl2+/-DBT. 0.008g of FeCl2.4H2O was also added to each of the tubes. Samples were then inoculated with colonies of the <i>Rhodococcus</i>.</p><br />
<br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<br />
<p>This week, while awaiting sequencing verification results which were required before we could begin the construction process, the desulfurization team initially aided in some of the tasks related to the other hydrocarbon groups. The success of the construction of <a href="http://partsregistry.org/Part:BBa_J13002"><i>P<sub>tetR</sub></i>-RBS</a><br />
with <a href="http://partsregistry.org/Part:BBa_K902057"><i>hpaC</i></a> was also explored by using forward and reverse primers of <a href="http://partsregistry.org/Part:BBa_R0040">BBa_R0040</a> (the promoter component of the composite part BBa_J13002). However, the eventual gel verification was inconclusive and sequencing results finally indicated an unsuccessful ligation. Additionally, the minimal media M9 preparation had been contaminated in the previous effort so this process was repeated to create tubes of each of the growth condition treatments detailed previously, and two repeats, one with an extra filtration step and one without was used to prepare the cultures.</p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<br />
<p> Mutagenic primers were redesigned after the initial ones were found to have premature stop codons. As part of the redesign process in constructing our overall gene circuits for desulfurization, a backbone switch of <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> into a chloramphenicol (Chlor) resistant vector was necessary. The subsequent transformed products were plated on a Chlor plate and selected colonies were used to prepare O/N cultures, then minipreped before finally being digested with enzymes EcoRI and PstI. The resulting gel verification images were inconclusive as they did not show the required banding pattern around 50bp. Meanwhile, colony PCR was run on colonies transformed with <i>katG-LAA</i> constructed into a <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> backbone, as well as the <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> +<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a> construct. <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></a> was shown to have been successfully amplified, so overnight cultures were prepared and subsequently miniprepped. On the other hand, the construct was not successful so a third attempt was carried out. Colony PCR treatments that used either <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> forward primers or <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
primers were used and the overall constructs were made either on a chlor-resistant, or ampicillin-resistant vectors. Preliminary images of the gel verification appeared to have confirmed the construct, although sequencing verification will be the final indicator of overall success. </p><br />
<br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<br />
<br />
<p>Sequencing results from the previous week's constructs were available confirming that we constructed KatGLAA in a chlor-resistant backbone. However, switching the plasmid backbone of <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> to <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> was not successful. The construction of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>+<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> was finally sent in for sequencing. Site-directed mutagenesis of the <i>dsz</i> operon was also initiated: <i>dszA</i> has four PstI cut sites; <i>dszB</i> has a PstI and a NotI site; <i>dszC</i> has two PstI cut sites. Site directed mutagenesis was started this week to change a single base pair in these genes in a way that eliminates the cut site but preserves the amino acid codons, so as to not mutate the protein coding sequence. Ohshiro 2007 demonstrated that replacing the Tyr residue at position 63 of <i>dszB</i> gene with a Phe increases the activity of the enzyme. Therefore we want to introduce the same mutation into our <i>dszB</i>.</p><p><br />
For the first attempt at mutagenesis we chose to mutate the second PstI site in <i>dszC</i> (PstI2). As a positive control for the procedure, we also performed the mutagenic PCR on a plasmid containing the β-galactosidase gene with a point mutation where the PCR would cause it to regain its function. For both mutagenesis protocols we used the Kappa Hifi kit. After confirming that the PCR worked by running some produce on a gel, the PCR products were DpnI digested, the purpose of which is to degrade the unmodified parental DNA (DpnI degrades methylated DNA only). Control PCR products were plated on an ampicillin plate containing IPTG and X-gal. The colonies that grew on the control plates were blue indicating that the mutagenesis had worked for the β-galactosidase gene. Minipreps of the O/N culture of <i>dszC</i> mutants were digested with PstI enzyme and the results indicated that the mutagenesis was successful.</p><p> <br />
Attempts to simultaneously perform all the mutations in <i>dszC</i> genes in one step using the Knight procedure failed (<a href="http://openwetware.org/wiki/Knight:Site-directed_mutagenesis/Multi_site">Knight Multi-site Mutagenesis Procedure</a>). What enables simultaneous mutations is that Taq ligase closes the gaps in PCR products after each cycle. In the protocol it instructs to use Taq ligase buffer only for the PCR/ligation protocol. We suspected that the reason this procedure did not work might be that the Kappa polymerase is not functional in Taq ligase buffer. Therefore we did some experiments on the controls in Taq ligase kit and kappa polymerase kit to find out which buffer that Kappa polymerase and Taq ligase both work best in. The result was that both enzymes work best in a buffer made of half Taq ligase buffer and half Kappa polymerase buffer. </p><br />
<br />
<br />
<br />
</html>[[File:Ucalgary2012 31.7.2012 dszc mutagenesis 5 20 and 50ng.jpg|thumb|500px|center|Figure 7: DszC PstI2 mutagenesis PCR with varying concentrations of template plasmid was performed. The gel shows the PCR products that were run on a gel. +C lanes show the PCR products of the control. The control was pWhitescript™ 4.5-kb control plasmid from Stratagene mutagenesis kit which was mutated with primers also from the kit. Bands indicate successful amplification of the plasmid.]]<br />
[[File:UCalgary 02.08.12 dszC psti digest mutagenesis.jpg|thumb|700px|center|Figure 8: The control and the mutated plasmids were digested with PstI restriction enzyme. The control is dszC biobrick. Other lanes show the plasmids purified from the cells transformed with PstI2 mutagenesis PCR products followed by digestion. The control shows three bands since it has three PstI cut sites. The other lanes show two bands which indicates one of the cut sites has been eliminated through mutagenesis. ]]<html><br />
<br />
</html>[[File:Ucalgary2012 2.8.2012 finding the right buffer for multisite mutagenesis.png|thumb|800px|center|Figure 9: The multisite mutagenesis using the Knight procedure was tried in different buffers to find out the buffer that Kappa works optimally at. All the PCRs were performed on the pWhitescript™ 4.5-kb control plasmid from Stratagene mutagenesis kit which contains the beta-galactosidase gene. Based on this gel, the optimal buffer is composed of 50% kappa buffer and 50% Taq ligase buffer.]]<html><br />
<br />
</html>[[File:Ucalgary 3.8.2012 optimal buffer for Taq ligase.png|thumb|800px|center|Figure 10: Using the control provided in the NEB Taq ligase buffer (BsteII digested lambda DNA), we tried to find if the Taq ligase enzyme can function in combinations of Kappa Hifi buffer and Taq ligase buffer. Lane two is the control which is only the digested lambda DNA. Lanes 1 and 3 show that some of the bands compared to control have been ligated together. Therefore, Taq ligase functions just as good in the buffer composed of 50% kappa hifi buffer and 50% Taq ligase as it would in its own buffer.]]<html><br />
<br />
<h2>Week 15 (August 6 - August 11)</h2><br />
<br />
<p>Sequencing results for <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A> <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> returned negative, so a 3-part ligation method was used to retry this construction. The following parts were ligated with the restriction enzymes indicated in brackets after each: <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>(EcoRI/SpeI) + <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> (XbaI/PstI) + <A HREF=" http://partsregistry.org/Part:PSB1K3">BBa_PSB1K3</A> (EcoRI/PstI). Also, the more conventional construction (only 1 insert) of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>(SpeI/PstI) + <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> (XbaI/PstI) was reattempted. Furthermore, 3-way ligations were also attempted for <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
+<A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A>+<A HREF="http://partsregistry.org/Part:PSB1K3">BBa_PSB1K3</A>, and <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a>+<a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
+<A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A>, as well as the two-way contruction of just <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> after the <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a>. After plating these transformations, colony PCRs were carried out and samples that gave an indication of being successful on the gels were used to prepare O/N cultures followed by miniprep. With regards to the site-directed mutagenesis side of the experimentation, <i>dszA</i>-PstI1 (the first PstI cut site in <i>dszA</i>) ,<i> dszB</i>-PstI and <i>dszC</i>(PstI2 mutated)-PstI1 mutagenesis were performed following the procedure explained in the previous week. The gel below shows the successful result of digest confirmation (Fig. 8). <br />
Multisite mutagenesis (Knight method) was repeated using the modified buffer (half Taq ligase buffer and half Kappa buffer). However it was not successful again. We also tried doing multisite mutagenesis using Pfu Turbo polymerase and following the Knight procedure without any buffer modifications. No successful results were observed. </p><br />
<br />
<br />
<br />
</html>[[File:Ucalgary2012 Digestion confirmation of mutagenesis in dszAPstI1, dszBPstI and dszCPstI1 (PstI2 site mutated)..jpg|thumb|800px|center|Figure 11: Confirmation of site-directed mutagensis to eliminate PstI cut sites in: <i>dszA</i>PstI1 cut site, <i>dszB</i>PstI cut site and <i>dszC</i>PstI1 (PstI2 site mutated). Note that in each case, the unmutated controls for each gene are to the left of each set of plasmids derived from mutagenic PCRs. Plasmids from cloned <i>dsz</i> genes are smaller than the bands to the right of them (which have undergone mutagenesis), indicating that in the plasmids on the right, the PstI cut site was lost. ]]<html><br />
<br />
<br />
<h2>Week 16 (August 12 - August 18)</h2><br />
<br />
<br />
<p>The progress in mutagenesis of <i>dsz</i> genes was continued from the previous week: <i>dszB</i>(PstI mutated)-Y63F and <i>dszA</i>(PstI1 mutated)-PstI3 mutagenesis. The gel below shows the digest confirmation.</p><br />
<br />
<br />
</html><br />
[[File:Ucalgary2012 15.08.2012 dszAPstI1&amp;3 dszB Psti y63f muta diges-1.jpg|thumb|800px|center|Figure 12: Digestion confirmation of biobricks of a) dszA (PstI1 mutated) PstI3 mutagenesis and b) dszB (PstI mutated) Y63F mutagenesis. Cutting dszA (PstI1 mutated) with PstI is expected to produce fragments of 2790, 333, 255, and 114bp. Cutting dszA (PstI1 and PstI3 mutated) is expected to produce fragments of 2730, 588, and 114bp. The primers for dszB-Y63F mutagenesis introduce an HpyAV cut site. pSB1C3 inteslf has two more HpyAV cut sites. Before the mutation bands of 2858 abd 310bp are expected, and after successful mutation bands of 1502, 1356, 310 are expected. Lane legend: 1- dszA (PstI1 mutated). 2-dszA (PstI1 mutated) digested with PstI. 3-dszA (PstI1 mutated) PstI3 mutagenesis c1 Digested with PstI. 4- dszA (PstI1 mutated) PstI3 mutagenesis c2 Digested with PstI. 5-dszA (PstI1 mutated) PstI3 mutagenesis c3 Digested with PstI. 6- dszA (PstI1 mutated) PstI3 mutagenesis c4 Digested with PstI. 7- Fermentas 1kb Plus Ladder. 8- Empty. 9- dszB (PstI mutated). 10-dszB (PstI mutated) digested with HpyAV. 11- dszB (PstI mutated) Y63F mutagenesis c1 digested with HpyAV. 12- dszB (PstI mutated) Y63F mutagenesis c2 digested with HpyAV. 13- dszB (PstI mutated) Y63F mutagenesis c3 digested with HpyAV. 14-dszB (PstI mutated) Y63F mutagenesis c4 digested with HpyAV.]]<html></p><br />
<br />
<p>We attempted a different approach to speed up the turnover time of the mutagenesis PCR. Briefly, after the PCR mutagenesis the PCR products were purified and then incubated with T4 polynucleotide kinase (PNK) and ligase. After heat inactivating the ligase and T4 PNK, the products were DpnI digested. Subsequently another round of DNA purification was performed. However, the results were unsatisfactory after the digest confirmation.</p><p><br />
Sequencing results came back. <i>dszA</i> (PstI1 and PstI3 mutated) and <i>dszB</i>(PstI and Y63F mutated) were good. However <i>dszC</i> (PstI1 and PstI2 mutated) had an insertion next to the PstI1 cut site. Mutagenesis was repeated on the <i>dszC</i>(PstI2 mutated).<br />
<i>dszB</i>(PstI and Y63F mutated)-NotI and <i>dszA</i>(PstI1 and PstI3 mutated)-PstI4 mutagenesis were also performed.</p><br />
<p> To investigate the desulfurisation capability of the <i>Rhodococcus</i> sp. from which we cloned the <i>dsz</i> operon, a desulfurization assay was prepared by inoculating different treatments of M9 media. We also prepared some solutions that will be needed for analysis in the following week: a conditioning agent composed of 100ml of 95% ethanol, 50ml glycerol, 30ml of 12M HCl (aq) and 70g of NaCl(s) was prepared. The assay relies on the turbidity of a sample containing sulphate ions which are precipitated (hence the turbidometric nature of the assay) upon adding BaCl2(s), therefore if the <i>dsz</i> pathway is active, we expect a more turbid solution to form than in control samples. </p><br />
<br />
<br />
<br />
<h2>Week 17 (August 19 - August 25)</h2><br />
<br />
<br />
<p>This week, progress was made in determining the desulfurization activity of our <i>Rhodococcus</i> strain as measured by the sulfate release using a turbidometric assay. We encountered several challenges in our prescribed protocol as the concentrations that we used to prepare the standard curve may have been too dilute, or the composition of out conditioning agent may have been flawed. Additionally, steps were taken to determine the decomposition of DBT to 2-HBP through Gas Chromatograph-Mass Spectroscopy (GC-MS) analysis, but due to a preparation error, the DBT was added to a growth solution of M9 media prematurely and the autoclaving process decomposed the DBT releasing a yellow colouration into the solution. These two approaches in determining the desulfurization capability of the <i>dsz</i> operon will be further investigated. </p><br />
<br />
<p>Since the <i>dszC</i> second mutagenesis had proven to be unsuccessful last week, the <i>dszC</i>(PstI2 mutated)PstI1 mutagenesis was repeated. Also <i>dszA</i>(PstI1,3,4 mutated) PstI2 mutagenesis was performed. <i>dszA</i> and <i>dszC</i> were sent for sequencing on Wednesday. <i>dszB</i> was sent for sequencing on Friday. Sequencing results of <i>dszA</i> and <i>dszC</i> were back by Friday. <i>dszC</i> was successful. However, <i>dszA</i> contained an insertion next to the binding site of PstI4 cut sit, so the last two mutations must be redone. <i>dszB</i>(PstI and Y63F mutated)-NotI-mutagenesis was also repeated in case the result of the sequencing was not successful. These constructions were repeated. <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>-<A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
-<i>dszC</i> constructions were attempted, however they were not successful as indicated by colony PCR. Constructions of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>/<i>hpaC</i> were carried out and also came back negative in sequencing, however <a href="http://partsregistry.org/Part:BBa_K902059">BBa_B0034<br />
/<i>katG-LAA</i> (BBa_K902059)</a> was sequence confirmed.</p><br />
</html>[[File:Ucalgary2012 20.8.2012 dszC(psti2)psti1 mutagenesis.png|thumb|700px|center|Figure 13: Another attempt to mutagenize dszC(PstI2 mutated)at PstI1 site. The gel shows the confirmation digestion. C is the control which is dszC(PstI2 mutated). Since the control has two PstI cut sites, two bands are observed on the gel. The mutagenesis has been successful since only one band is observed on the other lanes indicating that one of the cut sites has been eliminated during mutagenesis.]]<html><br />
<br />
</html>[[File:Ucalgary2012 21.8.2012 dszA final mutagenesis digest.png|thumb|700px|center|Figure 14: DszA (PstI1, PstI2, PstI3 mutated)PstI4 mutagenesis digestion confirmation gel. All the lanes show the results of the plasmids being cut with PstI enzyme. C indicates the lane containing the control which is dszA(PstI1, PstI3 and PstI4 mutated). The other lanes are the digestions of the plasmids that are mutagenized, from different colonies. Control plasmid has two PstI cut sites and therefore the two bands observed on the gel were expected. The other lanes show only one band of about 3500bp which is about the right size for dszA in a psb1c3 vector. Therefore one of the cutsites has been eliminated during mutagenesis.]]<html><br />
<br />
<br />
<br />
<br />
<h2>Week 18 (August 26 - September 1)</h2><br />
<br />
<p> <A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i> (BBa_K902052)</i></A> sequencing results came back as successful. <i>dszA</i>(PstI1,3 mutated)-PstI2-mutagenesis was performed and sent for sequencing. Also <i>dszA</i>(PstI1,2,3 mutated)-PstI4-mutagenesis was performed, and this was also sent for sequencing. </p><br />
<p>Constructions of <A HREF="http://partsregistry.org/Part:BBa_K902053">P<sub>tetR</sub>-RBS/<i>dszB</i> (BBa_K902053)</a> and <a href="http://partsregistry.org/Part:BBa_K902056">BBa_B0034<br />
/<i>dszC</i> (BBa_K902056)</a> were attempted, verification digested, and sent for sequencing. Sequencing results for these constructs came back as positive, along with successful mutagenesis of <A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i> (BBa_K902050)</A>.</p><br />
<p>At this point, all of the <i>dsz</i> genes have been successfully made biobrick compatible, and <i>hpaC</i> has been biobricked. We have also successfully constructed <a href="http://partsregistry.org/Part:BBa_K902059">BBa_B0034 with <i>katG-LAA</i></a> to be used in the optimization circuit, as well as <A HREF="http://partsregistry.org/Part:BBa_K902053"><i>P<sub>tetR</sub>-RBS</i>/<i>dszB</i> (BBa_K902053) </a>and <a href="http://partsregistry.org/Part:BBa_K902056">BBa_B0034/<i>dszC</i> (BBa_K902056)</a>.<br />
<p> Constructions of <A HREF="http://partsregistry.org/Part:BBa_J04500">P<sub>lacI</sub>-RBS</A>with <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> were performed. As well, attempts to construct <A HREF="http://partsregistry.org/Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></a>/<a href="http://partsregistry.org/Part:BBa_K902056">RBS-<i>dszC</i></a> as well as <A HREF="http://partsregistry.org/Part:BBa_J13002">P<sub>tetR</sub>-RBS</A>/<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
/<A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i></A>, and <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>/<A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> were also carried out. These parts are intended as construction intermediates towards building the final systems, as well as providing a way of testing the genes functionality (namely, to test HpaC for oxidoreductase activity and to test if over-expression of KatG in the cell will increase its ability to survive H<sub>2</sub>O<sub>2</sub> stress). Transformations of all these constructions were carried out at the end of the week.</p><br />
<br />
<h2>Week 19 (September 2- September 8)</h2><br />
<p>Confirmation digests on colonies of the previous constructions that gave bands of the expected size with cPCR were performed. Positive results were found for colonies of <A HREF="http://partsregistry.org/Part:BBa_J04500"><i>P<sub>lacI</sub>-RBS</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <A HREF="http://partsregistry.org/Part:BBa_J04500"><i>P<sub>lacI</sub>-RBS</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
/<A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i></A>. Sequencing was sent, and results indicated that the constructions of <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS/hpaC</i> (BBa_K902058)</a> were successful, meaning that after many months of trying we FINALLY have a promoter in front of the <i>hpaC</i> gene and can proceed to test the parts functionality. Attempts to construct <i>hpaC</i> with the (<A HREF="http://partsregistry.org/Part:BBa_J13002">evil TetR promotor, BBa_J13002</A>) were abandoned, as it was believed that this construction was failing due to toxicity of over-expressing the protein, and it was determined that this part was not necessary after all. <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub>-RBS/dszB</i>, (BBa_K902054)</a> also worked, though this was less exciting. <a href="http://partsregistry.org/Part:BBa_K902051">BBa_B0034<br />
/<i>dszA</i></a> came back as a bad read despite looking very good on the confirmation digest gel, so this part will be resent for sequencing. Constructions of <A HREF="http://partsregistry.org/Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> were reattempted, and confirmation digests for this part looked good, and so samples were sent for sequencing.</p><br />
<br />
<br />
<h2>Week 20 (September 9- September 15)</h2><br />
<p>Construction attempts on <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> were performed. Colonies grew for the constructions, however further confirmation results were dissapointing (only 2 clones of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a> appeared to have been successful). These clones were sent for sequencing, and constructions were reattempted.</p><p> However, when sequencing came back, somehow reads indicated that these clones were in fact a gene from the Denitrogenation project (which is 990bp and a completely different band then what we saw on the gel). We believe, somewhere, something has gone very wrong- further investigation into this will be carried out. In the meantime, the above constructions were reattempted, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902051">BBa_B0034/<i>dszA</i></A> was re-prepped in case a contaminant in the plasmid stock was to blame for the bad reads found in this batch of sequencing as well as the last. In addition, plasmid switches of multiple sequence confirmed parts into a <a href=" http://partsregistry.org/Part:pSB1C3"> pSB1C3</a> backbone were carried out. </p><br />
<br />
<h2>Week 21 (September 16- September 22)</h2><br />
<p>Colonies for the transforms of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> have been few and far between, and cPCR results are always discouraging. Sequencing results for other sections of the project have once again come back very confusing, and further research continues into the source of this madness. </p><br />
<p> The sulfur compound degradation assay was set up to test the desulfurization rate of the original <i>Rhodococcus baikonurensis </i> (refer to the protocol page).</p><br />
<br />
<br />
<h2>Week 22 (September 23- September 29)</h2><br />
<p>Attempts to construct <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, , and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> continue. In the meantime, <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> was tested for functionality. In order to do this, cultures of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> were grown up overnight in LB media. A strain carrying <A HREF="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</A> only was used as a negative control. The following morning, 20 &micro;L of each culture was inoculated into 3 mL of LB with various concentrations of hydrogen peroxide; 0 mM, 1 mM, 5 mM, and 10 mM. These cultures were then allowed to grow overnight, and culture turbidity was observed. It was found that the negative control exhibited no growth after 12h at 1 mM peroxide, however cultures with induced expression of catalase were turbid after 12 h of growth at this concentration (Fig. 10). This demonstrated the ability of the catalase to protect the cells from excessive peroxide concentrations.</p><p><br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 15: Catalase Assay. Overnight cultures of J04500 and J04500-<i>KatGLAA</i> were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbitity was observed.]]<html></p><br />
</p><br />
<p>In addition to this, activity of <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-hpaC</i></a> was tested. In order to do this, cultures of <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-hpaC</i></a> and <a href="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub>-dszB</i></a> were grown up overnight in LB with appropriate antibiotics. Following this, protein expression was induced with IPTG, after which the assay was carried out as described in the following figure and on the protocols page.</p><br />
<p> </html> <br />
[[File:Ucalgary2012 DesulfurizationGroup HpaC assayTake1.png|center|600px|thumb|Figure 16: HpaC Assay. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 2 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, 1 mL of cell lysate was transferred to a cuvette, and a spectrophotometer was blanked at 340 nm (maximal absorbance of NADH) with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p>When the assay was run, it was found that NADH does not convert readily to NAD+ on its own. When cell lysate containing the naturally expressed amounts of oxidoreductase was added, a decrease in absorbance could quickly be observed as the NADH was converted to NAD+. When cultures over-expressing HpaC were tested, the absorbance levels were found to start much lower than the control. We believe that this is because with the amount of cell lysate tested, when the HpaC protein is overexpressed the NADH is consumed almost immediately and therefore the data reflecting the drop in absorbance is missed. Further tests will use differing amounts of cell lysate in order to try to capture data that shows the drop in absorbance for HpaC cultures.</p><br />
<br />
<h2>Week 23 (September 30-October 3): Wiki-Freeze PANIC!!</h2><br />
<br />
<p>The GCMS results of the sulfur assay were received. DBT desulfurization was successful (figures below). Some compounds were not detected by GCMS since they were too polar. However, 2 additional compounds showed degradation in addition to DBT, indicating that the pathway has a wider substrate specificity!</p><br />
<p></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 17: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol in the protocols section for details). Samples were taken out at different time points and were run through GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria, run through the GCMS after 6 days of being in the incubator to account for abiotic degradation. ]]<html></p<br />
<br />
<p></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 18: The peak in this mass spectra demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound ('''A:''' dibenzothiophene, '''B:''' tetrahydro-4h-thiopyran-4-one, and '''C''': benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdown. Degradation is seen for DBT (the model studied compound) as well as other sulfur containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<p>The HpaC assay was repeated using 100 &micro;L of cell lysate instead of 1 mL of 2x concentrated cell lysate, following the previously used protocol. With this smaller amount, a clear sharp decrease in the absorbance of NADH can be observed, indicating a very fast conversion by HpaC, and further confirming that the part was functional.</p><br />
<br />
<p></html>[[File:Ucalgary2012Desulfurization-Hpacasay2.PNG|center|550px|thumb|Figure 20: HpaC Assay. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, 100 &micro;L of cell lysate was transferred to a cuvette, and a spectrophotometer was blanked at 340 nm (maximal absorbance of NADH) with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<h2>Week 24 (October 4- October 11)</h2><br />
<p>This week, before the Americas West Regional Competition, attempts to construct the previously mentioned parts continued. Unfortunately, they were unsuccessful.<br />
<br />
<h2>Week 25 (October 16- October 20)</h2><br />
<p>Traditional attempts to build the sulfur operon and its sub-parts were repeated, however attempts failed once more. During this week, it was decided that due to time consstraints, alternative approaches to construct the operon would be undertaken. Due to this, splice-overlap-extention (SOE) PCR primers (containing scar-sites where appropriate) were designed according to <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/soe">protocol</a></b> and ordered.</p><br />
<p><br><br />
<br />
<br />
Primer: 1a (BBK)-J13/DszB F - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGTCCCTATCAGTGATAGAGATTGACATCCC-3'<br />
<br><br><br />
Primer: 1b (BBK)-J04/DszB F - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 2 Jxx/B-(RBS/C) R - 5'-GGTGACAGTGTCATCTAGTATTTCTCCTCTTTCTAGTACTATCGGTGGCGATTGAGGCTGTTG-3'<br />
<br><br><br />
Primer: 3 (Jxx/B)-RBS/C F - 5'-CAACAGCCTCAATCGCCACCGATAGTACTAGAAAGAGGAGAAATACTAGATGACACTGTCACC-3'<br />
<br><br><br />
Primer: 4 RBC/C-(RBS/A) R - 5'-GCCAGATGCATTTGTCGTTGTTGAGTCATCTAGTATTTCTCCTCTTTCTAGTATCAGGAGGTGAAGCCGGGAATCG-3'<br />
<br><br><br />
Primer: 5 (RBS/C)-RBS/A F - 5'-CGATTCCCGGCTTCACCTCCTGATACTAGAAAGAGGAGAAATACTAGATGACTCAACAACGACAAATGCATCTGGC-3'<br />
<br><br><br />
Primer: 6 RBS/A-(J04/HpaC) R - 5'-GGGGAGAGGCGGTTTGCGTATTGCTAGTATCATGAAGGTTGTCCTTGCAGTTGTGG-3'<br />
<br><br><br />
Primer: 7 RBS/A-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATCATGAAGGTTGTCCTTGCAGTTGTGG-3'<br />
<br><br><br />
Primer: 8 (BBK)-J04/HpaC F - 5'-CGAATTCGCGGCCGCTTCTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 9 (RBS/A)-J04/HpaC F - 5'-CCACAACTGCAAGGACAACCTTCATGATACTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 10 J04/HpaC-(RBS/Kat) R - 5'-CTGACGTGCTCATCTAGTATTTCTCCTCTTTCTAGTATTAAATCGCAGCTTCCATTTCCAGCATCAC-3'<br />
<br><br><br />
Primer: 11 J04/HpaC-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTAAATCGCAGCTTCCATTTCCAGCATCAC-3'<br />
<br><br><br />
Primer: 12 (J04/HpaC)-RBS/Kat F - 5'-GTGATGCTGGAAATGGAAGCTGCGATTTAATACTAGAAAGAGGAGAAATACTAGATGAGCACGTCAG-3'<br />
<br><br><br />
Primer: 13 RBS/Kat-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTATTAAGCAGCCAGAGCGTAGTTTTCGTC-3'<br />
</p><br><br />
<p><br />
Colony PCR was done on potential colonies of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, , and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A>. Faint bands around the expected size were seen, and these were stabbed with a pipette tip and stirred into water in order to be used as template in a further round of PCR to get amplification of the part. The plan was that these amplicons could then be used as an insert in further constructions, however no amplification was seen. Further optimization of this procedure could possibly result in a short-cut to construction, however the time needed to do this is a luxury that we do not have.</p><p>Additional attempts to create inserts by doing PCR on ligation reactions in addition to gel extraction of colony PCR bands were attempted as well, however these attempts were unsuccessful as well. <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/soe">SOE PCR</a> and possible <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Gibson_Assembly">Gibson Assembly</a> appear to be the Sulfur Teams' last hope at this point, and we are eagerly awaiting our primers.<br />
</p><br />
<br />
<br />
<h2>Week 26 (October 21- October 26)</h2><br />
<p><br />
Much to our delight, SOE PCR primers were recieved this week. Therefore, alongside traditional construction of the aforementioned parts, SOE PCR was carried out according to <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/soe">protocol</a> and these parameters:</p><br />
<br><br />
<h4>Round 1:</h4><p><br />
<ul><br />
<li>Primers 1a and 2 with <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A> as a template = Product L1 (J13002/DszB with BBk prefix and B0034/DszC overlap)<br />
<br></li><li><br />
Primers 1b and 2 with <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A> as a template = Product L2 (J04500/<i>dszB</i> with BBk prefix and B0034/<i>dszC</i> overlap)<br />
<br></li><li><br />
Primers 3 and 4 with <A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a> as a template = Product L3 (B0034/<i>dszC</i> with <i>dszB</i> and B0034/<i>dszA</i> overlap)<br />
<br></li><li><br />
Primers 5 and 6 with <A HREF="http://partsregistry.org/Part:BBa_K902050"><i>dszA</i></a> as a template = Product L4 (B0034/<i>dszA</i> with <i>dszC</i> and J04500/<i>hpaC</i> overlap)<br />
<br></li><li><br />
Primers 5 and 7 with <A HREF="http://partsregistry.org/Part:BBa_K902050"><i>dszA</i></a> as a template = Product L5 (B0034/<i>dszA</i> with <i>dszC</i> overlap and BBk suffix)<br />
<br></li><li><br />
Primers 8 and 10 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L6 (J04500/<i>hpaC</i> with BBk prefix and B0034/<i>katG-LAA</i> overlap)<br />
<br></li><li><br />
Primers 9 and 10 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L7 (J04500/<i>hpaC</i> with <i>dszA</i> and B0034/<i>katG-LAA</i> overlap)<br />
<br></li><li><br />
Primers 9 and 11 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L8 (J04500/<i>hpaC</i> with <i>dszA</i> overlap and BBk suffix)<br />
<br></li><li><br />
Primers 12 and 13 with <A HREF="http://partsregistry.org/Part:BBa_K902059"><i>RBS-katG-LAA</i></a> as a template = Product L9 (B0034/<i>katG-LAA</i> with J04500/<i>hpaC</i> overlap and BBk suffix)<br />
</li></ul><br />
</p><br />
<p><br />
</html>[[File:Ucalgary2012 Spliceoverlapextentionpcr1.png|center|thumb|800px|Figure 21: Round 1 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with varying amounts of DNA template as indicated above the wells. Cycling conditions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 55%deg;C for 1 min, 72%deg;C for 2 min. 30 s.)x 31, Stage 3 (72%deg;C for 10 min.). Bands seen at ~1500 bp for L1 through L5, ~800 bp for L6-L8, and ~2200 bp for L9 indicate successful amplification of the desired product.]]<html><br />
</p><br />
<h4>Round 2</h4><br />
<p><br />
<ul><br />
<li>Primers 1a and 4 with templates L1 and L3 = Product X1 (J13002/<i>dszB</i>/B0034/<i>dszC</i> with BBk prefix and B0034/<i>dszA</i> overlap)</li><br />
<li>Primers 1b and 4 with templates L2 and L3 = Product X2 (J04500/<i>dszB</i>/B0034/<i>dszC</i> with BBk prefix and B0034/<i>dszA</i> overlap)</li><br />
<li>Primers 3 and 6 with templates L3 and L4 = Product X3 (B0034/<i>dszC</i>/B0034/<i>dszA</i> with <i>dszB</i> and J04500/<i>hpaC</i> overlap)</li><br />
<li>Primers 3 and 7 with templates L3 and L5 = Product X4 (B0034/<i>dszC</i>/B0034/<i>dszA</i> with <i>dszB</i> overlap and BBk suffix)</li><br />
<li>Primers 5 and 10 with templates L4 and L7 = Product X5 (B0034/<i>dszA</i>/J04500/<i>hpaC</i> with <i>dszC</i> and B0034/<i>katG-LAA</i> overlap)</li><br />
<li>Primers 5 and 11 with templates L4 and L8 = Product X6 (B0034/<i>dszA</i>/J04500/<i>hpaC</i> with <i>dszC</i> overlap and BBk suffix)</li><br />
<li>Primers 8 and 13 with templates L6 and L9 = Product X7 (J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with BBk prefix and suffix)</li><br />
<li>Primers 9 and 13 with templates L7 and L9 = Product X8 (J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with <i>dszA</i> overlap and BBk suffix)</li><br />
</ul></p><p><br />
</html>[[File:Ucalgary2012 Spliceoverlapextentionpcr2.png|center|thumb|800px|Figure 22: Round 2 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with varying amounts of gel-extracted DNA template from the previous PCR round as indicated above the wells. Bands were extracted using Qiagen QIAquick Gel Extraction Kit. Cycling conditions for the PCR reactions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 65%deg;C for 1 min, 72%deg;C for 3 min. 30 s.)x 31, Stage 3 (72%deg;C for 10 min.). Faint bands seen at ~3000bp indicate amplification of the desired product.]]<html> <br />
</p><br />
<h4>Round 3</h4><br />
<p><br />
<ul><br />
<li>Primers 1a and 10 with templates X1 and X5 = Product Z1 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with B0034/<i>katG-LAA</i> overlap and BBk prefix)</li><br />
<li>Primers 1a and 11 with templates X1 and X6 = Product Z2 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with BBk prefix and suffix)</li><br />
<li>Primers 1a and 7 with templates X1 and L5 = Product Z3 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 10 with templates X2 and X5 = Product Z4 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with B0034/<i>katG-LAA</i> overlap and BBk prefix)</li><br />
<li>Primers 1b and 7 with templates X2 and L5 = Product Z5 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 11 with templates X2 and X6 = Product Z6 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 6 with templates L2 and X3 = Product Z7(J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with J04500/<i>hpaC</i> overlap and BBk prefix)</li><br />
<li>Primers 1b and 7 with templates L2 and X4 = Product Z8 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1a and 6 with templates L1 and X3 = Product Z9 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with J04500/<i>hpaC</i> overlap and BBk prefix)</li><br />
<li>Primers 1a and 7 with templates L1 and X4 = Product Z10 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 5 and 13 with templates X5 and X8 = Product Z11 (B0034/<i>dszA</i>/J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with <i>dszC</i> overlap and BBk suffix)</li><br />
</ul></p><p><br />
</html>[[File:Ucalgary2012 sulfurSpliceoverlapextentionpcr3.png|center|thumb|800px|Figure 23: Round 3 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with 50 ng of gel-extracted DNA template from the previous PCR round. Bands were extracted using Qiagen QIAquick Gel Extraction Kit. Cycling conditions for the PCR reactions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 65%deg;C for 1 min, 72%deg;C for 6 min.)x 31, Stage 3 (72%deg;C for 10 min.). A faint band (~6000bp) can be seen in lane 4, indicating possible amplification of the desired product. Other reactions appear to have failed, as this bands are not present elsewhere.]]<html> <br />
</p><br />
<p><br />
Though it appears that S.O.E. PCR consists of crushed dreams and lies (or that extensive optimization would have to be performed in order to amplify the correct product), extraction was carried out on the single band seen, in the hopes that a subsequent round of PCR with the terminal primers for this product would possibly amplify it to a concentration that can be used in construction in the next week to assemble and test the sulfur operon constructs before competition. Conveniently, the first round of PCR creates <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Gibson_Assembly">Gibson Assembly</a> compatible products. Because a kit is available in the lab, this will be attempted in the following days as well. Meanwhile, a round of traditional construction appears to have finally created the parts <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902079"><i>P<sub>tetR</sub></i>-<i>dszB</i>-B0034-<i>dszC</i></A>, <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902080"><i>P<sub>lacI</sub></i>-<i>dszB</i>-B0034-<i>dszC</i></A>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902081"><i>P<sub>lacI</sub></i>-<i>hpaC</i>-B0034-<i>katG</i></A>. Though sequence confirmation could not be obtained before Wiki-Freeze, we are confident that these parts are correct due to the clear size difference seen between these parts and their sub-parts. A plasmid switch was attempted the night before DNA submission had to be sent, however due to technical issues the incubator stayed at a low temperature overnight, and cultures did not grow enough to obtain the first two parts in a pSB1C3 backbone. Because of the difficulty assembling these parts, the deadline for DNA submission, and the fact that having them in an alternate backbone in the registry would allow for easier and quicker assembly of the sulfur operon by others (no plasmid switches of large parts would have to be performed, as we have found that plasmid switching the larger constructs is quite difficult.) we decided to submit these parts in an ampicillin backbone. Further attempts to switch the backbone for these parts will continue, and hopefully we will be able to submit them in the standard backbone at a later date before the competition. In addition, a reconstruction of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902051">B0034-<i>dszA</i></A> was obtained and finally, at long last, sequence confirmed. Because of the verification of this part and the excellent sequence read when compared to those obtained for the previously submitted version, we decided to also resubmit this part to the registry.</p><p>Construction attempts of the operon will continue into the following week, as only 1 additional construction is needed to assemble a testable version of the operon. Hopefully, this data will be obtained before the competition, and documented on the respective parts pages.</p><br />
<p><br><br>This is the Desulfurization Team, signing off.</p><br />
</h><br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/OSCAR/DesulfurizationTeam:Calgary/Project/OSCAR/Desulfurization2012-10-27T01:17:11Z<p>Lisa.O: </p>
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<h2>Why Remove Sulfur?</h2><br />
<br />
<p align="justify"><br />
Sulfur is the third most abundant element in crude oil (Ma, 2010), and when sulfur containing hydrocarbons are burned they release S0<sub>2</sub> and S0<sub>3</sub> gasses into the atmosphere. Not only does this reduce the efficiency and value of our product, but it also contributes to global warming, acid rain, and various health issues due to the pollution (Reichmuth <i>et al</i>., 2000). Strict regulation on sulfur in fuels are now in place and low-sulfur gasoline is mandated across all of Canada (Source: Environment Canada). To upgrade the quality of our fuel we need to remove the sulfur but keep the hydrocarbon backbone.</p><br />
<br />
<h2>Our Vision</h2><p align="justify"><br />
Though a few pathways for biodesulfurization exist in the microbial world, most involve the destruction of part of the carbon skeleton (an example would be the Kodama pathway)(Soleimani <i>et al</i>., 2007). This would effectively reduce the quality of our product. With this in mind the pathway we have chosen is the 4S pathway found in <i>Rhodococcus spp</i>. It has been characterized and shown to remove sulfur from the model substrate dibenzothiophene (DBT) and convert it to 2-hydroxybiphenyl (2-HBP) in a non-destructive manner. DBT and its derivatives make up 70% of the organic sulfur compounds found in crude oil (Ma 2010), and are also some of the most difficult to remove through chemical means. By using the 4S pathway we will be able to upgrade our fuel and remove recalcitrant compounds at the same time. <br />
</p><br />
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</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|750px|thumb|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD.]]<html></p><br />
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<h2>4S pathway</h2><br />
<p align="justify"><br />
Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO<sub>2</sub>) through the addition of 2 oxygen atoms to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the sulfur, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-HBP. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.</p><p align="justify"><br />
The first three steps of the 4S pathway require FMNH<sub>2</sub> and subsequently reduces the reductive power of the cell. WIn order to regain this power an oxidoreductase (DszD) uses NADH to recycle the FMNH<sub>2</sub>, allowing the reaction to proceed. Without DszD the desulfurization pathway would grind to a halt.</p><p align="justify"><br />
The <i>dszA</i>,<i>B</i>, and <i>C</i> genes form an operon on the pSOX plasmid of <i>R. erythropolis</i>, while <i>dszD</i> is found in the chromosome. Naturally this pathway is slow, however using synthetic biology approaches this process can be optimized.</p><br />
<br />
<h2>Our Approach</h2><br />
<a name="Degradation"></a><h3>1) Find the genes!</h3><br />
<p align="justify">We isolated the plasmid containing the <i>dsz</i> genes from a desulfurising environmental isolate of <i>Rhodococcus</i> using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">modified miniprep procedure</a>. As the native promoter has been shown to be repressed by various sulfur-containing compounds (Li <i>et al</i>., 1996), we designed primers for just the coding sequences of the <i>A, B, </i> and <i>C</i> genes. As these genes all have some illegal cutsites in them we constructed them into the PSB1C3 vector and started our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis protocol</a>.</p><br />
<p align="justify"> We performed an experiment to measure the desulfurization rate of select organosulfur compounds by our <i>Rhodococcus</i> strain (Figures 4-6 below). These experiments monitored the degradation of the compounds by our strain over time. We discovered that the <i>dsz</i> operon is capable of desulfurizing a wider range of compounds than just the commonly studied DBT. This shows that this pathway could be a promising solution for degradation of a wide variety of sulfur containing toxins, including those that resemble naphthenic acids. </p> <br />
<br />
<p align="justify"></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 2: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through the GC/MS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GC/MS after 6 days of incubation. ]]<html></p><br />
<br />
<p align="justify"></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 3: The peak in this mass spectrum demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<p align="justify"><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound ('''A:''' dibenzothiophene, '''B:''' tetrahydro-4h-thiopyran-4-one, and '''C:''' benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdown. Degradation is seen for DBT as well as other sulfur-containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<h3>2) Mutagenesis: Biobrick Compatability and Increasing DszB Activity </h3><br />
<p align="justify">In total the <i>dszABC</i> genes had 7 PstI sites and 1 NotI site that needed to be mutated for the biobrick standard. The primers were designed such that the site was removed without the amino acid being changed. In addition, a point mutation of Y63F in DszB increased the activity of the protein (Oshiro <i>et al</i>., 2007), and was included in the mass mutagenesis we undertook. Mutagenesis was performed as described in <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">this protocol.</a></p><br />
<br />
<a name="catalase"></a><h3>3) Replacing DszD with HpaC & Introducing Catalase </h3><br />
<p align="justify"><br />
As FMNH<sub>2</sub> is consumed in the first three steps of the pathway it needs to be regenerated or the process will grind to a halt. This usually falls to the <i>dszD</i> gene, however it has been shown that the <i>hpaC</i> gene from <i>E. coli</i> performs the same function more efficiently (Gala´n <i>et al</i>., 2000). One problem arises from this though, as high levels of FMNH<sub>2</sub> cause the production of toxic hydrogen peroxide inside the cell (Gala´n <i>et al</i>. 2000). To address this issue we have included a catalase gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"> <i>P<sub>lacI</sub>-katG-LAA</i></a>) that will remove the peroxide that would be toxic to the cell.</p><br />
<br />
<p align="justify"></html>[[File:Ucalgary_sulfur_constructs_KatandHpaC.PNG|center|250px|thumb|Figure 7: Diagrammatic representation of the full "optimization circuit", consisting of the oxidoreductase HpaC and a catalase (KatG).]]<html></p><br />
<br />
<br />
<h3>Results</h3><br />
<p align="justify">To show that catalase activity increased <i>E. coli</i> survivability in peroxide we cultured the inducible catalase against a catalase-free control with varying levels of peroxide. After growing overnight the negative didn't grow in any culture except in the absence of peroxide, while the catalase cultures could tolerate peroxide. This is shown below.</p><p align="justify"><br />
<br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 8: Catalase Assay. Overnight cultures of P<sub>lacI</sub> and P<sub>lacI</sub>-KatGLAA were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbidity was observed. It was found that at 1 mM of peroxide, cultures with just the lacI promotor perished, however when KatG-LAA was expressed, the cells survived.]]<html></p><br />
<br />
<br />
<p align="justify">To test the action of HpaC to use NADH to recycle FMN into FMNH<sub>2</sub> cell lysates were exposed to NADH and it's absorbance at 340nm (Kamali <i>et al</i>., 2010) was measured over time. Both native HpaC expression and an induced <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> system were tested as well as a negative control. The results are shown below.</p><br />
<br />
<p align="justify"> </html> <br />
[[File:Ucalgary2012 HpaC assaycumulativeforthedatapage.png|center|850px|thumb|Figure 9: HpaC Assay with '''A)''' 2 mL cell lysate and '''B)''' 100 &micro;L cell lysate. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, various amounts of cell lysate were transferred to a cuvette, and a spectrophotometer was blanked at 340 nm with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p align="justify">The assay showed that NADH does not abiotically convert into NAD+, however the native expression of HpaC did show a steady decrease in the levels of NADH. The induced overexpression of HpaC caused extremely rapid conversion into NAD+ as reflected by a sharp drop in the absorbance of NADH (see figure B). This drop was much sharper than what was seen when native levels of oxidoreductases were tested, showing that the <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> was functional and that it would effectively recycle FMN.</p><br />
<br />
<br />
<a name="UBC"></a><h3>4) Optimizing Gene Order</h3><br />
<br />
<p align="justify">Further optimization of the system was achieved through reorganization of the reconstructed operon. Natively the genes are arranged ABC, however the catalytic efficiency of the protein products are 25:1:5 for A:B:C respectively (Li <i>et al</i>., 2008). By rearranging the genes into BCA there is stronger transcription of the weaker proteins, giving a more balanced system overall. These would all be constructed with the same strong ribosomal binding site, <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>.</p><p align="justify"><br />
<br />
</html>[[File:DszOperonOptimize.png|center|400px|thumb|Figure 10: Method of optimizing gene order. The top circuit represents that found natively in the organism, with the bottom circuit representing our modified version.]]<html><br />
</p><br />
</a><h2>Final Sulfur Constructs</h2><br />
<p align="justify">After all of the above considerations are met, four final constructs for our system will be made to allow us to test desulfurization under different conditions.</p><p align="justify"><br />
<br />
</html>[[File:WikiConstructs_ucalgary_sulfur_2012_final_systems.png|center|700px|thumb|Figure 11: First set of final constructs for the desulfurization operon, with constitutive Dsz expression and inducible expression of the optimization proteins; either HpaC on its own or coexpressed with KatG]]<html></p><br />
<br />
<p align="justify"><br />
The first two constructs have the modified <i>dsz</i> operon (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902052"><i>dszB</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><i>dszC</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902050"><i>dszA</i></a>) under the control of a constitutive TetR promotor (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>) This is to allow for the testing of the optimization circuit, which is under the control of a lacI promotor inducible by IPTG (<a href="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</a>). The set-up of these two constructs will therefore allow for the expression of the <i>dsz</i> genes with the ability to test and compare their desulfurization rates <br> A) On their own <br> B) With the addition of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> <br> C) With the addition of both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K137068"><i>katG-LAA</i></a></p><br />
<br />
<p align="justify">This will allow us to determine what the optimal construct and expression levels of the additional genes must be in order to have the most effective sulfur removal system.</p><br />
<br />
</html>[[File:WikiConstructs2 sulfur ucalgary induciblesytems.PNG|center|700px||thumb|Figure 12: Second set of final constructs for the desulfurization operon, with all genes under an IPTG inducible promotor.]]<html><br />
<br />
<p align="justify"><br />
Due to the large number of proteins being expressed in this system, the possibility of forming inclusion bodies is present. As such, a backup system was built where both the optimization circuit and the <i>dsz</i> operon were under the control of the inducible lacI promoter. This system would allow us to tune the expression of the genes, and determine which expression level is optimal for desulfurization in our bioreactor.</p> <br />
<br />
<p align="justify">Currently the final steps of construction of these constructs is underway, following which functionality tests will begin.</p><br />
<br />
<br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/FRED/DetectingTeam:Calgary/Project/FRED/Detecting2012-10-27T01:12:07Z<p>Lisa.O: </p>
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<div>{{Team:Calgary/TemplateProjectGreen|<br />
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<p align="justify"><br />
This year, our team wanted to identify a novel responsive element capable of detecting and quantifying different tailings ponds toxins (e.g. naphthenic acids, NAs) in solution. While numerous studies have begun to identify species of bacteria capable of surviving and sensing a variety of toxic compounds (e.g. NAs), the degradation pathways have not yet been fully characterized. Therefore, we needed to design and implement novel approaches to efficiently isolate the genetic elements that detect and potentially lead to the breakdown of these toxins.<br />
</p><br />
<h2>Transposons: What, How, Why?</h2><br />
<p align="justify"><br />
The transposable element (TE), Tn5, is a conservative transposon that can insert a segment of genes bordered by specific 19bp insertion sequences from one part of the genome (e.g. plasmid vector) randomly to another location like a chromosome (Reznikoff, 2008). The transposition event is catalyzed by a transposase enzyme encoded by <i>tnp</i> gene included in the TE. Using the appropriate selective pressure, the insertion can be maintained permanently in the genome.</p><br />
<br />
</html>[[File:Transposon.jpg|thumb|700px|center|Figure 1: "Transposition reaction from plasmid entry into the recipient cell to integration of the transposon into the genome. Modified from Transposons: Shifting Segments of the Genome" by McGraw Hill]]<html><br />
<br />
<br />
<p align="justify">By inserting a vector construct containing the TE with selectable markers (such as tetracyclin resistance and lacZ) into an organism with a desirable phenotype, we can find out what genetic elements (e.g. genes and promoters) are responsible for that particular function. This can happen via a random insertion of a TE containing a promoterless reporter gene downstream of promoter elements that creates a transcriptional fusion, providing activity in response to specific environmental stimuli. Using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">bipartite-mating (conjugation) method</a> to transfer the TE vector into the organism of choice is an efficient method for creating the massive number of mutants required.</p><br />
<p align="justify"><br />
Due to the complexity of biological systems, our team focused our efforts on utilizing a system for identification of promoter elements that respond specifically in the presence of environmental stimuli. Our hypothesis requires that the organisms we use respond specifically to particular toxins and result in upregulation of metabolic genes with little background effect in the cell. We recognize that any number of biological molecules may play a role in toxin sensing, such as enzymes, transcription factors, and even RNA elements (e.g. riboswitches). However, the identification of a promoter sequence takes us further in that we can better understand the degradation mechanism by elucidating the genes involved.<br />
</p><br />
<br />
<br />
<br><br />
<h2>Toxin-Degrading Organism Used</h2><br />
<p align="justify"><br />
<i>Pseudomonas spp. </i>have been isolated from oil sands tailings ponds and shown to biodegrade model and tailings-associated NAs and nitrogen- and sulfur-containing heterocyclic aromatic compounds (Ramos-Padrón <i>et al</i>. 2010; Herman <i>et al</i>., 1994; Del Rio <i>et al</i>., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins.</p><p> We wanted to use a commercially available strain of <i>Pseudomonas fluorescens</i> characterized for a response to toxins found in tailings pond water (TPW). The <i>P. fluorescens </i>PF-5 strain (Paulsen <i>et al</i>., 2005) is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, the genome sequence is available for this strain with annotations (Pseudomonas Genome Database V2, http://pseudomonas.com/). This allows us to use sequencing data from the mutants and identify where in the genome the TE insertion occurred, and what genes (if present) are located downstream of it.<br />
</p><br />
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<br><br />
<br />
<br />
<a name="library"></a><h2>Method Design</h2><br />
<h3>Mutant Library Generation</h3><br />
<p align="justify"><br />
To construct the promoter library, a pOT182 vector construct (containing a IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR transposable element) is introduced into commercially purchased <i>E. coli SM10</i> donor strain.</p><br />
<br />
<p align="justify"><br />
</html>[[File:Transposonproject Tn5OT182constructucalgary.png|thumb|750px|center|Figure 2: The transposable Tn5 element used in the pOT182 plasmid, containing a lacZ reporter gene, ampicillin and tetracycline resistance, an<br />
<i> E. coli</i> origin of replication for use during downstream sequencing protocols, and transposase. The genes are flanked by the transposon insertion elements]]<html><br />
</p><br />
<br />
<p align="justify">The plasmid contains a RP4 mob conjugation region and a p15A origin of replication (ori), and is engineered to only replicate in <i>E. coli</i>. The TE construct is transferred from the <i>E. coli</i> donor strain to the recipient <i>P. fluorescens </i> PF-5 using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in <i>P. fluorescens</i>, and selected by isolating the recipients that have a genomic TE insertion on Pseudomonas Isolation Agar/PIA with tetracycline. If a promoter element is fused upstream of the TE construct, then promoter activation will turn on the expression of lacZ, which can be detected by the degradation of a colorless compound, X-Gal, to an insoluble blue pigment product (an indoxyl compound) (Juers <i>et al</i>., 2012). If the fused promoter is activated in response to a stimulus, then the lacZ enzyme will be produced in response. Mutant strains sensitive to the particular toxic stimulus will appear as blue colonies on the selective plate.</p><br />
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<h3>Mutant Strain Characterization</h3><br />
<p align="justify">Mutants generated are characterized for their roles in the response to toxins with dose response experiments, and compared to general stress-inducing agents (e.g. H<font style="text-transform: lowercase;">2</font>O<font style="text-transform: lowercase;">2</font>) and compounds such as fatty acids to ensure the specificity of the response. These measurements help to determine thresholds of detection, robustness of the signal, and specificity of response. The dose response curves will also assess the usefulness of correlating the concentration of NA to the level of response, and the possibility of measuring NA concentrations in a sample, rather than simply by presence/absence.</p><br />
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<h3>Self-Cloning and Sequencing</h3><br />
<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman and Lamont, 1993). The TE used is a self-cloning construct because it contains all the elements required for plasmid replication (i.e. origin of replication) and selection (Tet resistance). Genomic DNA from a desirable mutant is isolated, and restriction digested with BglII (a restriction enzyme that does not cut within the TE but numerous times within the genome). The resulting fragments may contain the TE construct with flanking sequences. The genomic fragments are circularized by self-ligation and transformed into <i>E. coli</i>. Plasmids from the transformed cells contain the TE construct with the upstream and downstream flanking sequencing connected by the BglII restriction site. Sequencing primers designed against the 19 bp recognition sequence in the TE to sequence the isolated plasmids.</p><br />
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<p align="justify">For a detailed protocol, please consult our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">methods section</a>.</p><br />
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<h2>Results</h2><br />
<h3>Detection by Mutant <i>Pseudomonas fluorescens</i> PF-5</h3><br />
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<p align="justify">After mating experiments and plating on selective media (Pseudomonas isolation agar, with tetracycline and naphthenic acids), 24 responsive (blue) colonies were found. Screens were conducted on these blue colonies found on selective plates comparing a response in LB and LB with 100mg/L naphthenic acids (both with X-Gal). When results were observed it was found that 4 mutant strains are differentially regulated in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. These colonies were further screened to test the specificity of their responses.</p><br />
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<p align="justify"></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 3: Transposons: Shifting Segments of the Genome: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16hr. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><p align="justify"><br />
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Screens involving the use of different toxins at environmentally relevant concentrations were performed to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used as one testing condition to determine if the response is simply stress-induced.<br />
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<p align="justify"></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 4: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
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<p align="justify"></html>[[File:170-1data.png|thumb|650px|center|Figure 5: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
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<p align="justify"></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 6: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
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<p align="justify"></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 7: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<p align="justify"><br />
From these screens, it was seen that both colony 66-1 and colony 170-1 appear to respond to toxins when compared to a response in LB media. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure 8:Stress response screen on <i>P. fluorescens</i> Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 uL of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.]]<html></p><br />
<p><br />
These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.</p><br />
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<h2>Promoter Constructs Isolated</h2><br />
<p align="justify">To determine the location of the transposon insertion, we utilized the self-cloning properties of the transposon. By digesting the genome, religating, and transforming the ligated genomic fragments into <i>E. coli</i>, plasmids containing the transposon and flanking gene sequences were isolated. These plasmids have been isolated and sent for sequencing. However, we are having difficulty with getting sequencing reactions to produce a read. The results so far are a promising step towards finding a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds. </p><p> <br />
Our next steps were to test these strains in conjunction with our electrochemical detector as well as see if they could detect tailings toxins. <b>The results of this can be seen on the x page</b>.</p><br />
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}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T00:58:08Z<p>Lisa.O: </p>
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<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team began our project with human practices in mind. While we had established a research objective to produce a biosensor and bioreactor system, we wanted to ensure that our system was relevant to the industry where it would be employed. As well, we wanted to ensure that academic, government, and industry professionals' concerns were taken into consideration during the design process of our system. In order to best accomplish this, we conducted interviews with two leaders in oilsands reclamation. We approached a major oilsands company, Suncor, and talked to Christine Daly, an Ecologist who works in Environmental Cleanup. We then approached Ryan Radke, the president of BioAlberta. BioAlberta focuses on bringing biotechnology to our province and develop these in an industrial setting. His experience allowed us to better predict if our project would have any concerns amongst legislators and industrial leaders. <br />
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<h3>Talking with Suncor's Christine Daly on Biology in the Oil Sands</h3><br />
<p>We spoke with Christine Daly, an Aquatic Reclamation Research Coordinator at Suncor Energy Inc. Christine expressed an interest in our <a href="https://2011.igem.org/Team:Calgary">project in 2011</a> and was willing to discuss this year’s project design with us. One major point that was brought up early on in our design was that there is an opportunity for engineered organisms to outcompete existing tailings ponds bacteria, and we were pleased to hear that Christine had a similar concern. To address these concerns, we created our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">bioreactor</a> system, which would physically contain our bacteria, and also a <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">genetic killswitch mechanism</a>. Another interesting point brought up in this discussion was how the oil industry is currently looking into biology as one of many potential alternative methods to remediate the toxic components of tailings ponds and the oil sands in general. Research exists with other systems such as algal bioremediation, but practical implementations of biology in the oil sands appear to be rather few and far between. Oil industries do, however, appear to show an increased interest in biology (and in turn, synthetic biology) as a possible solution to various problems, a sentiment reflected in <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">our dialogue with the Oil Sands Leadership Initiative</a>.</p><br />
<p>The full interview can be viewed below.</p><br />
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<h3>BioAlberta's Ryan Radke on Biology in the Oil Sands</h3><br />
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<h2>Follow-Up Interviews</h2><br />
<p>Our second iteration of interviews were conducted once we had a more concrete product built. The purpose of these interviews was to see whether we had successfully addressed the concerns of the first iteration interviews. We also wanted to see whether any new issues with the design existed, which would provide us with potential future directions to take FRED and OSCAR. Kelly Roberge, an independent oil consultant, suggested we look into various ways to deal with the clay and silt particles that can enter our bioreactor system, which can be a major problem since mature fine tailings have a thick consistency that could clog the system.</p><br />
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<h3>Kelly Roberge, of K. Roberge Consulting Ltd. Discussing Bioreactor Improvements</h3><br />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our design at this point were issues with scale-up. These were things such as the amount of toxins that would need to be added to the system to provide constant production of our product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do towards this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be seen on the synergy page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we will have to look at the limitations in terms of the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to this grime and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
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<h3>William Sawchuk, of ARC Resources</h3><br />
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<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
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}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/ReferencesTeam:Calgary/Project/References2012-10-26T23:22:58Z<p>Lisa.O: </p>
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<li>Shu L, Chiou Y, Orville AM, Miller MA, Lipscomb JD, Que L. X-ray absorption spectroscopic studies of the Fe(II) active site of catechol 2,3-dioxygenase. Implications for the extradiol cleavage mechanism. Biochem 1995; 34:6649-6659.</li><br><br />
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<li>Slavcheva E, Shone B, Turnbull A. Review of napthenic acid corrosion in oilrefining. British Corrosion Journal 1999 Feb;34(2):125-131. </li><br><br />
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<li>So J. Mini-transposon Tn5gfp constructs for differential tagging of microorganisms. Biotech and Bioprocess Eng 1999;4(2):154-156. </li><br><br />
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<li>Soleimani M, Bassi A, Margaritis A. Biodesulfurization of refractory organic sulfur compounds in fossil fuels. Biotechnol Adv. 2007 Nov-Dec;25(6):570-96 </li><br><br />
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<li>Sukovich DJ. Hydrocarbon Biosynthesis by Bacteria: Genes and Hydrocarbon Products. PhD Dissertation, Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota. 2010; 1 – 208.</li><br><br />
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<li>Syncrude, (2012), Tailings Management. <a href="http://www.syncrude.ca/users/folder.asp?FolderID=5913 <br />
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<li>Timms-Wilson TM, Bailey MJ. Reliable use of green fluorescent protein in fluorescent pseudomonads. J Microbiol Methods 2001 Jul 30;46(1):77-80. </li><br><br />
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<li>TOMLAB Optimization, MatLab Optimization. <a href="http://tomopt.com/tomlab/">http://tomopt.com/tomlab/</a> </li><br><br />
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<li>United Nations Environment Programme: Sulfur Air Pollution. <a href="http://www.unep.org/transport/pcfv/pdf/Ethiopia-AirPollutionsulphur.pdf">http://www.unep.org/transport/pcfv/pdf/Ethiopia-AirPollutionsulphur.pdf </a> </li><br><br />
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<li>United States Environmental Protection Agency: Sulfur Dioxide. <a href="http://www.epa.gov/air/sulfurdioxide/">http://www.epa.gov/air/sulfurdioxide/</a> </li><br><br />
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<li>UW iGEM: Diesel Production Background. <a href="https://2011.igem.org/Team:Washington/Alkanes/Background">https://2011.igem.org/Team:Washington/Alkanes/Background</a> </li><br><br />
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<li>Vaillancourt FH, Bolin JT, Eltis LD. The ins and outs of ring-cleaving dioxygenases. Crit Rev Biochem Mol. 2006; 41:241-267. </li><br><br />
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<li>Venkitasubramanian P, Daniels L, Rosazza JPN. Reduction of Carboxylic Acids by Nocardia Aldehyde Oxidoreductase Requires a Phosphopantetheinylated Enzyme. Journal of Biological Chemistry 2007 Nov 13;282(1):478-485. </li><br><br />
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<li>Vitreschak AG, Rodionov DA, Mironov AA, Gelfand MS. Riboswitches: the oldest mechanism for the regulation of gene expression? Trends Genet 2004 Jan;20(1):44-50.</li></br><br />
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<li>Vogel U, Jensen KF. The RNA chain elongation rate in <i>Escherichia coli</i> depends on the growth rate. J Bacteriol. 1994 May;176(10):2807-13. </li><br><br />
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<li>Waters LS, Sandoval M, and Storz G. The<i> Escherichia coli</i> MntR miniregulon includes genes encoding a small protein and an efflux pump required for manganese homeostasis. J Bacteriol 2011 Nov; 193(21) 5887-97.</li></br><br />
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<li>Wright JK, Overath P. Purification of the lactose:H+ carrier of <i>Escherichia coli</i> and characterization of galactoside binding and transport. Eur J Biochem.1984 Feb 1;138(3):497-508.</li><br><br />
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<li>Xiong AS, Peng RH, Cheng ZM, Li Y, Liu JG, Zhuang J, Gao F, Xu F, Qiao YS, Zhang Z, Chen JM, Yao QH. Concurrent mutations in six amino acids in beta-glucuronidase improve its thermostability. Protein Eng Des Sel. 2007 Jul;20(7):319-25. Epub 2007 Jun 8. </li><br><br />
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<li>Xu P, Yu P, Li FP, Cai XF, Ma CQ. Microbial degradation of sulfur, nitrogen and oxygen heterocycles. Trends in Microbiology 2006; 14(9):398-405.</li><br><br />
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<li>Yoshimura F, Nikaido H. Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic solutes. J Bacteriol. 1982 Nov;152(2):636-42. </li><br><br />
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<li>Young R, Bremer H. Polypeptide-chain-elongation rate in <i>Escherichia coli</i> B/r as a function of growth rate. Biochem J. 1976 Nov 15;160(2):185-94. </li><br><br />
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<li>Zeuthen P, Knudsen KG, Whitehurst DD.Organic nitrogen compounds in gas oil blends, their hydrotreated products and the importance to hydrotreatment. Catalysis Today Feb 2001; 65(2-4):307-314.</li><br><br />
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<li>Zhang X, Wiseman S, Yu H, Liu H, Giesy JP, Hecker M. Assessing the toxicity of naphthenic acids using a microbial genome wide live cell reporter array system. Environ Sci Technol 2011 Mar 1;45(5):1984-1991.</li><br><br />
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<li>Zhang Y, Nelson M, Nietfeldt JW, Burbank DE, Van Etten JL.Characterization of Chlorella virus PBCV-1 CviAII restriction andmodification system. Nucleic Acids Res 1992;20(20):5351-5356.</li><br><br />
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<li>Zhu G, Pang K, Parkin G. New Modes for Coordination of Aromatic Heterocyclic Nitrogen Compounds to Molybdenum: Catalytic Hydrogenation of Quinoline, Isoquinoline, and Quinoxaline by Mo(PMe<sub>3</sub>)<sub>4</sub>H<sub>4</sub>. Journal of the American Chemical Society 2008; 130(5):1564-1565. </li><br><br />
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</ul><br />
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</html>}}}<br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/DesulfurizationTeam:Calgary/Notebook/Desulfurization2012-10-26T23:11:33Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Desulfurization Journal|<br />
<br />
CONTENT=<br />
<html><br />
<h2>Week 1 (May 1-4)</h2><br />
<p>During this week, literature search was performed.</p><br />
<br />
<br />
<br />
<br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
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<br />
<p>Along with the rest of the team, this week was dedicated to familiarizing ourselves on the protocols that will be utilized during this years project; specifically the polymerase chain reaction, gel verification, preparation of overnight cultures, as well as developing a procedural flowchart to transform competent cells with registry biobricks. With regards to our sub-group specific goals, we reviewed the current available literature around various industrial and laboratory approaches to desulfurization of organic groups, especially in the petroleum industry. This included a comparison of non-biological processes such as conventional hydrodesulfurization, which is currently employed in petroleum product refinery stages, and how a biological approach would supplement and perhaps even offer several advantages over these methods. Current limitations to biological desulfurization, however, include such factors as biocatalyst stability, enzyme specificity and desulfurization rate. We also identified the enzyme desulfinase (DszB) as being one of the bottlenecks in the desulfurization 4S pathway. Overall, our goals moving forward involve determining the specific pathways involved in the desulfurization process as well as the reaction conditions we would want to employ, and identifying specific model compounds in addition to dibenzothiophene (DBT) that we could use to test the effectivity of our biosystem in order to determine its functionality in the conversion of naphthenic acids to economically valuable hydrocarbons.</p><br />
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<br />
<h2>Week 3 (May 14-18)</h2><br />
<br />
<br />
<p> Building on the previous week's literature review, the 4S pathway was recognized as the preferred biological mechanism that we would explore in devising a desulfurization biosystem. Of specific interest is the dsz operon consisting of the genes for <i> dszA, dszB, </i>and<i> dszC</i> which selectively and non-destructively remove the sulfur from the hydrocarbon structure, and therefore preserves the carbon skeleton. In addition to these, another dsz gene exists.<i> dszD</i>, which codes for a FMN:NADH reductase, is an essential component of the pathway, but not part of the operon. Instead, it is on the genome. The enzyme produced by this gene is required to regenerate the FMNH2 consumed by the reactions carried out by DszA and DszC. <i>Rhodococcus erythropolis</i> IGTS8 is the most studied model organism in investigations of the 4S pathway, and has been shown in many different research endeavors to be capable of converting DBT to 2-HBP.</p><p><br />
</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|thumb|750px|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD. Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO2) through the addition of oxygen to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the heteroatom, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-hydroxybiphenyl. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.]]<html></p><p><br />
An alternative to the DszD gene is HpaC, an oxidoreductase encoded in the <i>E. coli</i> W genome. This enzyme has been shown to increase the rate of desulfurization. Following this, other protocols added to our growing lab methods 'toolkit' were a restriction digest protocol, PCR purification, and finally, DNA construction digest. Aims moving forward include obtaining strains of the <i> R. erythropolis </i>, while also executing a timeline devised to biobrick, test, and incorporate the genes necessary in the above processes in a biobrick circuit.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<br />
<br />
<p>This week was kicked off with a project development meeting with Emily and David, and we devised a protocol for biobricking the <i> hpaC </i> gene. Additionally, methods to place the genes coding for the 4 enzymes, DszA,B,C and HpaC into a single construct were explored. Within the lab, the PCR performed on the resuspended pUC18-hpaC was not successful initially. <br />
Furthermore, we ordered the substrates/compounds that we intend to use for desulfurization tests. Once the substrates and the <i>Rhodococcus</i> strain arrive we are going to test how effectively the bacteria can desulfurize different sulphur-containing compounds that resemble naphthenic acids. Finally, we came across a paper where a team had developed an improved efficiency DszB through site-directed mutagenesis in 2007. This was through a point mutation to the gene, converting a tyrosine at position 63 to a phenylalanine residue. A member of this team was contacted to request the plasmid that contains the mutated gene. The conversion step carried out by DszB is the major bottleneck in the 4S pathway and if a strain or sample containing this mutation was obtained, it would significantly bolster our later testing efforts on DBT, as well as other compounds such as thiophane.<br />
</p><br />
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<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<br />
<br />
<p>Since we wanted to make sure we would not run out of pUC18(plasmid containing the hpaC gene), we transformed some E.coli cells with it. We grew them on plates containing ampicillin (A), kanamycin (K), tetracycline (T) and chloramphenicol (C) antibiotics and they only grew on A. Therefore pUC18 has A resistance. We did a three sets of PCR with primers designed against <i>hpaC</i>, one using 1/10 dilution of pUC18, the other using 1/100 dilution of pUC18 and one with the colonies we had just obtained by transforming the <i>E.coli</i> cells. </p><br />
<p>The PCR worked and we saw bands of the same size for all three sets of PCR. (Unfortunately, the picture we saved is not a good one since some of the bands faded away under UV due to prolonged exposure. Following this, PCR purification was performed to obtain the pure <i>hpaC</i> with biobrick prefix and suffix attatched to gene, which would allow us to insert the sequence into a biobrick standard backbone. 3 sets of digestion, ligation, and transformation (using pairs of X&P enzymes, E&S enzymes and E&P enzymes) were carried out in order to insert the <i>hpaC </i> gene into the <a href=" http://partsregistry.org/Part:pSB1C3">pSB1C3</a><br />
vector. All the sets grew successfully.<br />
Following the above successes with<i> hpaC</i>, the arrival of our <i>Rhodococcus</i> strain afforded us the opportunity to begin investigation of the Dsz operon using the primers currently in our possession. This strain is an environmental isolate that has been shown to be an active desulfurizer. The gram-positive nature of the strain also dictated we explore various lysing strategies before the genes encoding the Dsz enzymes could be amplified for further purification and biobrick construction steps. PCR was carried out using <i>dszA</i> primers on three different treatments {microwave, lysate buffer, and a control} which yielded banding pattern around 1200 base pairs for the lysate treatment (2%SDS and 10% tritonX-100, plus heat for 5mins at 98C).<br />
</p> <br />
<br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<br />
<br />
<p> In order to confirm the <i>hpaC</i> biobrick construction, two sets of colony PCR were performed, choosing white colonies from the 3 plates we grew last week (white colonies indicate a loss of the RFP generator in the <a href=" http://partsregistry.org/Part:pSB1C3">pSB1C3</a><br />
backbone, and therefore allow for weeding out of the colonies which are simply the original plasmid vector). These reactions were carried out both with <i>hpaC</i> primers and with standard biobrick primers designed against the plasmid backbone. After running them on the gel we saw equal bands for the PCR reactions performed using <i>hpaC</i> primers (However, a PCR using biobrick primers was performed later and the same results were obtained). Colonies 1(-) and 5(-) were used to make overnight cultures, which were then miniprepped the following day to obtain the plasmid DNA of the putative <i>hpaC</i> biobrick. Digestions were performed on the miniprep products using EcoRI and PstI to look for part size as further verification for the genes presence in the plasmid. The results were good and two bands were observed on each column (one for vector and the other for <i>hpaC</i>)). <i>hpaC</i> was sent in for sequencing. </p><br />
<p><br />
<br />
<br />
<br />
</html>[[File:UCalgary2012_04.06.2012-desulfurisation_hpacverification.jpg|thumb|700px|center|Figure 2: HpaC verification cPCR. HpaC gene was inserted into the pSB1C33 vector and E. coli cells were transformed. In order to confirm that pSB1C3 contains the hpaC gene, two sets of colony PCR's were conducted. One with biobrick primers, and the other with hpaC primers. Bands indicate successful amplification at the approximate size of the hpaC gene (517 bp)]]<br />
[[File:Ucalgary2012 06.06.2012-digestion of hpaC with E and P.jpg|thumb|500px|center|Figure 3: HpaC confirmation digest. Plasmid was miniprepped and digested for 2h with EcoRI and SpeI before being run on a 1% gel for 1h and 120V. Bands between 500 and 700 bp indicate the hpaC part is present as an insert. Bands of about 2000bp show the size of the psb1c3 vector.]]<html></p><br />
<br />
<br />
<br />
<p> PCR reagents were prepared to re-test/confirm previous results of <i>dszA</i> amplification following two different lysing treatments (microwave + lysate buffer). This time, all three genes were amplified and gel verification showed clear banding patterns around 500bp range for all three genes for the microwave treatment. Remaining PCR products were run on a gel and extracted for further purification steps; however, presence of any genetic material were not confirmed through nanodropping which raised concerns about the composition of the purified products, the success of the initial amplification step, or perhaps even the lysis treatment. Further experimentation will have to be carried out to troubleshoot.</p><br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<br />
<br />
<p> This week, we focused on amplifying <i>dsz</i> genes from our <i>Rhodococcus</i> strain for construction into biobricks. We also wanted to purify the <a href=" http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"> BBa_K902057 <i>hpaC</i></a> and pUC18-<i>hpaC</i> plasmids to replenish our current stocks. For the <i>dsz</i> aspect, we were able to successfully grow extra plates of <i>Rhodococcus</i> strain which was used to inoculate PCR tubes. The PCR did not go well, with significant streaking and false positives with similar banding pattern to previous gels run in the previous week. A final gel verification of a random sample of a tube of PCR products from dszA,B,C respectively and two negative control treatments involving master mix only and the lysed cells only illustrated the lack of discrepancy between the supposed successful amplification and the lysed cells (with lysate buffer) alone. Because of this we decided to take a different approach involving plasmid isolation carried out before PCR, rather than applying the PCR reagents directly to a lysed culture sample.</p><br />
<p> <a href=" http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> verification through sequencing was successful, confirming the construction of our first biobrick. Subsequently, O/N cultures of the plasmid containing cultures were prepared and stored in glycerol at -80C. Furthermore, verification of catalase gene part (<a href=" http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a>), which was sent as a culture stab from the parts registry was initiated. Our newly identified biobricked-hpaC was used as a positive control, but the banding pattern was not very conclusive. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<br />
<br />
<p>PCR was reattempted on <i>Rhodoccocus</i> that was lysed using two different dilutions of the lysate buffer, but the gel verification confirmed the previous failure in using this approach. An alternative that involved preparation of an overnight culture of the <i>Rhodococcus</i> cells followed by a plasmid purification was followed. The plasmid purification eventually yielded plasmid samples with concentrations of 98.6ng/μL to 182.7ng/μL (4 samples obtained overall). Additionally, the catalase biobrick was used to transform some stock competent cells, and samples of some colonies were subsequently PCR'ed. Although, the gel verification showed some potential contamination, and the required banding patterns at around 2200bp was not obtained. </p><br />
<br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<br />
<br />
<p> PCR was attempted to amplify the genes of the <I>dsz</i> operon utilising an adapted PCR protocol with purified <i>Taq</i> polymerase that had been isolated from the host organism. Eventually, some banding pattern was obtained between 1200 and 1500 base pairs when a gradient thermocycler was used with melting temperatures ranging betweeen 55°C to 65°C. This was assumed to be indicative of successful amplification of <i>dszB</i>; however, further purification and gel verification results were inconclusive and no yield was obtained when placed tested using a nanodrop machine. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<br />
<br />
<p>Top 10 E.coli cells were transformed with <a href="http://partsregistry.org/Part:BBa_R0011">BBa_R0011</a><br />
(IPTG inducible promoter in psb1C3 backbone), and resulting colonies were tested using cPCR. <br />
Colony PCR was performed on cells containing the catalase biobrick. Catalase is 2217bp long but since biobrick primers add about 200bp, bands of 2400 bp were expected if the part was present in the biobrick. These bands were observed, indicating that the <a href=" http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a> gene was most likely present.</p><br />
<p></html>[[File:Ucalgary2012 4.7.2012 catalase colony pcr 2.jpg|thumb|500px|center|Figure 4: Colony PCR on potential colonies containing katG-LAA. Biobrick primers were used. Bands at 2200 indicate successful amplification of the catalase part within the biobrick backbone. Smaller bands are indicative of primer degradation and are nonspecific amplification. +C indicates the positive control, and RFP generator, which should amplify at around 1000 bp.]]<html></p> <p><br />
<br />
PCR using Phusion high fidelity polymerase was carried out on <i>dszA</i>, <i>dszB</i>, and <i>dszC</i> in a gradient thermocycler. Amplification of non-specific bands was present for <i>dszA</i> and <i>dszB</i>, however strong banding for the desired size of the gene was observed for both (around 1500 for <i>dszA</i>, 1100 for <i>dszB</i> </p><br />
<br />
<p></html>[[File:Ucalgary2012 6.7.2012.dszABphusionPCR.jpg|thumb|500px|center|Figure 5:DszA and DszB PCR from <i>Rhodococcus</i>. DszA amplicon is around 1300 bp, and is observed to run higher on the gel. DszB amplicon is expected to be 1098 bp, which is observed in addition to multiple nonspecific banding. +C indicates the positive control, and RFP generator, which when PCRed with biobrick primers should be around 1000 bp. As this control is running high as well, it is believed that both amplicons for DszA and DszB have been obtained. No contamination is observed in the NTC (no template control).]]<html><br />
</p> <br />
<p></html>[[File:Ucalgary2012 7.5.2012 dszC.png|thumb|500px|center|Figure 6:DszC PCR from <i>Rhodococcus</i>. The numbers above the figure show the colony number. DszC amplicon is around 1450 bp, and is observed to run at almost the right size on the gel. No contamination is observed in the NTC (no template control).]]<html><br />
<p><br />
Examining the sequences of the <i>dszABC</i> genes led to the discovery that all 4 had multiple illegal enzyme cut-sites in them that we have to eliminate before biobrick composite part construction can occur. <i>dszA</i> has four PstI cut sites, <i>dszB</i> has a PstI and a NotI and <i>dszC</i> has a PstI cut site. The Stratagene QuickChange mutagenesis procedure is going to be used to eliminate illegal cut sites with the only alteration being that Kapa HiFi polymerase would be used during the process. Primers needed for the mutagenesis were designed based on the procedure mentioned above. </p><br />
<br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<br />
<p> Following successful amplification of the <i>dsz</i> operon genes in the previous week, the genes were constructed into the <a href="http://partsregistry.org/Part:pSB1C3"> pSB1C3</a> vector. Colony PCR verifications were observed to be positive. Furthermore, the insertion of part <a href="http://partsregistry.org/Part:BBa_J13002"> BBa_J13002, (P<sub>tetR</sub>-RBS)</a><br />
in front of the previously biobricked <i>hpaC</i> was attempted. Overnight cultures were also prepared using two colonies each for <a href="http://partsregistry.org/Part:BBa_J13002"> <i>P<sub>tetR</sub>-RBS</i></a> and <a href="http://partsregistry.org/Part:BBa_R0011"> <i>P<sub>lacI</sub></i> </a> (an IPTG inducible promoter that we hope to build in front of <ahref="http://partsregistry.org/Part:BBa_B0034">an RBS site, BBa_B0034</a>). These cultures were then miniprepped to yield the respective parts.</p> <br />
<br />
<p>Additionally, <a href="http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a> was built into a <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> backbone. The construction and availability of all these parts will be critical in the construction of our overall circuit for biodesulfurization. Colonies which looked good on cPCR were used to prepare overnight cultures, and were miniprepped and sent in for sequencing verification the following day.<br />
On the side, M9 minimal media was also prepared to carry out growth experimentation and overall desulfurization capability of <i>Rhodococcus</i> when exposed to DBT. The various growth treatments were M9 Media and glucose only, M9+glucose+DBT, M9+glucose+MgSO4+/-DBT, M9+glucose+MgCl2+/-DBT. 0.008g of FeCl2.4H2O was also added to each of the tubes. Samples were then inoculated with colonies of the <i>Rhodococcus</i>.</p><br />
<br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<br />
<p>This week, while awaiting sequencing verification results which were required before we could begin the construction process, the desulfurization team initially aided in some of the tasks related to the other hydrocarbon groups. The success of the construction of <a href="http://partsregistry.org/Part:BBa_J13002"><i>P<sub>tetR</sub></i>-RBS</a><br />
with <a href="http://partsregistry.org/Part:BBa_K902057"><i>hpaC</i></a> was also explored by using forward and reverse primers of <a href="http://partsregistry.org/Part:BBa_R0040">BBa_R0040</a> (the promoter component of the composite part BBa_J13002). However, the eventual gel verification was inconclusive and sequencing results finally indicated an unsuccessful ligation. Additionally, the minimal media M9 preparation had been contaminated in the previous effort so this process was repeated to create tubes of each of the growth condition treatments detailed previously, and two repeats, one with an extra filtration step and one without was used to prepare the cultures.</p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<br />
<p> Mutagenic primers were redesigned after the initial ones were found to have premature stop codons. As part of the redesign process in constructing our overall gene circuits for desulfurization, a backbone switch of <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> into a chloramphenicol (Chlor) resistant vector was necessary. The subsequent transformed products were plated on a Chlor plate and selected colonies were used to prepare O/N cultures, then minipreped before finally being digested with enzymes EcoRI and PstI. The resulting gel verification images were inconclusive as they did not show the required banding pattern around 50bp. Meanwhile, colony PCR was run on colonies transformed with <i>katG-LAA</i> constructed into a <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> backbone, as well as the <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> +<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a> construct. <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></a> was shown to have been successfully amplified, so overnight cultures were prepared and subsequently miniprepped. On the other hand, the construct was not successful so a third attempt was carried out. Colony PCR treatments that used either <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> forward primers or <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
primers were used and the overall constructs were made either on a chlor-resistant, or ampicillin-resistant vectors. Preliminary images of the gel verification appeared to have confirmed the construct, although sequencing verification will be the final indicator of overall success. </p><br />
<br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<br />
<br />
<p>Sequencing results from the previous week's constructs were available confirming that we constructed KatGLAA in a chlor-resistant backbone. However, switching the plasmid backbone of <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> to <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> was not successful. The construction of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>+<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> was finally sent in for sequencing. Site-directed mutagenesis of the <i>dsz</i> operon was also initiated: <i>dszA</i> has four PstI cut sites; <i>dszB</i> has a PstI and a NotI site; <i>dszC</i> has two PstI cut sites. Site directed mutagenesis was started this week to change a single base pair in these genes in a way that eliminates the cut site but preserves the amino acid codons, so as to not mutate the protein coding sequence. Ohshiro 2007 demonstrated that replacing the Tyr residue at position 63 of <i>dszB</i> gene with a Phe increases the activity of the enzyme. Therefore we want to introduce the same mutation into our <i>dszB</i>.</p><p><br />
For the first attempt at mutagenesis we chose to mutate the second PstI site in <i>dszC</i> (PstI2). As a positive control for the procedure, we also performed the mutagenic PCR on a plasmid containing the β-galactosidase gene with a point mutation where the PCR would cause it to regain its function. For both mutagenesis protocols we used the Kappa Hifi kit. After confirming that the PCR worked by running some produce on a gel, the PCR products were DpnI digested, the purpose of which is to degrade the unmodified parental DNA (DpnI degrades methylated DNA only). Control PCR products were plated on an ampicillin plate containing IPTG and X-gal. The colonies that grew on the control plates were blue indicating that the mutagenesis had worked for the β-galactosidase gene. Minipreps of the O/N culture of <i>dszC</i> mutants were digested with PstI enzyme and the results indicated that the mutagenesis was successful.</p><p> <br />
Attempts to simultaneously perform all the mutations in <i>dszC</i> genes in one step using the Knight procedure failed (<a href="http://openwetware.org/wiki/Knight:Site-directed_mutagenesis/Multi_site">Knight Multi-site Mutagenesis Procedure</a>). What enables simultaneous mutations is that Taq ligase closes the gaps in PCR products after each cycle. In the protocol it instructs to use Taq ligase buffer only for the PCR/ligation protocol. We suspected that the reason this procedure did not work might be that the Kappa polymerase is not functional in Taq ligase buffer. Therefore we did some experiments on the controls in Taq ligase kit and kappa polymerase kit to find out which buffer that Kappa polymerase and Taq ligase both work best in. The result was that both enzymes work best in a buffer made of half Taq ligase buffer and half Kappa polymerase buffer. </p><br />
<br />
<br />
<br />
</html>[[File:Ucalgary2012 31.7.2012 dszc mutagenesis 5 20 and 50ng.jpg|thumb|500px|center|Figure 7: DszC PstI2 mutagenesis PCR with varying concentrations of template plasmid was performed. The gel shows the PCR products that were run on a gel. +C lanes show the PCR products of the control. The control was pWhitescript™ 4.5-kb control plasmid from Stratagene mutagenesis kit which was mutated with primers also from the kit. Bands indicate successful amplification of the plasmid.]]<br />
[[File:UCalgary 02.08.12 dszC psti digest mutagenesis.jpg|thumb|700px|center|Figure 8: The control and the mutated plasmids were digested with PstI restriction enzyme. The control is dszC biobrick. Other lanes show the plasmids purified from the cells transformed with PstI2 mutagenesis PCR products followed by digestion. The control shows three bands since it has three PstI cut sites. The other lanes show two bands which indicates one of the cut sites has been eliminated through mutagenesis. ]]<html><br />
<br />
</html>[[File:Ucalgary2012 2.8.2012 finding the right buffer for multisite mutagenesis.png|thumb|800px|center|Figure 9: The multisite mutagenesis using the Knight procedure was tried in different buffers to find out the buffer that Kappa works optimally at. All the PCRs were performed on the pWhitescript™ 4.5-kb control plasmid from Stratagene mutagenesis kit which contains the beta-galactosidase gene. Based on this gel, the optimal buffer is composed of 50% kappa buffer and 50% Taq ligase buffer.]]<html><br />
<br />
</html>[[File:Ucalgary 3.8.2012 optimal buffer for Taq ligase.png|thumb|800px|center|Figure 10: Using the control provided in the NEB Taq ligase buffer (BsteII digested lambda DNA), we tried to find if the Taq ligase enzyme can function in combinations of Kappa Hifi buffer and Taq ligase buffer. Lane two is the control which is only the digested lambda DNA. Lanes 1 and 3 show that some of the bands compared to control have been ligated together. Therefore, Taq ligase functions just as good in the buffer composed of 50% kappa hifi buffer and 50% Taq ligase as it would in its own buffer.]]<html><br />
<br />
<h2>Week 15 (August 6 - August 11)</h2><br />
<br />
<p>Sequencing results for <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A> <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> returned negative, so a 3-part ligation method was used to retry this construction. The following parts were ligated with the restriction enzymes indicated in brackets after each: <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>(EcoRI/SpeI) + <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> (XbaI/PstI) + <A HREF=" http://partsregistry.org/Part:PSB1K3">BBa_PSB1K3</A> (EcoRI/PstI). Also, the more conventional construction (only 1 insert) of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>(SpeI/PstI) + <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> (XbaI/PstI) was reattempted. Furthermore, 3-way ligations were also attempted for <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
+<A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A>+<A HREF="http://partsregistry.org/Part:PSB1K3">BBa_PSB1K3</A>, and <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a>+<a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
+<A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A>, as well as the two-way contruction of just <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> after the <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a>. After plating these transformations, colony PCRs were carried out and samples that gave an indication of being successful on the gels were used to prepare O/N cultures followed by miniprep. With regards to the site-directed mutagenesis side of the experimentation, <i>dszA</i>-PstI1 (the first PstI cut site in <i>dszA</i>) ,<i> dszB</i>-PstI and <i>dszC</i>(PstI2 mutated)-PstI1 mutagenesis were performed following the procedure explained in the previous week. The gel below shows the successful result of digest confirmation (Fig. 8). <br />
Multisite mutagenesis (Knight method) was repeated using the modified buffer (half Taq ligase buffer and half Kappa buffer). However it was not successful again. We also tried doing multisite mutagenesis using Pfu Turbo polymerase and following the Knight procedure without any buffer modifications. No successful results were observed. </p><br />
<br />
<br />
<br />
</html>[[File:Ucalgary2012 Digestion confirmation of mutagenesis in dszAPstI1, dszBPstI and dszCPstI1 (PstI2 site mutated)..jpg|thumb|800px|center|Figure 11: Confirmation of site-directed mutagensis to eliminate PstI cut sites in: <i>dszA</i>PstI1 cut site, <i>dszB</i>PstI cut site and <i>dszC</i>PstI1 (PstI2 site mutated). Note that in each case, the unmutated controls for each gene are to the left of each set of plasmids derived from mutagenic PCRs. Plasmids from cloned <i>dsz</i> genes are smaller than the bands to the right of them (which have undergone mutagenesis), indicating that in the plasmids on the right, the PstI cut site was lost. ]]<html><br />
<br />
<br />
<h2>Week 16 (August 12 - August 18)</h2><br />
<br />
<br />
<p>The progress in mutagenesis of <i>dsz</i> genes was continued from the previous week: <i>dszB</i>(PstI mutated)-Y63F and <i>dszA</i>(PstI1 mutated)-PstI3 mutagenesis. The gel below shows the digest confirmation.</p><br />
<br />
<br />
</html><br />
[[File:Ucalgary2012 15.08.2012 dszAPstI1&amp;3 dszB Psti y63f muta diges-1.jpg|thumb|800px|center|Figure 12: Digestion confirmation of biobricks of a) dszA (PstI1 mutated) PstI3 mutagenesis and b) dszB (PstI mutated) Y63F mutagenesis. Cutting dszA (PstI1 mutated) with PstI is expected to produce fragments of 2790, 333, 255, and 114bp. Cutting dszA (PstI1 and PstI3 mutated) is expected to produce fragments of 2730, 588, and 114bp. The primers for dszB-Y63F mutagenesis introduce an HpyAV cut site. pSB1C3 inteslf has two more HpyAV cut sites. Before the mutation bands of 2858 abd 310bp are expected, and after successful mutation bands of 1502, 1356, 310 are expected. Lane legend: 1- dszA (PstI1 mutated). 2-dszA (PstI1 mutated) digested with PstI. 3-dszA (PstI1 mutated) PstI3 mutagenesis c1 Digested with PstI. 4- dszA (PstI1 mutated) PstI3 mutagenesis c2 Digested with PstI. 5-dszA (PstI1 mutated) PstI3 mutagenesis c3 Digested with PstI. 6- dszA (PstI1 mutated) PstI3 mutagenesis c4 Digested with PstI. 7- Fermentas 1kb Plus Ladder. 8- Empty. 9- dszB (PstI mutated). 10-dszB (PstI mutated) digested with HpyAV. 11- dszB (PstI mutated) Y63F mutagenesis c1 digested with HpyAV. 12- dszB (PstI mutated) Y63F mutagenesis c2 digested with HpyAV. 13- dszB (PstI mutated) Y63F mutagenesis c3 digested with HpyAV. 14-dszB (PstI mutated) Y63F mutagenesis c4 digested with HpyAV.]]<html></p><br />
<br />
<p>We attempted a different approach to speed up the turnover time of the mutagenesis PCR. Briefly, after the PCR mutagenesis the PCR products were purified and then incubated with T4 polynucleotide kinase (PNK) and ligase. After heat inactivating the ligase and T4 PNK, the products were DpnI digested. Subsequently another round of DNA purification was performed. However, the results were unsatisfactory after the digest confirmation.</p><p><br />
Sequencing results came back. <i>dszA</i> (PstI1 and PstI3 mutated) and <i>dszB</i>(PstI and Y63F mutated) were good. However <i>dszC</i> (PstI1 and PstI2 mutated) had an insertion next to the PstI1 cut site. Mutagenesis was repeated on the <i>dszC</i>(PstI2 mutated).<br />
<i>dszB</i>(PstI and Y63F mutated)-NotI and <i>dszA</i>(PstI1 and PstI3 mutated)-PstI4 mutagenesis were also performed.</p><br />
<p> To investigate the desulfurisation capability of the <i>Rhodococcus</i> sp. from which we cloned the <i>dsz</i> operon, a desulfurization assay was prepared by inoculating different treatments of M9 media. We also prepared some solutions that will be needed for analysis in the following week: a conditioning agent composed of 100ml of 95% ethanol, 50ml glycerol, 30ml of 12M HCl (aq) and 70g of NaCl(s) was prepared. The assay relies on the turbidity of a sample containing sulphate ions which are precipitated (hence the turbidometric nature of the assay) upon adding BaCl2(s), therefore if the <i>dsz</i> pathway is active, we expect a more turbid solution to form than in control samples. </p><br />
<br />
<br />
<br />
<h2>Week 17 (August 19 - August 25)</h2><br />
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<br />
<p>This week, progress was made in determining the desulfurization activity of our <i>Rhodococcus</i> strain as measured by the sulfate release using a turbidometric assay. We encountered several challenges in our prescribed protocol as the concentrations that we used to prepare the standard curve may have been too dilute, or the composition of out conditioning agent may have been flawed. Additionally, steps were taken to determine the decomposition of DBT to 2-HBP through Gas Chromatograph-Mass Spectroscopy (GC-MS) analysis, but due to a preparation error, the DBT was added to a growth solution of M9 media prematurely and the autoclaving process decomposed the DBT releasing a yellow colouration into the solution. These two approaches in determining the desulfurization capability of the <i>dsz</i> operon will be further investigated. </p><br />
<br />
<p>Since the <i>dszC</i> second mutagenesis had proven to be unsuccessful last week, the <i>dszC</i>(PstI2 mutated)PstI1 mutagenesis was repeated. Also <i>dszA</i>(PstI1,3,4 mutated) PstI2 mutagenesis was performed. <i>dszA</i> and <i>dszC</i> were sent for sequencing on Wednesday. <i>dszB</i> was sent for sequencing on Friday. Sequencing results of <i>dszA</i> and <i>dszC</i> were back by Friday. <i>dszC</i> was successful. However, <i>dszA</i> contained an insertion next to the binding site of PstI4 cut sit, so the last two mutations must be redone. <i>dszB</i>(PstI and Y63F mutated)-NotI-mutagenesis was also repeated in case the result of the sequencing was not successful. These constructions were repeated. <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>-<A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
-<i>dszC</i> constructions were attempted, however they were not successful as indicated by colony PCR. Constructions of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>/<i>hpaC</i> were carried out and also came back negative in sequencing, however <a href="http://partsregistry.org/Part:BBa_K902059">BBa_B0034<br />
/<i>katG-LAA</i> (BBa_K902059)</a> was sequence confirmed.</p><br />
</html>[[File:Ucalgary2012 20.8.2012 dszC(psti2)psti1 mutagenesis.png|thumb|700px|center|Figure 13: Another attempt to mutagenize dszC(PstI2 mutated)at PstI1 site. The gel shows the confirmation digestion. C is the control which is dszC(PstI2 mutated). Since the control has two PstI cut sites, two bands are observed on the gel. The mutagenesis has been successful since only one band is observed on the other lanes indicating that one of the cut sites has been eliminated during mutagenesis.]]<html><br />
<br />
</html>[[File:Ucalgary2012 21.8.2012 dszA final mutagenesis digest.png|thumb|700px|center|Figure 14: DszA (PstI1, PstI2, PstI3 mutated)PstI4 mutagenesis digestion confirmation gel. All the lanes show the results of the plasmids being cut with PstI enzyme. C indicates the lane containing the control which is dszA(PstI1, PstI3 and PstI4 mutated). The other lanes are the digestions of the plasmids that are mutagenized, from different colonies. Control plasmid has two PstI cut sites and therefore the two bands observed on the gel were expected. The other lanes show only one band of about 3500bp which is about the right size for dszA in a psb1c3 vector. Therefore one of the cutsites has been eliminated during mutagenesis.]]<html><br />
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<br />
<br />
<br />
<h2>Week 18 (August 26 - September 1)</h2><br />
<br />
<p> <A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i> (BBa_K902052)</i></A> sequencing results came back as successful. <i>dszA</i>(PstI1,3 mutated)-PstI2-mutagenesis was performed and sent for sequencing. Also <i>dszA</i>(PstI1,2,3 mutated)-PstI4-mutagenesis was performed, and this was also sent for sequencing. </p><br />
<p>Constructions of <A HREF="http://partsregistry.org/Part:BBa_K902053">P<sub>tetR</sub>-RBS/<i>dszB</i> (BBa_K902053)</a> and <a href="http://partsregistry.org/Part:BBa_K902056">BBa_B0034<br />
/<i>dszC</i> (BBa_K902056)</a> were attempted, verification digested, and sent for sequencing. Sequencing results for these constructs came back as positive, along with successful mutagenesis of <A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i> (BBa_K902050)</A>.</p><br />
<p>At this point, all of the <i>dsz</i> genes have been successfully made biobrick compatible, and <i>hpaC</i> has been biobricked. We have also successfully constructed <a href="http://partsregistry.org/Part:BBa_K902059">BBa_B0034 with <i>katG-LAA</i></a> to be used in the optimization circuit, as well as <A HREF="http://partsregistry.org/Part:BBa_K902053"><i>P<sub>tetR</sub>-RBS</i>/<i>dszB</i> (BBa_K902053) </a>and <a href="http://partsregistry.org/Part:BBa_K902056">BBa_B0034/<i>dszC</i> (BBa_K902056)</a>.<br />
<p> Constructions of <A HREF="http://partsregistry.org/Part:BBa_J04500">P<sub>lacI</sub>-RBS</A>with <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> were performed. As well, attempts to construct <A HREF="http://partsregistry.org/Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></a>/<a href="http://partsregistry.org/Part:BBa_K902056">RBS-<i>dszC</i></a> as well as <A HREF="http://partsregistry.org/Part:BBa_J13002">P<sub>tetR</sub>-RBS</A>/<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
/<A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i></A>, and <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>/<A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> were also carried out. These parts are intended as construction intermediates towards building the final systems, as well as providing a way of testing the genes functionality (namely, to test HpaC for oxidoreductase activity and to test if over-expression of KatG in the cell will increase its ability to survive H<sub>2</sub>O<sub>2</sub> stress). Transformations of all these constructions were carried out at the end of the week.</p><br />
<br />
<h2>Week 19 (September 2- September 8)</h2><br />
<p>Confirmation digests on colonies of the previous constructions that gave bands of the expected size with cPCR were performed. Positive results were found for colonies of <A HREF="http://partsregistry.org/Part:BBa_J04500"><i>P<sub>lacI</sub>-RBS</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <A HREF="http://partsregistry.org/Part:BBa_J04500"><i>P<sub>lacI</sub>-RBS</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
/<A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i></A>. Sequencing was sent, and results indicated that the constructions of <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS/hpaC</i> (BBa_K902058)</a> were successful, meaning that after many months of trying we FINALLY have a promoter in front of the <i>hpaC</i> gene and can proceed to test the parts functionality. Attempts to construct <i>hpaC</i> with the (<A HREF="http://partsregistry.org/Part:BBa_J13002">evil TetR promotor, BBa_J13002</A>) were abandoned, as it was believed that this construction was failing due to toxicity of over-expressing the protein, and it was determined that this part was not necessary after all. <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub>-RBS/dszB</i>, (BBa_K902054)</a> also worked, though this was less exciting. <a href="http://partsregistry.org/Part:BBa_K902051">BBa_B0034<br />
/<i>dszA</i></a> came back as a bad read despite looking very good on the confirmation digest gel, so this part will be resent for sequencing. Constructions of <A HREF="http://partsregistry.org/Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> were reattempted, and confirmation digests for this part looked good, and so samples were sent for sequencing.</p><br />
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<br />
<h2>Week 20 (September 9- September 15)</h2><br />
<p>Construction attempts on <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> were performed. Colonies grew for the constructions, however further confirmation results were dissapointing (only 2 clones of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a> appeared to have been successful). These clones were sent for sequencing, and constructions were reattempted.</p><p> However, when sequencing came back, somehow reads indicated that these clones were in fact a gene from the Denitrogenation project (which is 990bp and a completely different band then what we saw on the gel). We believe, somewhere, something has gone very wrong- further investigation into this will be carried out. In the meantime, the above constructions were reattempted, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902051">BBa_B0034/<i>dszA</i></A> was re-prepped in case a contaminant in the plasmid stock was to blame for the bad reads found in this batch of sequencing as well as the last. In addition, plasmid switches of multiple sequence confirmed parts into a <a href=" http://partsregistry.org/Part:pSB1C3"> pSB1C3</a> backbone were carried out. </p><br />
<br />
<h2>Week 21 (September 16- September 22)</h2><br />
<p>Colonies for the transforms of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> have been few and far between, and cPCR results are always discouraging. Sequencing results for other sections of the project have once again come back very confusing, and further research continues into the source of this madness. </p><br />
<p> The sulfur compound degradation assay was set up to test the desulfurization rate of the original <i>Rhodococcus baikonurensis </i> (refer to the protocol page).</p><br />
<br />
<br />
<h2>Week 22 (September 23- September 29)</h2><br />
<p>Attempts to construct <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, , and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> continue. In the meantime, <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> was tested for functionality. In order to do this, cultures of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> were grown up overnight in LB media. A strain carrying <A HREF="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</A> only was used as a negative control. The following morning, 20 &micro;L of each culture was inoculated into 3 mL of LB with various concentrations of hydrogen peroxide; 0 mM, 1 mM, 5 mM, and 10 mM. These cultures were then allowed to grow overnight, and culture turbidity was observed. It was found that the negative control exhibited no growth after 12h at 1 mM peroxide, however cultures with induced expression of catalase were turbid after 12 h of growth at this concentration (Fig. 10). This demonstrated the ability of the catalase to protect the cells from excessive peroxide concentrations.</p><p><br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 15: Catalase Assay. Overnight cultures of J04500 and J04500-<i>KatGLAA</i> were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbitity was observed.]]<html></p><br />
</p><br />
<p>In addition to this, activity of <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-hpaC</i></a> was tested. In order to do this, cultures of <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-hpaC</i></a> and <a href="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub>-dszB</i></a> were grown up overnight in LB with appropriate antibiotics. Following this, protein expression was induced with IPTG, after which the assay was carried out as described in the following figure and on the protocols page.</p><br />
<p> </html> <br />
[[File:Ucalgary2012 DesulfurizationGroup HpaC assayTake1.png|center|600px|thumb|Figure 16: HpaC Assay. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 2 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, 1 mL of cell lysate was transferred to a cuvette, and a spectrophotometer was blanked at 340 nm (maximal absorbance of NADH) with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p>When the assay was run, it was found that NADH does not convert readily to NAD+ on its own. When cell lysate containing the naturally expressed amounts of oxidoreductase was added, a decrease in absorbance could quickly be observed as the NADH was converted to NAD+. When cultures over-expressing HpaC were tested, the absorbance levels were found to start much lower than the control. We believe that this is because with the amount of cell lysate tested, when the HpaC protein is overexpressed the NADH is consumed almost immediately and therefore the data reflecting the drop in absorbance is missed. Further tests will use differing amounts of cell lysate in order to try to capture data that shows the drop in absorbance for HpaC cultures.</p><br />
<br />
<h2>Week 23 (September 30-October 3): Wiki-Freeze PANIC!!</h2><br />
<br />
<p>The GCMS results of the sulfur assay were received. DBT desulfurization was successful (figures below). Some compounds were not detected by GCMS since they were too polar. However, 2 additional compounds showed degradation in addition to DBT, indicating that the pathway has a wider substrate specificity!</p><br />
<p></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 17: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol in the protocols section for details). Samples were taken out at different time points and were run through GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria, run through the GCMS after 6 days of being in the incubator to account for abiotic degradation. ]]<html></p<br />
<br />
<p></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 18: The peak in this mass spectra demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound ('''A:''' dibenzothiophene, '''B:''' tetrahydro-4h-thiopyran-4-one, and '''C''': benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdownn. Degradation is seen for DBT (the model studied compound) as well as other sulfur containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<p>The HpaC assay was repeated using 100 &micro;L of cell lysate instead of 1 mL of 2x concentrated cell lysate, following the previously used protocol. With this smaller amount, a clear sharp decrease in the absorbance of NADH can be observed, indicating a very fast conversion by HpaC, and further confirming that the part was functional.</p><br />
<br />
<p></html>[[File:Ucalgary2012Desulfurization-Hpacasay2.PNG|center|550px|thumb|Figure 20: HpaC Assay. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, 100 &micro;L of cell lysate was transferred to a cuvette, and a spectrophotometer was blanked at 340 nm (maximal absorbance of NADH) with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<h2>Week 24 (October 4- October 11)</h2><br />
<p>This week, before the Americas West Regional Competition, attempts to construct the previously mentioned parts continued. Unfortunately, they were unsuccessful.<br />
<br />
<h2>Week 25 (October 16- October 20)</h2><br />
<p>Traditional attempts to build the sulfur operon and its sub-parts were repeated, however attempts failed once more. During this week, it was decided that due to time consstraints, alternative approaches to construct the operon would be undertaken. Due to this, splice-overlap-extention (SOE) PCR primers (containing scar-sites where appropriate) were designed according to <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/soe">protocol</a></b> and ordered.</p><br />
<p><br><br />
<br />
<br />
Primer: 1a (BBK)-J13/DszB F - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGTCCCTATCAGTGATAGAGATTGACATCCC-3'<br />
<br><br><br />
Primer: 1b (BBK)-J04/DszB F - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 2 Jxx/B-(RBS/C) R - 5'-GGTGACAGTGTCATCTAGTATTTCTCCTCTTTCTAGTACTATCGGTGGCGATTGAGGCTGTTG-3'<br />
<br><br><br />
Primer: 3 (Jxx/B)-RBS/C F - 5'-CAACAGCCTCAATCGCCACCGATAGTACTAGAAAGAGGAGAAATACTAGATGACACTGTCACC-3'<br />
<br><br><br />
Primer: 4 RBC/C-(RBS/A) R - 5'-GCCAGATGCATTTGTCGTTGTTGAGTCATCTAGTATTTCTCCTCTTTCTAGTATCAGGAGGTGAAGCCGGGAATCG-3'<br />
<br><br><br />
Primer: 5 (RBS/C)-RBS/A F - 5'-CGATTCCCGGCTTCACCTCCTGATACTAGAAAGAGGAGAAATACTAGATGACTCAACAACGACAAATGCATCTGGC-3'<br />
<br><br><br />
Primer: 6 RBS/A-(J04/HpaC) R - 5'-GGGGAGAGGCGGTTTGCGTATTGCTAGTATCATGAAGGTTGTCCTTGCAGTTGTGG-3'<br />
<br><br><br />
Primer: 7 RBS/A-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATCATGAAGGTTGTCCTTGCAGTTGTGG-3'<br />
<br><br><br />
Primer: 8 (BBK)-J04/HpaC F - 5'-CGAATTCGCGGCCGCTTCTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 9 (RBS/A)-J04/HpaC F - 5'-CCACAACTGCAAGGACAACCTTCATGATACTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 10 J04/HpaC-(RBS/Kat) R - 5'-CTGACGTGCTCATCTAGTATTTCTCCTCTTTCTAGTATTAAATCGCAGCTTCCATTTCCAGCATCAC-3'<br />
<br><br><br />
Primer: 11 J04/HpaC-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTAAATCGCAGCTTCCATTTCCAGCATCAC-3'<br />
<br><br><br />
Primer: 12 (J04/HpaC)-RBS/Kat F - 5'-GTGATGCTGGAAATGGAAGCTGCGATTTAATACTAGAAAGAGGAGAAATACTAGATGAGCACGTCAG-3'<br />
<br><br><br />
Primer: 13 RBS/Kat-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTATTAAGCAGCCAGAGCGTAGTTTTCGTC-3'<br />
</p><br><br />
<p><br />
Colony PCR was done on potential colonies of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, , and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A>. Faint bands around the expected size were seen, and these were stabbed with a pipette tip and stirred into water in order to be used as template in a further round of PCR to get amplification of the part. The plan was that these amplicons could then be used as an insert in further constructions, however no amplification was seen. Further optimization of this procedure could possibly result in a short-cut to construction, however the time needed to do this is a luxury that we do not have.</p><p>Additional attempts to create inserts by doing PCR on ligation reactions in addition to gel extraction of colony PCR bands were attempted as well, however these attempts were unsuccessful as well. <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/soe">SOE PCR</a> and possible <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Gibson_Assembly">Gibson Assembly</a> appear to be the Sulfur Teams' last hope at this point, and we are eagerly awaiting our primers.<br />
</p><br />
<br />
<br />
<h2>Week 26 (October 21- October 26)</h2><br />
<p><br />
Much to our delight, SOE PCR primers were recieved this week. Therefore, alongside traditional construction of the aforementioned parts, SOE PCR was carried out according to <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/soe">protocol</a> and these parameters:</p><br />
<br><br />
<h4>Round 1:</h4><p><br />
<ul><br />
<li>Primers 1a and 2 with <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A> as a template = Product L1 (J13002/DszB with BBk prefix and B0034/DszC overlap)<br />
<br></li><li><br />
Primers 1b and 2 with <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A> as a template = Product L2 (J04500/<i>dszB</i> with BBk prefix and B0034/<i>dszC</i> overlap)<br />
<br></li><li><br />
Primers 3 and 4 with <A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a> as a template = Product L3 (B0034/<i>dszC</i> with <i>dszB</i> and B0034/<i>dszA</i> overlap)<br />
<br></li><li><br />
Primers 5 and 6 with <A HREF="http://partsregistry.org/Part:BBa_K902050"><i>dszA</i></a> as a template = Product L4 (B0034/<i>dszA</i> with <i>dszC</i> and J04500/<i>hpaC</i> overlap)<br />
<br></li><li><br />
Primers 5 and 7 with <A HREF="http://partsregistry.org/Part:BBa_K902050"><i>dszA</i></a> as a template = Product L5 (B0034/<i>dszA</i> with <i>dszC</i> overlap and BBk suffix)<br />
<br></li><li><br />
Primers 8 and 10 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L6 (J04500/<i>hpaC</i> with BBk prefix and B0034/<i>katG-LAA</i> overlap)<br />
<br></li><li><br />
Primers 9 and 10 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L7 (J04500/<i>hpaC</i> with <i>dszA</i> and B0034/<i>katG-LAA</i> overlap)<br />
<br></li><li><br />
Primers 9 and 11 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L8 (J04500/<i>hpaC</i> with <i>dszA</i> overlap and BBk suffix)<br />
<br></li><li><br />
Primers 12 and 13 with <A HREF="http://partsregistry.org/Part:BBa_K902059"><i>RBS-katG-LAA</i></a> as a template = Product L9 (B0034/<i>katG-LAA</i> with J04500/<i>hpaC</i> overlap and BBk suffix)<br />
</li></ul><br />
</p><br />
<p><br />
</html>[[File:Ucalgary2012 Spliceoverlapextentionpcr1.png|center|thumb|800px|Figure 21: Round 1 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with varying amounts of DNA template as indicated above the wells. Cycling conditions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 55%deg;C for 1 min, 72%deg;C for 2 min. 30 s.)x 31, Stage 3 (72%deg;C for 10 min.). Bands seen at ~1500 bp for L1 through L5, ~800 bp for L6-L8, and ~2200 bp for L9 indicate successful amplification of the desired product.]]<html><br />
</p><br />
<h4>Round 2</h4><br />
<p><br />
<ul><br />
<li>Primers 1a and 4 with templates L1 and L3 = Product X1 (J13002/<i>dszB</i>/B0034/<i>dszC</i> with BBk prefix and B0034/<i>dszA</i> overlap)</li><br />
<li>Primers 1b and 4 with templates L2 and L3 = Product X2 (J04500/<i>dszB</i>/B0034/<i>dszC</i> with BBk prefix and B0034/<i>dszA</i> overlap)</li><br />
<li>Primers 3 and 6 with templates L3 and L4 = Product X3 (B0034/<i>dszC</i>/B0034/<i>dszA</i> with <i>dszB</i> and J04500/<i>hpaC</i> overlap)</li><br />
<li>Primers 3 and 7 with templates L3 and L5 = Product X4 (B0034/<i>dszC</i>/B0034/<i>dszA</i> with <i>dszB</i> overlap and BBk suffix)</li><br />
<li>Primers 5 and 10 with templates L4 and L7 = Product X5 (B0034/<i>dszA</i>/J04500/<i>hpaC</i> with <i>dszC</i> and B0034/<i>katG-LAA</i> overlap)</li><br />
<li>Primers 5 and 11 with templates L4 and L8 = Product X6 (B0034/<i>dszA</i>/J04500/<i>hpaC</i> with <i>dszC</i> overlap and BBk suffix)</li><br />
<li>Primers 8 and 13 with templates L6 and L9 = Product X7 (J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with BBk prefix and suffix)</li><br />
<li>Primers 9 and 13 with templates L7 and L9 = Product X8 (J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with <i>dszA</i> overlap and BBk suffix)</li><br />
</ul></p><p><br />
</html>[[File:Ucalgary2012 Spliceoverlapextentionpcr2.png|center|thumb|800px|Figure 22: Round 2 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with varying amounts of gel-extracted DNA template from the previous PCR round as indicated above the wells. Bands were extracted using Qiagen QIAquick Gel Extraction Kit. Cycling conditions for the PCR reactions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 65%deg;C for 1 min, 72%deg;C for 3 min. 30 s.)x 31, Stage 3 (72%deg;C for 10 min.). Faint bands seen at ~3000bp indicate amplification of the desired product.]]<html> <br />
</p><br />
<h4>Round 3</h4><br />
<p><br />
<ul><br />
<li>Primers 1a and 10 with templates X1 and X5 = Product Z1 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with B0034/<i>katG-LAA</i> overlap and BBk prefix)</li><br />
<li>Primers 1a and 11 with templates X1 and X6 = Product Z2 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with BBk prefix and suffix)</li><br />
<li>Primers 1a and 7 with templates X1 and L5 = Product Z3 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 10 with templates X2 and X5 = Product Z4 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with B0034/<i>katG-LAA</i> overlap and BBk prefix)</li><br />
<li>Primers 1b and 7 with templates X2 and L5 = Product Z5 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 11 with templates X2 and X6 = Product Z6 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 6 with templates L2 and X3 = Product Z7(J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with J04500/<i>hpaC</i> overlap and BBk prefix)</li><br />
<li>Primers 1b and 7 with templates L2 and X4 = Product Z8 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1a and 6 with templates L1 and X3 = Product Z9 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with J04500/<i>hpaC</i> overlap and BBk prefix)</li><br />
<li>Primers 1a and 7 with templates L1 and X4 = Product Z10 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 5 and 13 with templates X5 and X8 = Product Z11 (B0034/<i>dszA</i>/J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with <i>dszC</i> overlap and BBk suffix)</li><br />
</ul></p><p><br />
</html>[[File:Ucalgary2012 sulfurSpliceoverlapextentionpcr3.png|center|thumb|800px|Figure 23: Round 3 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with 50 ng of gel-extracted DNA template from the previous PCR round. Bands were extracted using Qiagen QIAquick Gel Extraction Kit. Cycling conditions for the PCR reactions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 65%deg;C for 1 min, 72%deg;C for 6 min.)x 31, Stage 3 (72%deg;C for 10 min.). A faint band (~6000bp) can be seen in lane 4, indicating possible amplification of the desired product. Other reactions appear to have failed, as this bands are not present elsewhere.]]<html> <br />
</p><br />
<p><br />
Though it appears that S.O.E. PCR consists of crushed dreams and lies (or that extensive optimization would have to be performed in order to amplify the correct product), extraction was carried out on the single band seen, in the hopes that a subsequent round of PCR with the terminal primers for this product would possibly amplify it to a concentration that can be used in construction in the next week to assemble and test the sulfur operon constructs before competition. Conveniently, the first round of PCR creates <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Gibson_Assembly">Gibson Assembly</a> compatible products. Because a kit is available in the lab, this will be attempted in the following days as well. Meanwhile, a round of traditional construction appears to have finally created the parts <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902079"><i>P<sub>tetR</sub></i>-<i>dszB</i>-B0034-<i>dszC</i></A>, <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902080"><i>P<sub>lacI</sub></i>-<i>dszB</i>-B0034-<i>dszC</i></A>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902081"><i>P<sub>lacI</sub></i>-<i>hpaC</i>-B0034-<i>katG</i></A>. Though sequence confirmation could not be obtained before Wiki-Freeze, we are confident that these parts are correct due to the clear size difference seen between these parts and their sub-parts. A plasmid switch was attempted the night before DNA submission had to be sent, however due to technical issues the incubator stayed at a low temperature overnight, and cultures did not grow enough to obtain the first two parts in a pSB1C3 backbone. Because of the difficulty assembling these parts, the deadline for DNA submission, and the fact that having them in an alternate backbone in the registry would allow for easier and quicker assembly of the sulfur operon by others (no plasmid switches of large parts would have to be performed, as we have found that plasmid switching the larger constructs is quite difficult.) we decided to submit these parts in an ampicillin backbone. Further attempts to switch the backbone for these parts will continue, and hopefully we will be able to submit them in the standard backbone at a later date before the competition. In addition, a reconstruction of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902051">B0034-<i>dszA</i></A> was obtained and finally, at long last, sequence confirmed. Because of the verification of this part and the excellent sequence read when compared to those obtained for the previously submitted version, we decided to also resubmit this part to the registry.</p><p>Construction attempts of the operon will continue into the following week, as only 1 additional construction is needed to assemble a testable version of the operon. Hopefully, this data will be obtained before the competition, and documented on the respective parts pages.</p><br />
<p><br><br>This is the Desulfurization Team, signing off.</p><br />
</h><br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/DesulfurizationTeam:Calgary/Notebook/Desulfurization2012-10-26T23:06:04Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Desulfurization Journal|<br />
<br />
CONTENT=<br />
<html><br />
<h2>Week 1 (May 1-4)</h2><br />
<p>During this week, literature search was performed.</p><br />
<br />
<br />
<br />
<br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<br />
<br />
<p>Along with the rest of the team, this week was dedicated to familiarizing ourselves on the protocols that will be utilized during this years project; specifically the polymerase chain reaction, gel verification, preparation of overnight cultures, as well as developing a procedural flowchart to transform competent cells with registry biobricks. With regards to our sub-group specific goals, we reviewed the current available literature around various industrial and laboratory approaches to desulfurization of organic groups, especially in the petroleum industry. This included a comparison of non-biological processes such as conventional hydrodesulfurization, which is currently employed in petroleum product refinery stages, and how a biological approach would supplement and perhaps even offer several advantages over these methods. Current limitations to biological desulfurization, however, include such factors as biocatalyst stability, enzyme specificity and desulfurization rate. We also identified the enzyme desulfinase (DszB) as being one of the bottlenecks in the desulfurization 4S pathway. Overall, our goals moving forward involve determining the specific pathways involved in the desulfurization process as well as the reaction conditions we would want to employ, and identifying specific model compounds in addition to dibenzothiophene (DBT) that we could use to test the effectivity of our biosystem in order to determine its functionality in the conversion of naphthenic acids to economically valuable hydrocarbons.</p><br />
<br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<br />
<br />
<p> Building on the previous week's literature review, the 4S pathway was recognized as the preferred biological mechanism that we would explore in devising a desulfurization biosystem. Of specific interest is the dsz operon consisting of the genes for <i> dszA, dszB, </i>and<i> dszC</i> which selectively and non-destructively remove the sulfur from the hydrocarbon structure, and therefore preserves the carbon skeleton. In addition to these, another dsz gene exists.<i> dszD</i>, which codes for a FMN:NADH reductase, is an essential component of the pathway, but not part of the operon. Instead, it is on the genome. The enzyme produced by this gene is required to regenerate the FMNH2 consumed by the reactions carried out by DszA and DszC. <i>Rhodococcus erythropolis</i> IGTS8 is the most studied model organism in investigations of the 4S pathway, and has been shown in many different research endeavors to be capable of converting DBT to 2-HBP.</p><p><br />
</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|thumb|750px|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD. Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO2) through the addition of oxygen to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the heteroatom, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-hydroxybiphenyl. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.]]<html></p><p><br />
An alternative to the DszD gene is HpaC, an oxidoreductase encoded in the <i>E. coli</i> W genome. This enzyme has been shown to increase the rate of desulfurization. Following this, other protocols added to our growing lab methods 'toolkit' were a restriction digest protocol, PCR purification, and finally, DNA construction digest. Aims moving forward include obtaining strains of the <i> R. erythropolis </i>, while also executing a timeline devised to biobrick, test, and incorporate the genes necessary in the above processes in a biobrick circuit.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<br />
<br />
<p>This week was kicked off with a project development meeting with Emily and David, and we devised a protocol for biobricking the <i> hpaC </i> gene. Additionally, methods to place the genes coding for the 4 enzymes, DszA,B,C and HpaC into a single construct were explored. Within the lab, the PCR performed on the resuspended pUC18-hpaC was not successful initially. <br />
Furthermore, we ordered the substrates/compounds that we intend to use for desulfurization tests. Once the substrates and the <i>Rhodococcus</i> strain arrive we are going to test how effectively the bacteria can desulfurize different sulphur-containing compounds that resemble naphthenic acids. Finally, we came across a paper where a team had developed an improved efficiency DszB through site-directed mutagenesis in 2007. This was through a point mutation to the gene, converting a tyrosine at position 63 to a phenylalanine residue. A member of this team was contacted to request the plasmid that contains the mutated gene. The conversion step carried out by DszB is the major bottleneck in the 4S pathway and if a strain or sample containing this mutation was obtained, it would significantly bolster our later testing efforts on DBT, as well as other compounds such as thiophane.<br />
</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<br />
<br />
<p>Since we wanted to make sure we would not run out of pUC18(plasmid containing the hpaC gene), we transformed some E.coli cells with it. We grew them on plates containing ampicillin (A), kanamycin (K), tetracycline (T) and chloramphenicol (C) antibiotics and they only grew on A. Therefore pUC18 has A resistance. We did a three sets of PCR with primers designed against <i>hpaC</i>, one using 1/10 dilution of pUC18, the other using 1/100 dilution of pUC18 and one with the colonies we had just obtained by transforming the <i>E.coli</i> cells. </p><br />
<p>The PCR worked and we saw bands of the same size for all three sets of PCR. (Unfortunately, the picture we saved is not a good one since some of the bands faded away under UV due to prolonged exposure. Following this, PCR purification was performed to obtain the pure <i>hpaC</i> with biobrick prefix and suffix attatched to gene, which would allow us to insert the sequence into a biobrick standard backbone. 3 sets of digestion, ligation, and transformation (using pairs of X&P enzymes, E&S enzymes and E&P enzymes) were carried out in order to insert the <i>hpaC </i> gene into the <a href=" http://partsregistry.org/Part:pSB1C3">pSB1C3</a><br />
vector. All the sets grew successfully.<br />
Following the above successes with<i> hpaC</i>, the arrival of our <i>Rhodococcus</i> strain afforded us the opportunity to begin investigation of the Dsz operon using the primers currently in our possession. This strain is an environmental isolate that has been shown to be an active desulfurizer. The gram-positive nature of the strain also dictated we explore various lysing strategies before the genes encoding the Dsz enzymes could be amplified for further purification and biobrick construction steps. PCR was carried out using <i>dszA</i> primers on three different treatments {microwave, lysate buffer, and a control} which yielded banding pattern around 1200 base pairs for the lysate treatment (2%SDS and 10% tritonX-100, plus heat for 5mins at 98C).<br />
</p> <br />
<br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<br />
<br />
<p> In order to confirm the <i>hpaC</i> biobrick construction, two sets of colony PCR were performed, choosing white colonies from the 3 plates we grew last week (white colonies indicate a loss of the RFP generator in the <a href=" http://partsregistry.org/Part:pSB1C3">pSB1C3</a><br />
backbone, and therefore allow for weeding out of the colonies which are simply the original plasmid vector). These reactions were carried out both with <i>hpaC</i> primers and with standard biobrick primers designed against the plasmid backbone. After running them on the gel we saw equal bands for the PCR reactions performed using <i>hpaC</i> primers (However, a PCR using biobrick primers was performed later and the same results were obtained). Colonies 1(-) and 5(-) were used to make overnight cultures, which were then miniprepped the following day to obtain the plasmid DNA of the putative <i>hpaC</i> biobrick. Digestions were performed on the miniprep products using EcoRI and PstI to look for part size as further verification for the genes presence in the plasmid. The results were good and two bands were observed on each column (one for vector and the other for <i>hpaC</i>)). <i>hpaC</i> was sent in for sequencing. </p><br />
<p><br />
<br />
<br />
<br />
</html>[[File:UCalgary2012_04.06.2012-desulfurisation_hpacverification.jpg|thumb|700px|center|Figure 2: HpaC verification cPCR. HpaC gene was inserted into the pSB1C33 vector and E. coli cells were transformed. In order to confirm that pSB1C3 contains the hpaC gene, two sets of colony PCR's were conducted. One with biobrick primers, and the other with hpaC primers. Bands indicate successful amplification at the approximate size of the hpaC gene (517 bp)]]<br />
[[File:Ucalgary2012 06.06.2012-digestion of hpaC with E and P.jpg|thumb|500px|center|Figure 3: HpaC confirmation digest. Plasmid was miniprepped and digested for 2h with EcoRI and SpeI before being run on a 1% gel for 1h and 120V. Bands between 500 and 700 bp indicate the hpaC part is present as an insert. Bands of about 2000bp show the size of the psb1c3 vector.]]<html></p><br />
<br />
<br />
<br />
<p> PCR reagents were prepared to re-test/confirm previous results of <i>dszA</i> amplification following two different lysing treatments (microwave + lysate buffer). This time, all three genes were amplified and gel verification showed clear banding patterns around 500bp range for all three genes for the microwave treatment. Remaining PCR products were run on a gel and extracted for further purification steps; however, presence of any genetic material were not confirmed through nanodropping which raised concerns about the composition of the purified products, the success of the initial amplification step, or perhaps even the lysis treatment. Further experimentation will have to be carried out to troubleshoot.</p><br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<br />
<br />
<p> This week, we focused on amplifying <i>dsz</i> genes from our <i>Rhodococcus</i> strain for construction into biobricks. We also wanted to purify the <a href=" http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"> BBa_K902057 <i>hpaC</i></a> and pUC18-<i>hpaC</i> plasmids to replenish our current stocks. For the <i>dsz</i> aspect, we were able to successfully grow extra plates of <i>Rhodococcus</i> strain which was used to inoculate PCR tubes. The PCR did not go well, with significant streaking and false positives with similar banding pattern to previous gels run in the previous week. A final gel verification of a random sample of a tube of PCR products from dszA,B,C respectively and two negative control treatments involving master mix only and the lysed cells only illustrated the lack of discrepancy between the supposed successful amplification and the lysed cells (with lysate buffer) alone. Because of this we decided to take a different approach involving plasmid isolation carried out before PCR, rather than applying the PCR reagents directly to a lysed culture sample.</p><br />
<p> <a href=" http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> verification through sequencing was successful, confirming the construction of our first biobrick. Subsequently, O/N cultures of the plasmid containing cultures were prepared and stored in glycerol at -80C. Furthermore, verification of catalase gene part (<a href=" http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a>), which was sent as a culture stab from the parts registry was initiated. Our newly identified biobricked-hpaC was used as a positive control, but the banding pattern was not very conclusive. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<br />
<br />
<p>PCR was reattempted on <i>Rhodoccocus</i> that was lysed using two different dilutions of the lysate buffer, but the gel verification confirmed the previous failure in using this approach. An alternative that involved preparation of an overnight culture of the <i>Rhodococcus</i> cells followed by a plasmid purification was followed. The plasmid purification eventually yielded plasmid samples with concentrations of 98.6ng/μL to 182.7ng/μL (4 samples obtained overall). Additionally, the catalase biobrick was used to transform some stock competent cells, and samples of some colonies were subsequently PCR'ed. Although, the gel verification showed some potential contamination, and the required banding patterns at around 2200bp was not obtained. </p><br />
<br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<br />
<br />
<p> PCR was attempted to amplify the genes of the <I>dsz</i> operon utilising an adapted PCR protocol with purified <i>Taq</i> polymerase that had been isolated from the host organism. Eventually, some banding pattern was obtained between 1200 and 1500 base pairs when a gradient thermocycler was used with melting temperatures ranging betweeen 55°C to 65°C. This was assumed to be indicative of successful amplification of <i>dszB</i>; however, further purification and gel verification results were inconclusive and no yield was obtained when placed tested using a nanodrop machine. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<br />
<br />
<p>Top 10 E.coli cells were transformed with <a href="http://partsregistry.org/Part:BBa_R0011">BBa_R0011</a><br />
(IPTG inducible promoter in psb1C3 backbone), and resulting colonies were tested using cPCR. <br />
Colony PCR was performed on cells containing the catalase biobrick. Catalase is 2217bp long but since biobrick primers add about 200bp, bands of 2400 bp were expected if the part was present in the biobrick. These bands were observed, indicating that the <a href=" http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a> gene was most likely present.</p><br />
<p></html>[[File:Ucalgary2012 4.7.2012 catalase colony pcr 2.jpg|thumb|500px|center|Figure 4: Colony PCR on potential colonies containing katG-LAA. Biobrick primers were used. Bands at 2200 indicate successful amplification of the catalase part within the biobrick backbone. Smaller bands are indicative of primer degradation and are nonspecific amplification. +C indicates the positive control, and RFP generator, which should amplify at around 1000 bp.]]<html></p> <p><br />
<br />
PCR using Phusion high fidelity polymerase was carried out on <i>dszA</i>, <i>dszB</i>, and <i>dszC</i> in a gradient thermocycler. Amplification of non-specific bands was present for <i>dszA</i> and <i>dszB</i>, however strong banding for the desired size of the gene was observed for both (around 1500 for <i>dszA</i>, 1100 for <i>dszB</i> </p><br />
<br />
<p></html>[[File:Ucalgary2012 6.7.2012.dszABphusionPCR.jpg|thumb|500px|center|Figure 5:DszA and DszB PCR from <i>Rhodococcus</i>. DszA amplicon is around 1300 bp, and is observed to run higher on the gel. DszB amplicon is expected to be 1098 bp, which is observed in addition to multiple nonspecific banding. +C indicates the positive control, and RFP generator, which when PCRed with biobrick primers should be around 1000 bp. As this control is running high as well, it is believed that both amplicons for DszA and DszB have been obtained. No contamination is observed in the NTC (no template control).]]<html><br />
</p> <br />
<p></html>[[File:Ucalgary2012 7.5.2012 dszC.png|thumb|500px|center|Figure 6:DszC PCR from <i>Rhodococcus</i>. The numbers above the figure show the colony number. DszC amplicon is around 1450 bp, and is observed to run at almost the right size on the gel. No contamination is observed in the NTC (no template control).]]<html><br />
<p><br />
Examining the sequences of the <i>dszABC</i> genes led to the discovery that all 4 had multiple illegal enzyme cut-sites in them that we have to eliminate before biobrick composite part construction can occur. <i>dszA</i> has four PstI cut sites, <i>dszB</i> has a PstI and a NotI and <i>dszC</i> has a PstI cut site. The Stratagene QuickChange mutagenesis procedure is going to be used to eliminate illegal cut sites with the only alteration being that Kapa HiFi polymerase would be used during the process. Primers needed for the mutagenesis were designed based on the procedure mentioned above. </p><br />
<br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<br />
<p> Following successful amplification of the <i>dsz</i> operon genes in the previous week, the genes were constructed into the <a href="http://partsregistry.org/Part:pSB1C3"> pSB1C3</a> vector. Colony PCR verifications were observed to be positive. Furthermore, the insertion of part <a href="http://partsregistry.org/Part:BBa_J13002"> BBa_J13002, (P<sub>tetR</sub>-RBS)</a><br />
in front of the previously biobricked <i>hpaC</i> was attempted. Overnight cultures were also prepared using two colonies each for <a href="http://partsregistry.org/Part:BBa_J13002"> <i>P<sub>tetR</sub>-RBS</i></a> and <a href="http://partsregistry.org/Part:BBa_R0011"> <i>P<sub>lacI</sub></i> </a> (an IPTG inducible promoter that we hope to build in front of <ahref="http://partsregistry.org/Part:BBa_B0034">an RBS site, BBa_B0034</a>). These cultures were then miniprepped to yield the respective parts.</p> <br />
<br />
<p>Additionally, <a href="http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a> was built into a <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> backbone. The construction and availability of all these parts will be critical in the construction of our overall circuit for biodesulfurization. Colonies which looked good on cPCR were used to prepare overnight cultures, and were miniprepped and sent in for sequencing verification the following day.<br />
On the side, M9 minimal media was also prepared to carry out growth experimentation and overall desulfurization capability of <i>Rhodococcus</i> when exposed to DBT. The various growth treatments were M9 Media and glucose only, M9+glucose+DBT, M9+glucose+MgSO4+/-DBT, M9+glucose+MgCl2+/-DBT. 0.008g of FeCl2.4H2O was also added to each of the tubes. Samples were then inoculated with colonies of the <i>Rhodococcus</i>.</p><br />
<br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<br />
<p>This week, while awaiting sequencing verification results which were required before we could begin the construction process, the desulfurization team initially aided in some of the tasks related to the other hydrocarbon groups. The success of the construction of <a href="http://partsregistry.org/Part:BBa_J13002"><i>P<sub>tetR</sub></i>-RBS</a><br />
with <a href="http://partsregistry.org/Part:BBa_K902057"><i>hpaC</i></a> was also explored by using forward and reverse primers of <a href="http://partsregistry.org/Part:BBa_R0040">BBa_R0040</a> (the promoter component of the composite part BBa_J13002). However, the eventual gel verification was inconclusive and sequencing results finally indicated an unsuccessful ligation. Additionally, the minimal media M9 preparation had been contaminated in the previous effort so this process was repeated to create tubes of each of the growth condition treatments detailed previously, and two repeats, one with an extra filtration step and one without was used to prepare the cultures.</p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<br />
<p> Mutagenic primers were redesigned after the initial ones were found to have premature stop codons. As part of the redesign process in constructing our overall gene circuits for desulfurization, a backbone switch of <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> into a chloramphenicol (Chlor) resistant vector was necessary. The subsequent transformed products were plated on a Chlor plate and selected colonies were used to prepare O/N cultures, then minipreped before finally being digested with enzymes EcoRI and PstI. The resulting gel verification images were inconclusive as they did not show the required banding pattern around 50bp. Meanwhile, colony PCR was run on colonies transformed with <i>katG-LAA</i> constructed into a <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> backbone, as well as the <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> +<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a> construct. <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></a> was shown to have been successfully amplified, so overnight cultures were prepared and subsequently miniprepped. On the other hand, the construct was not successful so a third attempt was carried out. Colony PCR treatments that used either <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> forward primers or <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
primers were used and the overall constructs were made either on a chlor-resistant, or ampicillin-resistant vectors. Preliminary images of the gel verification appeared to have confirmed the construct, although sequencing verification will be the final indicator of overall success. </p><br />
<br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<br />
<br />
<p>Sequencing results from the previous week's constructs were available confirming that we constructed KatGLAA in a chlor-resistant backbone. However, switching the plasmid backbone of <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> to <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> was not successful. The construction of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>+<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> was finally sent in for sequencing. Site-directed mutagenesis of the <i>dsz</i> operon was also initiated: <i>dszA</i> has four PstI cut sites; <i>dszB</i> has a PstI and a NotI site; <i>dszC</i> has two PstI cut sites. Site directed mutagenesis was started this week to change a single base pair in these genes in a way that eliminates the cut site but preserves the amino acid codons, so as to not mutate the protein coding sequence. Ohshiro 2007 demonstrated that replacing the Tyr residue at position 63 of <i>dszB</i> gene with a Phe increases the activity of the enzyme. Therefore we want to introduce the same mutation into our <i>dszB</i>.</p><p><br />
For the first attempt at mutagenesis we chose to mutate the second PstI site in <i>dszC</i> (PstI2). As a positive control for the procedure, we also performed the mutagenic PCR on a plasmid containing the β-galactosidase gene with a point mutation where the PCR would cause it to regain its function. For both mutagenesis protocols we used the Kappa Hifi kit. After confirming that the PCR worked by running some produce on a gel, the PCR products were DpnI digested, the purpose of which is to degrade the unmodified parental DNA (DpnI degrades methylated DNA only). Control PCR products were plated on an ampicillin plate containing IPTG and X-gal. The colonies that grew on the control plates were blue indicating that the mutagenesis had worked for the β-galactosidase gene. Minipreps of the O/N culture of <i>dszC</i> mutants were digested with PstI enzyme and the results indicated that the mutagenesis was successful.</p><p> <br />
Attempts to simultaneously perform all the mutations in <i>dszC</i> genes in one step using the Knight procedure failed (<a href="http://openwetware.org/wiki/Knight:Site-directed_mutagenesis/Multi_site">Knight Multi-site Mutagenesis Procedure</a>). What enables simultaneous mutations is that Taq ligase closes the gaps in PCR products after each cycle. In the protocol it instructs to use Taq ligase buffer only for the PCR/ligation protocol. We suspected that the reason this procedure did not work might be that the Kappa polymerase is not functional in Taq ligase buffer. Therefore we did some experiments on the controls in Taq ligase kit and kappa polymerase kit to find out which buffer that Kappa polymerase and Taq ligase both work best in. The result was that both enzymes work best in a buffer made of half Taq ligase buffer and half Kappa polymerase buffer. </p><br />
<br />
<br />
<br />
</html>[[File:Ucalgary2012 31.7.2012 dszc mutagenesis 5 20 and 50ng.jpg|thumb|500px|center|Figure 7: DszC PstI2 mutagenesis PCR with varying concentrations of template plasmid was performed. The gel shows the PCR products that were run on a gel. +C lanes show the PCR products of the control. The control was pWhitescript™ 4.5-kb control plasmid from Stratagene mutagenesis kit which was mutated with primers also from the kit. Bands indicate successful amplification of the plasmid.]]<br />
[[File:UCalgary 02.08.12 dszC psti digest mutagenesis.jpg|thumb|700px|center|Figure 8: The control and the mutated plasmids were digested with PstI restriction enzyme. The control is dszC biobrick. Other lanes show the plasmids purified from the cells transformed with PstI2 mutagenesis PCR products followed by digestion. The control shows three bands since it has three PstI cut sites. The other lanes show two bands which indicates one of the cut sites has been eliminated through mutagenesis. ]]<html><br />
<br />
</html>[[File:Ucalgary2012 2.8.2012 finding the right buffer for multisite mutagenesis.png|thumb|800px|center|Figure 9: The multisite mutagenesis using the Knight procedure was tried in different buffers to find out the buffer that Kappa works optimally at. All the PCRs were performed on the pWhitescript™ 4.5-kb control plasmid from Stratagene mutagenesis kit which contains the beta-galactosidase gene. Based on this gel, the optimal buffer is composed of 50% kappa buffer and 50% Taq ligase buffer.]]<html><br />
<br />
</html>[[File:Ucalgary 3.8.2012 optimal buffer for Taq ligase.png|thumb|800px|center|Figure 10: Using the control provided in the NEB Taq ligase buffer (BsteII digested lambda DNA), we tried to find if the Taq ligase enzyme can function in combinations of Kappa Hifi buffer and Taq ligase buffer. Lane two is the control which is only the digested lambda DNA. Lanes 1 and 3 show that some of the bands compared to control have been ligated together. Therefore, Taq ligase functions just as good in the buffer composed of 50% kappa hifi buffer and 50% Taq ligase as it would in its own buffer.]]<html><br />
<br />
<h2>Week 15 (August 6 - August 11)</h2><br />
<br />
<p>Sequencing results for <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A> <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> returned negative, so a 3-part ligation method was used to retry this construction. The following parts were ligated with the restriction enzymes indicated in brackets after each: <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>(EcoRI/SpeI) + <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> (XbaI/PstI) + <A HREF=" http://partsregistry.org/Part:PSB1K3">BBa_PSB1K3</A> (EcoRI/PstI). Also, the more conventional construction (only 1 insert) of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>(SpeI/PstI) + <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> (XbaI/PstI) was reattempted. Furthermore, 3-way ligations were also attempted for <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
+<A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A>+<A HREF="http://partsregistry.org/Part:PSB1K3">BBa_PSB1K3</A>, and <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a>+<a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
+<A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A>, as well as the two-way contruction of just <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> after the <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a>. After plating these transformations, colony PCRs were carried out and samples that gave an indication of being successful on the gels were used to prepare O/N cultures followed by miniprep. With regards to the site-directed mutagenesis side of the experimentation, <i>dszA</i>-PstI1 (the first PstI cut site in <i>dszA</i>) ,<i> dszB</i>-PstI and <i>dszC</i>(PstI2 mutated)-PstI1 mutagenesis were performed following the procedure explained in the previous week. The gel below shows the successful result of digest confirmation (Fig. 8). <br />
Multisite mutagenesis (Knight method) was repeated using the modified buffer (half Taq ligase buffer and half Kappa buffer). However it was not successful again. We also tried doing multisite mutagenesis using Pfu Turbo polymerase and following the Knight procedure without any buffer modifications. No successful results were observed. </p><br />
<br />
<br />
<br />
</html>[[File:Ucalgary2012 Digestion confirmation of mutagenesis in dszAPstI1, dszBPstI and dszCPstI1 (PstI2 site mutated)..jpg|thumb|800px|center|Figure 11: Confirmation of site-directed mutagensis to eliminate PstI cut sites in: <i>dszA</i>PstI1 cut site, <i>dszB</i>PstI cut site and <i>dszC</i>PstI1 (PstI2 site mutated). Note that in each case, the unmutated controls for each gene are to the left of each set of plasmids derived from mutagenic PCRs. Plasmids from cloned <i>dsz</i> genes are smaller than the bands to the right of them (which have undergone mutagenesis), indicating that in the plasmids on the right, the PstI cut site was lost. ]]<html><br />
<br />
<br />
<h2>Week 16 (August 12 - August 18)</h2><br />
<br />
<br />
<p>The progress in mutagenesis of <i>dsz</i> genes was continued from the previous week: <i>dszB</i>(PstI mutated)-Y63F and <i>dszA</i>(PstI1 mutated)-PstI3 mutagenesis. The gel below shows the digest confirmation.</p><br />
<br />
<br />
</html><br />
[[File:Ucalgary2012 15.08.2012 dszAPstI1&amp;3 dszB Psti y63f muta diges-1.jpg|thumb|800px|center|Figure 12: Digestion confirmation of biobricks of a) dszA (PstI1 mutated) PstI3 mutagenesis and b) dszB (PstI mutated) Y63F mutagenesis. Cutting dszA (PstI1 mutated) with PstI is expected to produce fragments of 2790, 333, 255, and 114bp. Cutting dszA (PstI1 and PstI3 mutated) is expected to produce fragments of 2730, 588, and 114bp. The primers for dszB-Y63F mutagenesis introduce an HpyAV cut site. pSB1C3 inteslf has two more HpyAV cut sites. Before the mutation bands of 2858 abd 310bp are expected, and after successful mutation bands of 1502, 1356, 310 are expected. Lane legend: 1- dszA (PstI1 mutated). 2-dszA (PstI1 mutated) digested with PstI. 3-dszA (PstI1 mutated) PstI3 mutagenesis c1 Digested with PstI. 4- dszA (PstI1 mutated) PstI3 mutagenesis c2 Digested with PstI. 5-dszA (PstI1 mutated) PstI3 mutagenesis c3 Digested with PstI. 6- dszA (PstI1 mutated) PstI3 mutagenesis c4 Digested with PstI. 7- Fermentas 1kb Plus Ladder. 8- Empty. 9- dszB (PstI mutated). 10-dszB (PstI mutated) digested with HpyAV. 11- dszB (PstI mutated) Y63F mutagenesis c1 digested with HpyAV. 12- dszB (PstI mutated) Y63F mutagenesis c2 digested with HpyAV. 13- dszB (PstI mutated) Y63F mutagenesis c3 digested with HpyAV. 14-dszB (PstI mutated) Y63F mutagenesis c4 digested with HpyAV.]]<html></p><br />
<br />
<p>We attempted a different approach to speed up the turnover time of the mutagenesis PCR. Briefly, after the PCR mutagenesis the PCR products were purified and then incubated with T4 polynucleotide kinase (PNK) and ligase. After heat inactivating the ligase and T4 PNK, the products were DpnI digested. Subsequently another round of DNA purification was performed. However, the results were unsatisfactory after the digest confirmation.</p><p><br />
Sequencing results came back. <i>dszA</i> (PstI1 and PstI3 mutated) and <i>dszB</i>(PstI and Y63F mutated) were good. However <i>dszC</i> (PstI1 and PstI2 mutated) had an insertion next to the PstI1 cut site. Mutagenesis was repeated on the <i>dszC</i>(PstI2 mutated).<br />
<i>dszB</i>(PstI and Y63F mutated)-NotI and <i>dszA</i>(PstI1 and PstI3 mutated)-PstI4 mutagenesis were also performed.</p><br />
<p> To investigate the desulfurisation capability of the <i>Rhodococcus</i> sp. from which we cloned the <i>dsz</i> operon, a desulfurization assay was prepared by inoculating different treatments of M9 media. We also prepared some solutions that will be needed for analysis in the following week: a conditioning agent composed of 100ml of 95% ethanol, 50ml glycerol, 30ml of 12M HCl (aq) and 70g of NaCl(s) was prepared. The assay relies on the turbidity of a sample containing sulphate ions which are precipitated (hence the turbidometric nature of the assay) upon adding BaCl2(s), therefore if the <i>dsz</i> pathway is active, we expect a more turbid solution to form than in control samples. </p><br />
<br />
<br />
<br />
<h2>Week 17 (August 19 - August 25)</h2><br />
<br />
<br />
<p>This week, progress was made in determining the desulfurization activity of our <i>Rhodococcus</i> strain as measured by the sulfate release using a turbidometric assay. We encountered several challenges in our prescribed protocol as the concentrations that we used to prepare the standard curve may have been too dilute, or the composition of out conditioning agent may have been flawed. Additionally, steps were taken to determine the decomposition of DBT to 2-HBP through Gas Chromatograph-Mass Spectroscopy (GC-MS) analysis, but due to a preparation error, the DBT was added to a growth solution of M9 media prematurely and the autoclaving process decomposed the DBT releasing a yellow colouration into the solution. These two approaches in determining the desulfurization capability of the <i>dsz</i> operon will be further investigated. </p><br />
<br />
<p>Since the <i>dszC</i> second mutagenesis had proven to be unsuccessful last week, the <i>dszC</i>(PstI2 mutated)PstI1 mutagenesis was repeated. Also <i>dszA</i>(PstI1,3,4 mutated) PstI2 mutagenesis was performed. <i>dszA</i> and <i>dszC</i> were sent for sequencing on Wednesday. <i>dszB</i> was sent for sequencing on Friday. Sequencing results of <i>dszA</i> and <i>dszC</i> were back by Friday. <i>dszC</i> was successful. However, <i>dszA</i> contained an insertion next to the binding site of PstI4 cut sit, so the last two mutations must be redone. <i>dszB</i>(PstI and Y63F mutated)-NotI-mutagenesis was also repeated in case the result of the sequencing was not successful. These constructions were repeated. <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>-<A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
-<i>dszC</i> constructions were attempted, however they were not successful as indicated by colony PCR. Constructions of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>/<i>hpaC</i> were carried out and also came back negative in sequencing, however <a href="http://partsregistry.org/Part:BBa_K902059">BBa_B0034<br />
/<i>katG-LAA</i> (BBa_K902059)</a> was sequence confirmed.</p><br />
</html>[[File:Ucalgary2012 20.8.2012 dszC(psti2)psti1 mutagenesis.png|thumb|700px|center|Figure 13: Another attempt to mutagenize dszC(PstI2 mutated)at PstI1 site. The gel shows the confirmation digestion. C is the control which is dszC(PstI2 mutated). Since the control has two PstI cut sites, two bands are observed on the gel. The mutagenesis has been successful since only one band is observed on the other lanes indicating that one of the cut sites has been eliminated during mutagenesis.]]<html><br />
<br />
</html>[[File:Ucalgary2012 21.8.2012 dszA final mutagenesis digest.png|thumb|700px|center|Figure 14: DszA (PstI1, PstI2, PstI3 mutated)PstI4 mutagenesis digestion confirmation gel. All the lanes show the results of the plasmids being cut with PstI enzyme. C indicates the lane containing the control which is dszA(PstI1, PstI3 and PstI4 mutated). The other lanes are the digestions of the plasmids that are mutagenized, from different colonies. Control plasmid has two PstI cut sites and therefore the two bands observed on the gel were expected. The other lanes show only one band of about 3500bp which is about the right size for dszA in a psb1c3 vector. Therefore one of the cutsites has been eliminated during mutagenesis.]]<html><br />
<br />
<br />
<br />
<br />
<h2>Week 18 (August 26 - September 1)</h2><br />
<br />
<p> <A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i> (BBa_K902052)</i></A> sequencing results came back as successful. <i>dszA</i>(PstI1,3 mutated)-PstI2-mutagenesis was performed and sent for sequencing. Also <i>dszA</i>(PstI1,2,3 mutated)-PstI4-mutagenesis was performed, and this was also sent for sequencing. </p><br />
<p>Constructions of <A HREF="http://partsregistry.org/Part:BBa_K902053">P<sub>tetR</sub>-RBS/<i>dszB</i> (BBa_K902053)</a> and <a href="http://partsregistry.org/Part:BBa_K902056">BBa_B0034<br />
/<i>dszC</i> (BBa_K902056)</a> were attempted, verification digested, and sent for sequencing. Sequencing results for these constructs came back as positive, along with successful mutagenesis of <A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i> (BBa_K902050)</A>.</p><br />
<p>At this point, all of the <i>dsz</i> genes have been successfully made biobrick compatible, and <i>hpaC</i> has been biobricked. We have also successfully constructed <a href="http://partsregistry.org/Part:BBa_K902059">BBa_B0034 with <i>katG-LAA</i></a> to be used in the optimization circuit, as well as <A HREF="http://partsregistry.org/Part:BBa_K902053"><i>P<sub>tetR</sub>-RBS</i>/<i>dszB</i> (BBa_K902053) </a>and <a href="http://partsregistry.org/Part:BBa_K902056">BBa_B0034/<i>dszC</i> (BBa_K902056)</a>.<br />
<p> Constructions of <A HREF="http://partsregistry.org/Part:BBa_J04500">P<sub>lacI</sub>-RBS</A>with <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> were performed. As well, attempts to construct <A HREF="http://partsregistry.org/Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></a>/<a href="http://partsregistry.org/Part:BBa_K902056">RBS-<i>dszC</i></a> as well as <A HREF="http://partsregistry.org/Part:BBa_J13002">P<sub>tetR</sub>-RBS</A>/<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
/<A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i></A>, and <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>/<A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> were also carried out. These parts are intended as construction intermediates towards building the final systems, as well as providing a way of testing the genes functionality (namely, to test HpaC for oxidoreductase activity and to test if over-expression of KatG in the cell will increase its ability to survive H<sub>2</sub>O<sub>2</sub> stress). Transformations of all these constructions were carried out at the end of the week.</p><br />
<br />
<h2>Week 19 (September 2- September 8)</h2><br />
<p>Confirmation digests on colonies of the previous constructions that gave bands of the expected size with cPCR were performed. Positive results were found for colonies of <A HREF="http://partsregistry.org/Part:BBa_J04500"><i>P<sub>lacI</sub>-RBS</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <A HREF="http://partsregistry.org/Part:BBa_J04500"><i>P<sub>lacI</sub>-RBS</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
/<A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i></A>. Sequencing was sent, and results indicated that the constructions of <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS/hpaC</i> (BBa_K902058)</a> were successful, meaning that after many months of trying we FINALLY have a promoter in front of the <i>hpaC</i> gene and can proceed to test the parts functionality. Attempts to construct <i>hpaC</i> with the (<A HREF="http://partsregistry.org/Part:BBa_J13002">evil TetR promotor, BBa_J13002</A>) were abandoned, as it was believed that this construction was failing due to toxicity of over-expressing the protein, and it was determined that this part was not necessary after all. <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub>-RBS/dszB</i>, (BBa_K902054)</a> also worked, though this was less exciting. <a href="http://partsregistry.org/Part:BBa_K902051">BBa_B0034<br />
/<i>dszA</i></a> came back as a bad read despite looking very good on the confirmation digest gel, so this part will be resent for sequencing. Constructions of <A HREF="http://partsregistry.org/Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> were reattempted, and confirmation digests for this part looked good, and so samples were sent for sequencing.</p><br />
<br />
<br />
<h2>Week 20 (September 9- September 15)</h2><br />
<p>Construction attempts on <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> were performed. Colonies grew for the constructions, however further confirmation results were dissapointing (only 2 clones of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a> appeared to have been successful). These clones were sent for sequencing, and constructions were reattempted.</p><p> However, when sequencing came back, somehow reads indicated that these clones were in fact a gene from the Denitrogenation project (which is 990bp and a completely different band then what we saw on the gel). We believe, somewhere, something has gone very wrong- further investigation into this will be carried out. In the meantime, the above constructions were reattempted, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902051">BBa_B0034/<i>dszA</i></A> was re-prepped in case a contaminant in the plasmid stock was to blame for the bad reads found in this batch of sequencing as well as the last. In addition, plasmid switches of multiple sequence confirmed parts into a <a href=" http://partsregistry.org/Part:pSB1C3"> pSB1C3</a> backbone were carried out. </p><br />
<br />
<h2>Week 21 (September 16- September 22)</h2><br />
<p>Colonies for the transforms of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> have been few and far between, and cPCR results are always discouraging. Sequencing results for other sections of the project have once again come back very confusing, and further research continues into the source of this madness. </p><br />
<p> The sulfur compound degradation assay was set up to test the desulfurization rate of the original <i>Rhodococcus baikonurensis </i> (refer to the protocol page).</p><br />
<br />
<br />
<h2>Week 22 (September 23- September 29)</h2><br />
<p>Attempts to construct <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, , and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> continue. In the meantime, <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> was tested for functionality. In order to do this, cultures of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> were grown up overnight in LB media. A strain carrying <A HREF="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</A> only was used as a negative control. The following morning, 20 &micro;L of each culture was inoculated into 3 mL of LB with various concentrations of hydrogen peroxide; 0 mM, 1 mM, 5 mM, and 10 mM. These cultures were then allowed to grow overnight, and culture turbidity was observed. It was found that the negative control exhibited no growth after 12h at 1 mM peroxide, however cultures with induced expression of catalase were turbid after 12 h of growth at this concentration (Fig. 10). This demonstrated the ability of the catalase to protect the cells from excessive peroxide concentrations.</p><p><br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 15: Catalase Assay. Overnight cultures of J04500 and J04500-<i>KatGLAA</i> were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbitity was observed.]]<html></p><br />
</p><br />
<p>In addition to this, activity of <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-hpaC</i></a> was tested. In order to do this, cultures of <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-hpaC</i></a> and <a href="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub>-dszB</i></a> were grown up overnight in LB with appropriate antibiotics. Following this, protein expression was induced with IPTG, after which the assay was carried out as described in the following figure and on the protocols page.</p><br />
<p> </html> <br />
[[File:Ucalgary2012 DesulfurizationGroup HpaC assayTake1.png|center|600px|thumb|Figure 16: HpaC Assay. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 2 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, 1 mL of cell lysate was transferred to a cuvette, and a spectrophotometer was blanked at 340 nm (maximal absorbance of NADH) with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p>When the assay was run, it was found that NADH does not convert readily to NAD+ on its own. When cell lysate containing the naturally expressed amounts of oxidoreductase was added, a decrease in absorbance could quickly be observed as the NADH was converted to NAD+. When cultures over-expressing HpaC were tested, the absorbance levels were found to start much lower than the control. We believe that this is because with the amount of cell lysate tested, when the HpaC protein is overexpressed the NADH is consumed almost immediately and therefore the data reflecting the drop in absorbance is missed. Further tests will use differing amounts of cell lysate in order to try to capture data that shows the drop in absorbance for HpaC cultures.</p><br />
<br />
<h2>Week 23 (September 30-October 3): Wiki-Freeze PANIC!!</h2><br />
<br />
<p>The GCMS results of the sulfur assay were received. DBT desulfurization was successful (figures below). Some compounds were not detected by GCMS since they were too polar. However, 2 additional compounds showed degradation in addition to DBT, indicating that the pathway has a wider substrate specificity!</p><br />
<p></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 17: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol in the protocols section for details). Samples were taken out at different time points and were run through GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria, run through the GCMS after 6 days of being in the incubator to account for abiotic degradation. ]]<html></p<br />
<br />
<p></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 18: The peak in this mass spectra demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound ('''A:''' dibenzothiophene, '''B:''' tetrahydro-4h-thiopyran-4-one, and '''C''': benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdownn. Degradation is seen for DBT (the model studied compound) as well as other sulfur containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<p>The HpaC assay was repeated using 100 &micro;L of cell lysate instead of 1 mL of 2x concentrated cell lysate, following the previously used protocol. With this smaller amount, a clear sharp decrease in the absorbance of NADH can be observed, indicating a very fast conversion by HpaC, and further confirming that the part was functional.</p><br />
<br />
<p></html>[[File:Ucalgary2012Desulfurization-Hpacasay2.PNG|center|550px|thumb|Figure 20: HpaC Assay. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, 100 &micro;L of cell lysate was transferred to a cuvette, and a spectrophotometer was blanked at 340 nm (maximal absorbance of NADH) with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<h2>Week 24 (October 4- October 11)</h2><br />
<p>This week, before the Americas West Regional Competition, attempts to construct the previously mentioned parts continued. Unfortunately, they were unsuccessful.<br />
<br />
<h2>Week 25 (October 16- October 20)</h2><br />
<p>Traditional attempts to build the sulfur operon and its sub-parts were repeated, however attempts failed once more. During this week, it was decided that due to time consstraints, alternative approaches to construct the operon would be undertaken. Due to this, splice-overlap-extention (SOE) PCR primers (containing scar-sites where appropriate) were designed according to <b>protocol</b> and ordered.</p><br />
<p><br><br />
<br />
<br />
Primer: 1a (BBK)-J13/DszB F - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGTCCCTATCAGTGATAGAGATTGACATCCC-3'<br />
<br><br><br />
Primer: 1b (BBK)-J04/DszB F - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 2 Jxx/B-(RBS/C) R - 5'-GGTGACAGTGTCATCTAGTATTTCTCCTCTTTCTAGTACTATCGGTGGCGATTGAGGCTGTTG-3'<br />
<br><br><br />
Primer: 3 (Jxx/B)-RBS/C F - 5'-CAACAGCCTCAATCGCCACCGATAGTACTAGAAAGAGGAGAAATACTAGATGACACTGTCACC-3'<br />
<br><br><br />
Primer: 4 RBC/C-(RBS/A) R - 5'-GCCAGATGCATTTGTCGTTGTTGAGTCATCTAGTATTTCTCCTCTTTCTAGTATCAGGAGGTGAAGCCGGGAATCG-3'<br />
<br><br><br />
Primer: 5 (RBS/C)-RBS/A F - 5'-CGATTCCCGGCTTCACCTCCTGATACTAGAAAGAGGAGAAATACTAGATGACTCAACAACGACAAATGCATCTGGC-3'<br />
<br><br><br />
Primer: 6 RBS/A-(J04/HpaC) R - 5'-GGGGAGAGGCGGTTTGCGTATTGCTAGTATCATGAAGGTTGTCCTTGCAGTTGTGG-3'<br />
<br><br><br />
Primer: 7 RBS/A-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATCATGAAGGTTGTCCTTGCAGTTGTGG-3'<br />
<br><br><br />
Primer: 8 (BBK)-J04/HpaC F - 5'-CGAATTCGCGGCCGCTTCTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 9 (RBS/A)-J04/HpaC F - 5'-CCACAACTGCAAGGACAACCTTCATGATACTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 10 J04/HpaC-(RBS/Kat) R - 5'-CTGACGTGCTCATCTAGTATTTCTCCTCTTTCTAGTATTAAATCGCAGCTTCCATTTCCAGCATCAC-3'<br />
<br><br><br />
Primer: 11 J04/HpaC-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTAAATCGCAGCTTCCATTTCCAGCATCAC-3'<br />
<br><br><br />
Primer: 12 (J04/HpaC)-RBS/Kat F - 5'-GTGATGCTGGAAATGGAAGCTGCGATTTAATACTAGAAAGAGGAGAAATACTAGATGAGCACGTCAG-3'<br />
<br><br><br />
Primer: 13 RBS/Kat-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTATTAAGCAGCCAGAGCGTAGTTTTCGTC-3'<br />
</p><br><br />
<p><br />
Colony PCR was done on potential colonies of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, , and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A>. Faint bands around the expected size were seen, and these were stabbed with a pipette tip and stirred into water in order to be used as template in a further round of PCR to get amplification of the part. The plan was that these amplicons could then be used as an insert in further constructions, however no amplification was seen. Further optimization of this procedure could possibly result in a short-cut to construction, however the time needed to do this is a luxury that we do not have.</p><p>Additional attempts to create inserts by doing PCR on ligation reactions in addition to gel extraction of colony PCR bands were attempted as well, however these attempts were unsuccessful as well. SOE PCR and possible <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Gibson_Assembly">Gibson Assembly</a> appear to be the Sulfur Teams' last hope at this point, and we are eagerly awaiting our primers.<br />
</p><br />
<br />
<br />
<h2>Week 26 (October 21- October 26)</h2><br />
<p><br />
Much to our delight, SOE PCR primers were recieved this week. Therefore, alongside traditional construction of the aforementioned parts, SOE PCR was carried out according to <b>protocol</b> and these parameters:</p><br />
<br><br />
<h4>Round 1:</h4><p><br />
<ul><br />
<li>Primers 1a and 2 with <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A> as a template = Product L1 (J13002/DszB with BBk prefix and B0034/DszC overlap)<br />
<br></li><li><br />
Primers 1b and 2 with <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A> as a template = Product L2 (J04500/<i>dszB</i> with BBk prefix and B0034/<i>dszC</i> overlap)<br />
<br></li><li><br />
Primers 3 and 4 with <A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a> as a template = Product L3 (B0034/<i>dszC</i> with <i>dszB</i> and B0034/<i>dszA</i> overlap)<br />
<br></li><li><br />
Primers 5 and 6 with <A HREF="http://partsregistry.org/Part:BBa_K902050"><i>dszA</i></a> as a template = Product L4 (B0034/<i>dszA</i> with <i>dszC</i> and J04500/<i>hpaC</i> overlap)<br />
<br></li><li><br />
Primers 5 and 7 with <A HREF="http://partsregistry.org/Part:BBa_K902050"><i>dszA</i></a> as a template = Product L5 (B0034/<i>dszA</i> with <i>dszC</i> overlap and BBk suffix)<br />
<br></li><li><br />
Primers 8 and 10 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L6 (J04500/<i>hpaC</i> with BBk prefix and B0034/<i>katG-LAA</i> overlap)<br />
<br></li><li><br />
Primers 9 and 10 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L7 (J04500/<i>hpaC</i> with <i>dszA</i> and B0034/<i>katG-LAA</i> overlap)<br />
<br></li><li><br />
Primers 9 and 11 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L8 (J04500/<i>hpaC</i> with <i>dszA</i> overlap and BBk suffix)<br />
<br></li><li><br />
Primers 12 and 13 with <A HREF="http://partsregistry.org/Part:BBa_K902059"><i>RBS-katG-LAA</i></a> as a template = Product L9 (B0034/<i>katG-LAA</i> with J04500/<i>hpaC</i> overlap and BBk suffix)<br />
</li></ul><br />
</p><br />
<p><br />
</html>[[File:Ucalgary2012 Spliceoverlapextentionpcr1.png|center|thumb|800px|Figure 21: Round 1 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with varying amounts of DNA template as indicated above the wells. Cycling conditions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 55%deg;C for 1 min, 72%deg;C for 2 min. 30 s.)x 31, Stage 3 (72%deg;C for 10 min.). Bands seen at ~1500 bp for L1 through L5, ~800 bp for L6-L8, and ~2200 bp for L9 indicate successful amplification of the desired product.]]<html><br />
</p><br />
<h4>Round 2</h4><br />
<p><br />
<ul><br />
<li>Primers 1a and 4 with templates L1 and L3 = Product X1 (J13002/<i>dszB</i>/B0034/<i>dszC</i> with BBk prefix and B0034/<i>dszA</i> overlap)</li><br />
<li>Primers 1b and 4 with templates L2 and L3 = Product X2 (J04500/<i>dszB</i>/B0034/<i>dszC</i> with BBk prefix and B0034/<i>dszA</i> overlap)</li><br />
<li>Primers 3 and 6 with templates L3 and L4 = Product X3 (B0034/<i>dszC</i>/B0034/<i>dszA</i> with <i>dszB</i> and J04500/<i>hpaC</i> overlap)</li><br />
<li>Primers 3 and 7 with templates L3 and L5 = Product X4 (B0034/<i>dszC</i>/B0034/<i>dszA</i> with <i>dszB</i> overlap and BBk suffix)</li><br />
<li>Primers 5 and 10 with templates L4 and L7 = Product X5 (B0034/<i>dszA</i>/J04500/<i>hpaC</i> with <i>dszC</i> and B0034/<i>katG-LAA</i> overlap)</li><br />
<li>Primers 5 and 11 with templates L4 and L8 = Product X6 (B0034/<i>dszA</i>/J04500/<i>hpaC</i> with <i>dszC</i> overlap and BBk suffix)</li><br />
<li>Primers 8 and 13 with templates L6 and L9 = Product X7 (J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with BBk prefix and suffix)</li><br />
<li>Primers 9 and 13 with templates L7 and L9 = Product X8 (J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with <i>dszA</i> overlap and BBk suffix)</li><br />
</ul></p><p><br />
</html>[[File:Ucalgary2012 Spliceoverlapextentionpcr2.png|center|thumb|800px|Figure 22: Round 2 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with varying amounts of gel-extracted DNA template from the previous PCR round as indicated above the wells. Bands were extracted using Qiagen QIAquick Gel Extraction Kit. Cycling conditions for the PCR reactions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 65%deg;C for 1 min, 72%deg;C for 3 min. 30 s.)x 31, Stage 3 (72%deg;C for 10 min.). Faint bands seen at ~3000bp indicate amplification of the desired product.]]<html> <br />
</p><br />
<h4>Round 3</h4><br />
<p><br />
<ul><br />
<li>Primers 1a and 10 with templates X1 and X5 = Product Z1 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with B0034/<i>katG-LAA</i> overlap and BBk prefix)</li><br />
<li>Primers 1a and 11 with templates X1 and X6 = Product Z2 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with BBk prefix and suffix)</li><br />
<li>Primers 1a and 7 with templates X1 and L5 = Product Z3 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 10 with templates X2 and X5 = Product Z4 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with B0034/<i>katG-LAA</i> overlap and BBk prefix)</li><br />
<li>Primers 1b and 7 with templates X2 and L5 = Product Z5 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 11 with templates X2 and X6 = Product Z6 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 6 with templates L2 and X3 = Product Z7(J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with J04500/<i>hpaC</i> overlap and BBk prefix)</li><br />
<li>Primers 1b and 7 with templates L2 and X4 = Product Z8 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1a and 6 with templates L1 and X3 = Product Z9 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with J04500/<i>hpaC</i> overlap and BBk prefix)</li><br />
<li>Primers 1a and 7 with templates L1 and X4 = Product Z10 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 5 and 13 with templates X5 and X8 = Product Z11 (B0034/<i>dszA</i>/J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with <i>dszC</i> overlap and BBk suffix)</li><br />
</ul></p><p><br />
</html>[[File:Ucalgary2012 sulfurSpliceoverlapextentionpcr3.png|center|thumb|800px|Figure 23: Round 3 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with 50 ng of gel-extracted DNA template from the previous PCR round. Bands were extracted using Qiagen QIAquick Gel Extraction Kit. Cycling conditions for the PCR reactions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 65%deg;C for 1 min, 72%deg;C for 6 min.)x 31, Stage 3 (72%deg;C for 10 min.). A faint band (~6000bp) can be seen in lane 4, indicating possible amplification of the desired product. Other reactions appear to have failed, as this bands are not present elsewhere.]]<html> <br />
</p><br />
<p><br />
Though it appears that S.O.E. PCR consists of crushed dreams and lies (or that extensive optimization would have to be performed in order to amplify the correct product), extraction was carried out on the single band seen, in the hopes that a subsequent round of PCR with the terminal primers for this product would possibly amplify it to a concentration that can be used in construction in the next week to assemble and test the sulfur operon constructs before competition. Conveniently, the first round of PCR creates <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Gibson_Assembly">Gibson Assembly</a> compatible products. Because a kit is available in the lab, this will be attempted in the following days as well. Meanwhile, a round of traditional construction appears to have finally created the parts <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902079"><i>P<sub>tetR</sub></i>-<i>dszB</i>-B0034-<i>dszC</i></A>, <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902080"><i>P<sub>lacI</sub></i>-<i>dszB</i>-B0034-<i>dszC</i></A>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902081"><i>P<sub>lacI</sub></i>-<i>hpaC</i>-B0034-<i>katG</i></A>. Though sequence confirmation could not be obtained before Wiki-Freeze, we are confident that these parts are correct due to the clear size difference seen between these parts and their sub-parts. A plasmid switch was attempted the night before DNA submission had to be sent, however due to technical issues the incubator stayed at a low temperature overnight, and cultures did not grow enough to obtain the first two parts in a pSB1C3 backbone. Because of the difficulty assembling these parts, the deadline for DNA submission, and the fact that having them in an alternate backbone in the registry would allow for easier and quicker assembly of the sulfur operon by others (no plasmid switches of large parts would have to be performed, as we have found that plasmid switching the larger constructs is quite difficult.) we decided to submit these parts in an ampicillin backbone. Further attempts to switch the backbone for these parts will continue, and hopefully we will be able to submit them in the standard backbone at a later date before the competition. In addition, a reconstruction of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902051">B0034-<i>dszA</i></A> was obtained and finally, at long last, sequence confirmed. Because of the verification of this part and the excellent sequence read when compared to those obtained for the previously submitted version, we decided to also resubmit this part to the registry.</p><p>Construction attempts of the operon will continue into the following week, as only 1 additional construction is needed to assemble a testable version of the operon. Hopefully, this data will be obtained before the competition, and documented on the respective parts pages.</p><br />
<p><br><br>This is the Desulfurization Team, signing off.</p><br />
</h><br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/soeTeam:Calgary/Notebook/Protocols/soe2012-10-26T22:49:38Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE= Splice Overlap Extension (SOE) PCR Protocol|<br />
CONTENT = <html><br />
<br />
<p>Primers are designed against a part template sequence in order to add sequence complementary to another part that you wish to join together. In addition to standard primer design considerations, the primers should have a melting temperature of 60&deg;C with one part, and 60&deg;C with the other. <br />
</p><br />
<p>Reactions should be set up as follows:</p><br />
<h4>Reaction Mixture</h4><br />
<ul><br />
<li>5 μL 10x PCR Buffer with MgSO<sub>4</sub></li><br />
<li> 5μL 10x dNTPs</li><br />
<li>50 pmol each primer*</li><br />
<li>50-125ng Template DNA**</li><br />
<li>0.5 μL <i>Pfu</i> Polymerase</li><br />
<li>Water up to 50 μL total volume</li><br />
</ul><br />
<br><br />
<p>*Primers should be added according to the desired product you want to amplify.<br>**During the first round of PCR, template DNA will be the part itself. Subsequent rounds of PCR will use the appropriate PCR fragments from this round of PCR which you want to build together as template. These should be added at equal concentrations, along with appropriate flanking primers. Using varying concentrations of DNA template can help to optimize the reaction.</p><br />
<h4>Cycling Conditions</h4><ul><br />
<li>Stage 1: 95%deg;C for 2 min.</li><br />
<li>Stage 2: 94&deg;C for 1 min., 55-65&deg;C for 1 min.***, 72&deg;C for 1min/kb</li><br />
<li>Stage 3: 72&deg;C for 10 min., hold at 4&deg;C</li><br />
</ul><br />
<p><br />
***Annealing temperature must be adjusted according to melting temperatures of specific primers used.</p><br />
<p>Following each PCR reaction, gel purification of the desired band should be carried out (using the QIAquick Gel Purification Kit or another kit). Products can subsequently be used in further PCR, or in constructions once a full circuit is assembled.</p><br />
<br />
<br />
<br />
<br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/soeTeam:Calgary/Notebook/Protocols/soe2012-10-26T22:48:51Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE= Splice Overlap Extension (SOE) PCR Protocol|<br />
CONTENT = <html><br />
<br />
<p>Primers are designed against a part template sequence in order to add sequence complementary to another part that you wish to join together. In addition to standard primer design considerations, the primers should have a melting temperature of 60&deg;C with one part, and 60&deg;C with the other. <br />
<p>Image</p><br />
<p>Reactions should be set up as follows:</p><br />
<h4>Reaction Mixture</h4><br />
<ul><br />
<li>5 μL 10x PCR Buffer with MgSO<sub>4</sub></li><br />
<li> 5μL 10x dNTPs</li><br />
<li>50 pmol each primer*</li><br />
<li>50-125ng Template DNA**</li><br />
<li>0.5 μL <i>Pfu</i> Polymerase</li><br />
<li>Water up to 50 μL total volume</li><br />
</ul><br />
<br><br />
<p>*Primers should be added according to the desired product you want to amplify.<br>**During the first round of PCR, template DNA will be the part itself. Subsequent rounds of PCR will use the appropriate PCR fragments from this round of PCR which you want to build together as template. These should be added at equal concentrations, along with appropriate flanking primers. Using varying concentrations of DNA template can help to optimize the reaction.</p><br />
<h4>Cycling Conditions</h4><ul><br />
<li>Stage 1: 95%deg;C for 2 min.</li><br />
<li>Stage 2: 94&deg;C for 1 min., 55-65&deg;C for 1 min.***, 72&deg;C for 1min/kb</li><br />
<li>Stage 3: 72&deg;C for 10 min., hold at 4&deg;C</li><br />
</ul><br />
<p><br />
***Annealing temperature must be adjusted according to melting temperatures of specific primers used.</p><br />
<p>Following each PCR reaction, gel purification of the desired band should be carried out (using the QIAquick Gel Purification Kit or another kit). Products can subsequently be used in further PCR, or in constructions once a full circuit is assembled.</p><br />
<br />
<br />
<br />
<br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreenTeam:Calgary/Notebook/Protocols/tnscreen2012-10-26T22:09:07Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Transposon-Mediated Mutant Library Generation|<br />
CONTENT=<html><br />
<h2>Materials</h2><br />
<ul><br />
<li><i>E.coli</i> SM10 pOT182</li><br />
<li><i>Pseudomonas fluorescens</i> PF-5</li><br />
<li>LB Broth </li><br />
<li>Pseudomonas isolation agar (PIA)</li><br />
<li>Mix naphthenic acid solution (Acros, 0.91g/mL) or any other toxin</li><br />
<li>Tetracycline (stock concentration at 5mg/mL)</li><br />
<li>X-Gal</li><br />
<li>Hydrogen peroxide</li><br />
<li>Decanoic acid</li><br />
</ul><br />
<br><br />
<h2>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Day 1</h2><br />
<ol><br />
<li>Around 3 or 4pm, prepare an <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">overnight culture</a> of donor <i>E.coli</i> SM10 pOT182 in 2mL of LB with 10μg/mL of tetracycline, and incubate overnight at 37&deg;C. </li><br />
<li>Subculture the recipient <i>Pseudomonas fluorescens</i> PF-5 as well in 5mL LB+50mg/L NA media, and incubate at 30&deg;C, shaking at 110rpm. </li><br />
<ul><li>Note: this protocol is designed to isolate NA sensing promoters. To isolate promoters sensitive to other compounds, simply replace NA with the compound of interest.</li></ul><br />
<h2>Day 2</h2><br />
<li>Make the selection media (Pseudomonas isolation agar/PIA) for the next day. The selection media contains PIA, tetracycline, NAs, and X-Gal.</li><br />
<li>To make the media, mix 45g of the PIA powder in 1L of milliQ or nanopure water containing 20mL of glycerol. Add 54.95µL of the Acros NAs. Label the bottle properly, mix well using a magnetic stir bar, and autoclave.</li><br />
<li>After autoclaving, allow agar to cool to about 55&deg;C in a water bath. Add tetracycline to a final concentration of 50μg/mL, and the X-Gal to a final concentration of 20 μg/mL. Mix the media well by slowly stirring (magnetically) while adding X-Gal and tetracycline.</li><br />
<li>Pour the agar solution into labelled petri dishes, allow to solidify (in about 1hr), and store at 4&deg;C in the dark to prevent antibiotic degradation.</li><br />
<li>At 2pm, re-subculture <i>E. coli</i> SM10 in 2mL of LB (no tetracycline) to remove the tetracyline. Incubate by shaking at 37&deg;C for 2hr.</li><br />
<li>At 4pm, prepare the following in 2mL tubes:<br />
<br />
<ul><br />
<li>Negative control: 500µL <i>Pseudomonas fluorescens</i> PF-5 (from the culture prepared in Day 1)</li><br />
<li>Negative control: 500µL of the <i>E.coli</i> SM10 (the re-subcultured sample) </li><br />
<li>Experimental sample: 500µL of the <i>E.coli</i> SM10 (the re-subcultured sample) and 500µL of of the <i>Pseudomonas fluorescens</i> PF-5 cultures</li><br />
</ul><br />
<br />
<li>Mix all samples by vortexing and centrifuge all the tubes at max speed for 5min.</li><br />
<li>Discard the supernatant and resuspend the cell pellet in 50μL of LB.</li><br />
<li>Pipette all 50μL onto a LB (no additives) plate. A single LB plate can be divided in three sections (make sure to label all the sections). Draw on the back of the plate like so:</li><br />
</html>[[ File:CalgaryiGEM2012 Matingplate.png|center|thumb|400px|Figure 1: Mating spot setup on a a LB plate]]<html><br />
<li>Let the plate sit on the bench at room temp until all the spots have dried. <li>Incubate overnight at 37&deg;C.</li><br />
<h2>Day3</h2><br />
<li>In the morning, scrape up the mating spots using a sterile pipette tip, and resuspend in 500μL sterile distilled water.</li><br />
<li>Mix thoroughly by vortexing or pipetting up and down. Do this for each spot from the LB plate.</li><br />
<li>For the mating spots (with both <i>E.coli</i> and <i>Pseudomonas fluorescens</i>), make serial dilutions from the resuspension (1/10, 1/100, and 1/1000).</li><br />
<li>Make spread plates using 100 µl of the undiluted mixture and each of the dilutions on PIA selection plates.</li><br />
<li>Plate 100μL of the resuspended <i>Pseudomonas fluorescens</i> PF-5 (alone) spot onto a PIA plate as well (no dilution required). Do the same for the <i>E.coli</i> SM10 spot.</li><br />
<li>Incubate overnight at 30&deg;C. Check the next morning for growth.</li><br />
<h2>Day4</h2><br />
<li>Check the plates for growth. Do not allow the plates to overgrow in order to isolate single colonies.</li><br />
<li>Ensure no growth is present on the plates containing the <li>Pseudomonas fluorescens</i> PF-5 (alone) and <i>E.coli</i> SM10 (alone) spots (At least at a reasonably low level indicating a small amount of spontaneous mutations).</li><br />
<li>On the experimental sample plates, isolate all blue colonies in <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">overnight cultures</a> (in LB+10μg/mL tetracycline+50mg/L NAs). Make <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/glycerolstock">glycerol stocks</a> from the overnight cultures.</li><br />
<h2>Day5</h2><br />
<li>The strains isolated from the blue colonies are further screened in a 96 well plate. Each strain is inoculated into the following set of media (300μL) in the plate:</li><br />
<ul><br />
<li>LB alone</li><br />
<li>LB with 200μg/mL X-Gal</li><br />
<li> LB with 50mg/L NAs and 200μg/mL X-Gal </li><br />
</ul><br />
<li>Make sure the plate setup is recorded properly, and make an initial (0hr) reading using a spectrophotometer at 615nm to measure the X-Gal degradation product concentration.</li><br />
<li>Incubate the plate at 30&deg;C in a shaker set at 140rpm for 16hr.</li><br />
<br />
<h2>Day6</h2><br />
<li>Measure the absorbance of the plate once again at 615nm at 16hr.</li><br />
<li>Based on the absorbance readings, record any colony that has a higher absorbance in the LB+NA+X-Gal sample than the LB+X-Gal sample. Preferably, the colony should show no growth in the LB+X-Gal sample.</li><br />
<li>Ensure the selected colonies are <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/glycerolstock">glycerol stocked</a>, and subculture these colonies in LB+10μg/mL tetracycline+50mg/L NAs.</li><br />
<br />
<h2>Day7</h2><br />
<li>Using the selected strains from the previous day, perform a more detailed assay for each strain using undiluted, 1/10, 1/100, and 1/1000 of the culture in LB+50mg/L NAs (duplicates for each dilution). </li><br />
<li>Also test the colonies in LB medium with 10μM, 50μM, and 100μM hydrogen peroxide, as well a fatty acid (e.g. decanoic acid) to ensure the response is not stress-induced, and specific to NAs.</li><br />
<li>The assay can be set up again in a 96-well plate.</li><br />
<li>Incubate the plate at 30&deg;C in a shaker set at 140rpm for 12h-24h.</li><br />
<h2>Day8</h2><br />
<li>Keep strains that respond specifically to NAs (or any other toxin), i.e. X-Gal degradation and growth in LB+NAs, but no/less X-gal production in LB+ varying amounts of hydrogen peroxide or LB+ decanoic acid.</li><br />
<li>For these strains, <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/gemomicprep">purify genomic DNA</a> from overnight cultures. Measure the DNA concentration, and run on a gel to confirm that the DNA is not degraded.</li><br />
<li>Completely <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/construction">digest</a> 600ng of the genomic DNA with BglII. (This restriction enzyme cuts frequently (6-cutter) in the <i>Pseudomonas fluorescens</i> PF-5 genome and not in the transposon). Run the reaction overnight at 37&deg;C for 16hr.<br />
<br />
<h2>Day 9</h2><br />
<li> Inactivate the BglII enzyme by performing a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/pcrpurification">enzymatic reaction cleanup</a> (using a PCR purification kit, as the enzyme cannot be heat inactivated).</li><br />
<li>Using 50ng of DNA, perform a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/construction">self-ligation</a> to circularize all the genomic fragments.</li><br />
<li>Transform the ligation mixture (all 20µL) into <i>E. coli</i> DH5α or Top10. Plate on LB+10μg/mL tetracyline plates.</li><br />
<br />
<h2>Day 10</h2><br />
<li>Prepare <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">overnight cultures</a> of the transformed cells in 5mL of LB+10μg/mL tetracyline.</li><br />
<br />
<h2>Day 11</h2><br />
<li>Purify the plasmid from the overnight cultures by <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">miniprep</a>.</li><br />
<li>Run the isolated plasmids on a gel to ensure the right size (at least the size of the transposon).</li><br />
<li>Sequence the plasmids using sequencing primers targeted against the insertion sequences of the transposon.</li><br />
<li>When sequencing results are received, identify the flanking sequences to the transposon insertion. The sequence outside the transposon (in the plasmid) should have a BglII restriction site. This would allow us to determine where the sequence upstream to the insertion occurs, and where the sequence downstream of the insertion ends.</li><br />
<li>Blast the flanking sequences against the <i>Pseudomonas fluorescens</i> genome to determine the position of the genomic insertion.</li><br />
</ol><br />
<br />
<br />
<br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreenTeam:Calgary/Notebook/Protocols/tnscreen2012-10-26T22:05:57Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Transposon-Mediated Mutant Library Generation|<br />
CONTENT=<html><br />
<h2>Materials</h2><br />
<ul><br />
<li><i>E.coli</i> SM10 pOT182</li><br />
<li><i>Pseudomonas fluorescens</i> PF-5</li><br />
<li>LB Broth </li><br />
<li>Pseudomonas isolation agar (PIA)</li><br />
<li>Mix naphthenic acid solution (Acros, 0.91g/mL) or any other toxin</li><br />
<li>Tetracycline (stock concentration at 5mg/mL)</li><br />
<li>X-Gal</li><br />
<li>Hydrogen peroxide</li><br />
<li>Decanoic acid</li><br />
</ul><br />
<br><br />
<h2>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Day 1</h2><br />
<ol><br />
<li>Around 3 or 4pm, prepare an <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">overnight culture</a> of donor <i>E.coli</i> SM10 pOT182 in 2mL of LB with 10μg/mL of tetracycline, and incubate overnight at 37&deg;C. </li><br />
<li>Subculture the recipient <i>Pseudomonas fluorescens</i> PF-5 as well in 5mL LB+50mg/L NA media, and incubate at 30&deg;C, shaking at 110rpm. </li><br />
<ul><li>Note: this protocol is designed to isolate NA sensing promoters. To isolate promoters sensitive to other compounds, simply replace NA with the compound of interest.</li></ul><br />
<h2>Day 2</h2><br />
<li>Make the selection media (Pseudomonas isolation agar/PIA) for the next day. The selection media contains PIA, tetracycline, NAs, and X-Gal.</li><br />
<li>To make the media, mix 45g of the PIA powder in 1L of milliQ or nanopure water containing 20mL of glycerol. Add 54.95µL of the Acros NAs. Label the bottle properly, mix well using a magnetic stir bar, and autoclave.</li><br />
<li>After autoclaving, allow agar to cool to about 55&deg;C in a water bath. Add tetracycline to a final concentration of 50μg/mL, and the X-Gal to a final concentration of 20 μg/mL. Mix the media well by slowly stirring (magnetically) while adding X-Gal and tetracycline.</li><br />
<li>Pour the agar solution into labelled petri dishes, allow to solidify (in about 1hr), and store at 4&deg;C in the dark to prevent antibiotic degradation.</li><br />
<li>At 2pm, re-subculture <i>E. coli</i> SM10 in 2mL of LB (no tetracycline) to remove the tetracyline. Incubate by shaking at 37&deg;C for 2hr.</li><br />
<li>At 4pm, prepare the following in 2mL tubes:<br />
<br />
<ul><br />
<li>Negative control: 500µL <i>Pseudomonas fluorescens</i> PF-5 (from the culture prepared in Day 1)</li><br />
<li>Negative control: 500µL of the <i>E.coli</i> SM10 (the re-subcultured sample) </li><br />
<li>Experimental sample: 500µL of the <i>E.coli</i> SM10 (the re-subcultured sample) and 500µL of of the <i>Pseudomonas fluorescens</i> PF-5 cultures</li><br />
</ul><br />
<br />
<li>Mix all samples by vortexing and centrifuge all the tubes at max speed for 5min.</li><br />
<li>Discard the supernatant and resuspend the cell pellet in 50μL of LB.</li><br />
<li>Pipette all 50μL onto a LB (no additives) plate. A single LB plate can be divided in three sections (make sure to label all the sections). Draw on the back of the plate like so:</li><br />
</html>[[ File:CalgaryiGEM2012 Matingplate.png|center|thumb|400px|Figure 1: Mating spot setup on a a LB plate]]<html><br />
<li>Let the plate sit on the bench at room temp until all the spots have dried. <li>Incubate overnight at 37&deg;C.</li><br />
<h2>Day3</h2><br />
<li>In the morning, scrape up the mating spots using a sterile pipette tip, and resuspend in 500μL sterile distilled water.</li><br />
<li>Mix thoroughly by vortexing or pipetting up and down. Do this for each spot from the LB plate.</li><br />
<li>For the mating spots (with both <i>E.coli</i> and <i>Pseudomonas fluorescens</i>), make serial dilutions from the resuspension (1/10, 1/100, and 1/1000).</li><br />
<li>Make spread plates using 100 µl of the undiluted mixture and each of the dilutions on PIA selection plates.</li><br />
<li>Plate 100μL of the resuspended <i>Pseudomonas fluorescens</i> PF-5 (alone) spot onto a PIA plate as well (no dilution required). Do the same for the <i>E.coli</i> SM10 spot.</li><br />
<li>Incubate overnight at 30&deg;C. Check the next morning for growth.</li><br />
<h2>Day4</h2><br />
<li>Check the plates for growth. Do not allow the plates to overgrow in order to isolate single colonies.</li><br />
<li>Ensure no growth is present on the plates containing the <li>Pseudomonas fluorescens</i> PF-5 (alone) and <i>E.coli</i> SM10 (alone) spots (At least at a reasonably low level indicating a small amount of spontaneous mutations).</li><br />
<li>On the experimental sample plates, isolate all blue colonies in <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">overnight cultures</a> (in LB+10μg/mL tetracycline+50mg/L NAs). Make <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/glycerolstock">glycerol stocks</a> from the overnight cultures.</li><br />
<h2>Day5</h2><br />
<li>The strains isolated from the blue colonies are further screened in a 96 well plate. Each strain is inoculated into the following set of media (200μL) in the plate:</li><br />
<ul><br />
<li>LB alone</li><br />
<li>LB with 200μg/mL X-Gal</li><br />
<li> LB with 50mg/L and 200μg/mL X-Gal </li><br />
</ul><br />
<li>Make sure the plate setup is recorded properly, and make an initial (0hr) reading using a spectrophotometer at 615nm to measure the X-Gal degradation product concentration.</li><br />
<li>Incubate the plate at 30&deg;C in a shaker set at 140rpm for 16hr.</li><br />
<br />
<h2>Day6</h2><br />
<li>Measure the absorbance of the plate once again at 615nm at 16hr.</li><br />
<li>Based on the absorbance readings, record any colony that has a higher absorbance in the LB+NA+X-Gal sample than the LB+X-Gal sample. Preferably, the colony should show no coloration in the LB+X-Gal sample.</li><br />
<li>Ensure the selected colonies are <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/glycerolstock">glycerol stocked</a>, and subculture these colonies in LB+10μg/mL tetracycline+50mg/L NAs.</li><br />
<br />
<h2>Day7</h2><br />
<li>Using the selected strains from the previous day, perform a more detailed assay for each strain using undiluted, 1/10, 1/100, and 1/1000 of the culture in LB+50mg/L NAs (duplicates for each dilution). </li><br />
<li>Also test the colonies in LB medium with comparable concentrations of other toxins (DBT, carbazole). In addition, 10μM, 50μM, and 100μM hydrogen peroxide should be tested, as well a fatty acid (e.g. decanoic acid) to ensure the response is not stress-induced, and specific to chemical toxins.</li><br />
<li>The assay can be set up again in a 96-well plate.</li><br />
<li>Incubate the plate at 30&deg;C in a shaker set at 140rpm for 12h-24h.</li><br />
<h2>Day8</h2><br />
<li>Keep strains that respond specifically to toxins, i.e. X-Gal degradation and growth in LB+NAs, but no/less X-gal production in LB+ varying amounts of hydrogen peroxide or LB+ decanoic acid.</li><br />
<li>For these strains, <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/gemomicprep">purify genomic DNA</a> from overnight cultures. Measure the DNA concentration, and run on a gel to confirm that the DNA is not degraded.</li><br />
<li>Completely <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/construction">digest</a> 600ng of the genomic DNA with BglII. (This restriction enzyme cuts frequently (6-cutter) in the <i>Pseudomonas fluorescens</i> Pf5 genome and not in the transposon). Run the reaction overnight at 37&deg;C for 16hr.<br />
<br />
<h2>Day 9</h2><br />
<li> Inactivate the BglII enzyme by performing a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/pcrpurification">enzymatic reaction cleanup</a> (using a PCR purification kit, as the enzyme cannot be heat inactivated).</li><br />
<li>Using 50ng of DNA, perform a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/construction">self-ligation</a> to circularize all the genomic fragments.</li><br />
<li>Transform the ligation mixture (all 20µL) into <i>E. coli</i> DH5α or Top10. Plate on LB+10μg/mL tetracyline plates.</li><br />
<br />
<h2>Day 10</h2><br />
<li>Prepare <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">overnight cultures</a> of the transformed cells in 5mL of LB+10μg/mL tetracyline.</li><br />
<br />
<h2>Day 11</h2><br />
<li>Purify the plasmid from the overnight cultures by <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">miniprep</a>.</li><br />
<li>Run the isolated plasmids on a gel to ensure the right size (at least the size of the transposon).</li><br />
<li>Sequence the plasmids using sequencing primers targeted against the insertion sequences of the transposon.</li><br />
<li>When sequencing results are received, identify the flanking sequences to the transposon insertion. The sequence outside the transposon (in the plasmid) should have a BglII restriction site. This would allow us to determine where the sequence upstream to the insertion occurs, and where the sequence downstream of the insertion ends.</li><br />
<li>Blast the flanking sequences against the <i>Pseudomonas fluorescens</i> genome to determine the position of the genomic insertion.</li><br />
</ol><br />
<br />
<br />
<br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreenTeam:Calgary/Notebook/Protocols/tnscreen2012-10-26T22:02:13Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Transposon-Mediated Mutant Library Generation|<br />
CONTENT=<html><br />
<h2>Materials</h2><br />
<ul><br />
<li><i>E.coli</i> SM10 pOT182</li><br />
<li><i>Pseudomonas fluorescens</i> PF-5</li><br />
<li>LB Broth </li><br />
<li>Pseudomonas isolation agar (PIA)</li><br />
<li>Mix naphthenic acid solution (Acros, 0.91g/mL) or any other toxin</li><br />
<li>Tetracycline (stock concentration at 5mg/mL)</li><br />
<li>X-Gal</li><br />
<li>Hydrogen peroxide</li><br />
<li>Decanoic acid</li><br />
</ul><br />
<br><br />
<h2>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Day 1</h2><br />
<ol><br />
<li>Around 3 or 4pm, prepare an <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">overnight culture</a> of donor <i>E.coli</i> SM10 pOT182 in 2mL of LB with 10μg/mL of tetracycline, and incubate overnight at 37&deg;C. </li><br />
<li>Subculture the recipient <i>Pseudomonas fluorescens</i> PF-5 as well in 5mL LB+50mg/L NA media, and incubate at 30&deg;C, shaking at 110rpm. </li><br />
<ul><li>Note: this protocol is designed to isolate NA sensing promoters. To isolate promoters sensitive to other compounds, simply replace NA with the compound of interest.</li></ul><br />
<h2>Day 2</h2><br />
<li>Make the selection media (Pseudomonas isolation agar/PIA) for the next day. The selection media contains PIA, tetracycline, NAs, and X-Gal.</li><br />
<li>To make the media, mix 45g of the PIA powder in 1L of milliQ or nanopure water containing 20mL of glycerol. Add 54.95µL of the Acros NAs. Label the bottle properly, mix well using a magnetic stir bar, and autoclave.</li><br />
<li>After autoclaving, allow agar to cool to about 55&deg;C in a water bath. Add tetracycline to a final concentration of 50μg/mL, and the X-Gal to a final concentration of 20 μg/mL. Mix the media well by slowly stirring (magnetically) while adding X-Gal and tetracycline.</li><br />
<li>Pour the agar solution into labelled petri dishes, allow to solidify (in about 1hr), and store at 4&deg;C in the dark to prevent antibiotic degradation.</li><br />
<li>At 2pm, re-subculture <i>E. coli</i> SM10 in 2mL of LB (no tetracycline) to remove the tetracyline. Incubate by shaking at 37&deg;C for 2hr.</li><br />
<li>At 4pm, prepare the following in 2mL tubes:<br />
<br />
<ul><br />
<li>Negative control: 500µL <i>Pseudomonas fluorescens</i> PF-5 (from the culture prepared in Day 1)</li><br />
<li>Negative control: 500µL of the <i>E.coli</i> SM10 (the re-subcultured sample) </li><br />
<li>Experimental sample: 500µL of the <i>E.coli</i> SM10 (the re-subcultured sample) and 500µL of of the <i>Pseudomonas fluorescens</i> PF-5 cultures</li><br />
</ul><br />
<br />
<li>Mix all samples by vortexing and centrifuge all the tubes at max speed for 5min.</li><br />
<li>Discard the supernatant and resuspend the cell pellet in 50μL of LB.</li><br />
<li>Pipette all 50μL onto a LB (no additives) plate. A single LB plate can be divided in three sections (make sure to label all the sections). Draw on the back of the plate like so:</li><br />
</html>[[ File:CalgaryiGEM2012 Matingplate.png|center|thumb|400px|Figure 1: Mating spot setup on a a LB plate]]<html><br />
<li>Let the plate sit on the bench at room temp until all the spots have dried. <li>Incubate overnight at 37&deg;C.</li><br />
<h2>Day3</h2><br />
<li>In the morning, scrape up the mating spots using a sterile pipette tip, and resuspend in 500μL sterile distilled water.</li><br />
<li>Mix thoroughly by vortexing or pipetting up and down. Do this for each spot from the LB plate.</li><br />
<li>For the mating spots (with both <i>E.coli</i> and <i>Pseudomonas fluorescens</i>), make serial dilutions from the resuspension (1/10, 1/100, and 1/1000).</li><br />
<li>Make spread plates using 100 µl of the undiluted mixture and each of the dilutions on PIA selection plates.</li><br />
<li>Plate 100μL of the resuspended <i>Pseudomonas fluorescens</i> PF-5 (alone) spot onto a PIA plate as well (no dilution required). Do the same for the <i>E.coli</i> SM10 spot.</li><br />
<li>Incubate overnight at 30&deg;C. Check the next morning for growth.</li><br />
<h2>Day4</h2><br />
<li>Check the plates for growth. Do not allow the plates to overgrow in order to isolate single colonies.</li><br />
<li>Ensure no growth is present on the plates containing the <li>Pseudomonas fluorescens</i> PF-5 (alone) and <i>E.coli</i> SM10 (alone) spots (At least at a reasonably low level indicating a small amount of spontaneous mutations).</li><br />
<li>On the experimental sample plates, isolate all blue colonies in <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">overnight cultures</a> (in LB+10μg/mL tetracycline+50mg/L NAs). Make <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/glycerolstock">glycerol stocks</a> from the overnight cultures.</li><br />
<h2>Day5</h2><br />
<li>The strains isolated from the blue colonies are further screened in a 96 well plate. Each strain is inoculated into the following set of media (300μL) in the plate:</li><br />
<ul><br />
<li>LB alone</li><br />
<li>LB with 200μg/mL X-Gal</li><br />
<li> LB with 50mg/L and 200μg/mL X-Gal </li><br />
</ul><br />
<li>Make sure the plate setup is recorded properly, and make an initial (0hr) reading using a spectrophotometer at 635nm to measure the X-Gal degradation product concentration.</li><br />
<li>Incubate the plate at 30&deg;C in a shaker set at 140rpm for 16hr.</li><br />
<br />
<h2>Day6</h2><br />
<li>Measure the absorbance of the plate once again at 635nm at 16hr.</li><br />
<li>Based on the absorbance readings, record any colony that has a higher absorbance in the LB+NA+X-Gal sample than the LB+X-Gal sample. Preferably, the colony should show no growth in the LB+X-Gal sample.</li><br />
<li>Ensure the selected colonies are <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/glycerolstock">glycerol stocked</a>, and subculture these colonies in LB+10μg/mL tetracycline+50mg/L NAs.</li><br />
<br />
<h2>Day7</h2><br />
<li>Using the selected strains from the previous day, perform a more detailed assay for each strain using undiluted, 1/10, 1/100, and 1/1000 of the culture in LB+50mg/L NAs (duplicates for each dilution). </li><br />
<li>Also test the colonies in LB medium with 10μM, 50μM, and 100μM hydrogen peroxide, as well a fatty acid (e.g. decanoic acid) to ensure the response is not stress-induced, and specific to NAs.</li><br />
<li>The assay can be set up again in a 96-well plate.</li><br />
<li>Incubate the plate at 30&deg;C in a shaker set at 140rpm for 12h-24h.</li><br />
<h2>Day8</h2><br />
<li>Keep strains that respond specifically to NAs (or any other toxin), i.e. X-Gal degradation and growth in LB+NAs, but no/less X-gal production in LB+ varying amounts of hydrogen peroxide or LB+ decanoic acid.</li><br />
<li>For these strains, <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/gemomicprep">purify genomic DNA</a> from overnight cultures. Measure the DNA concentration, and run on a gel to confirm that the DNA is not degraded.</li><br />
<li>Completely <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/construction">digest</a> 600ng of the genomic DNA with BglII. (This restriction enzyme cuts frequently (6-cutter) in the <i>Pseudomonas fluorescens</i> PF-5 genome and not in the transposon). Run the reaction overnight at 37&deg;C for 16hr.<br />
<br />
<h2>Day 9</h2><br />
<li> Inactivate the BglII enzyme by performing a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/pcrpurification">enzymatic reaction cleanup</a> (using a PCR purification kit, as the enzyme cannot be heat inactivated).</li><br />
<li>Using 50ng of DNA, perform a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/construction">self-ligation</a> to circularize all the genomic fragments.</li><br />
<li>Transform the ligation mixture (all 20µL) into <i>E. coli</i> DH5α or Top10. Plate on LB+10μg/mL tetracyline plates.</li><br />
<br />
<h2>Day 10</h2><br />
<li>Prepare <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">overnight cultures</a> of the transformed cells in 5mL of LB+10μg/mL tetracyline.</li><br />
<br />
<h2>Day 11</h2><br />
<li>Purify the plasmid from the overnight cultures by <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">miniprep</a>.</li><br />
<li>Run the isolated plasmids on a gel to ensure the right size (at least the size of the transposon).</li><br />
<li>Sequence the plasmids using sequencing primers targeted against the insertion sequences of the transposon.</li><br />
<li>When sequencing results are received, identify the flanking sequences to the transposon insertion. The sequence outside the transposon (in the plasmid) should have a BglII restriction site. This would allow us to determine where the sequence upstream to the insertion occurs, and where the sequence downstream of the insertion ends.</li><br />
<li>Blast the flanking sequences against the <i>Pseudomonas fluorescens</i> genome to determine the position of the genomic insertion.</li><br />
</ol><br />
<br />
<br />
<br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/hpacTeam:Calgary/Notebook/Protocols/hpac2012-10-26T21:58:46Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=HpaC Assay|<br />
CONTENT=<html><br />
<p>The theory behind this assay depends on the absorbance of NADH at 340 nm. HpaC produces FMNH<sub>2</sub> from FMN in order to recycle reducing power. In the process, NADH is converted to NAD<sup>+</sup>. Because of this, the amount of NADH in solution will drop over time, which can be monitored by recording the absorbance at 340nm over time. Note: NADH and FMN stock solutions were aliquoted and stored in the -80&deg;C freezer to prevent degradation.</p><br><p><br />
<ol><br />
<br />
<li>Grow up cultures of <i>E. coli J04500-hpaC </i>and <i>J04500-dszB</i> overnight at 37*C in LB with appropriate antibiotics.</li><br />
<li> The following morning, subculture 1 mL of each into 3 mL fresh LB with antibiotics, and add 200 uM IPTG to induce protein expression. Grow these cultures for 2h at 37*C.</li><br />
<li>Take out 1 mL of cells, spin down to pellet. Discard supernatant, and wash 2x with 50 mM Tris-HCl (pH 7.5). Discard supernatant, and resuspend in 1 mL of 50 mM Tris-HCl (pH 7.5). </li><br />
<li>Freeze-thaw Cells (ethanol over dry ice, then 37*C waterbath) 5 times in order to lyse.</li><br />
<li>Pipette different volumes of supernatant into a cuvette, and bring up to 998uL with Tris-HCl (pH 7.5).</li><br />
<li>Blank the spectrophotometer at 340nm with this sample, add 140uM NADH and 20uM FMN, invert quickly to mix, and read absorbance at 340 nm every 15s for 10 minutes. As a control, add 1 mL of Tris-HCl (pH 7.5), blank the spectrophotometer, add 140uM NADH and 20uM FMN, invert to mix, and read every 15s for 10 min.</li><br />
<br />
</ol><br />
<br><br><br><br><br><br><br><br />
<br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/hpacTeam:Calgary/Notebook/Protocols/hpac2012-10-26T21:58:27Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=HpaC Assay|<br />
CONTENT=<html><br />
<p>The theory behind this assay depends on the absorbance of NADH at 340 nm. HpaC produces FMNH<sub>2</sub> from FMN in order to recycle reducing power. In the process, NADH is converted to NAD<sup>+</sup>. Because of this, the amount of NADH in solution will drop over time, which can be monitored by recording the absorbance at 340nm over time. Note: NADH and FMN stock solutions were aliquoted and stored in the -80&deg;C freezer to prevent degradation.<br />
<ol><br />
<br />
<li>Grow up cultures of <i>E. coli J04500-hpaC </i>and <i>J04500-dszB</i> overnight at 37*C in LB with appropriate antibiotics.</li><br />
<li> The following morning, subculture 1 mL of each into 3 mL fresh LB with antibiotics, and add 200 uM IPTG to induce protein expression. Grow these cultures for 2h at 37*C.</li><br />
<li>Take out 1 mL of cells, spin down to pellet. Discard supernatant, and wash 2x with 50 mM Tris-HCl (pH 7.5). Discard supernatant, and resuspend in 1 mL of 50 mM Tris-HCl (pH 7.5). </li><br />
<li>Freeze-thaw Cells (ethanol over dry ice, then 37*C waterbath) 5 times in order to lyse.</li><br />
<li>Pipette different volumes of supernatant into a cuvette, and bring up to 998uL with Tris-HCl (pH 7.5).</li><br />
<li>Blank the spectrophotometer at 340nm with this sample, add 140uM NADH and 20uM FMN, invert quickly to mix, and read absorbance at 340 nm every 15s for 10 minutes. As a control, add 1 mL of Tris-HCl (pH 7.5), blank the spectrophotometer, add 140uM NADH and 20uM FMN, invert to mix, and read every 15s for 10 min.</li><br />
<br />
</ol><br />
<br><br><br><br><br><br><br><br />
<br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/Protocols/catalaseTeam:Calgary/Notebook/Protocols/catalase2012-10-26T21:55:02Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
TITLE=Catalase Assay|<br />
CONTENT=<html><br />
<p> The purpose of this protocol is to test the ability of KatG-LAA to detoxify hydrogen peroxide. This was important, as catalase will be encorporated into the final sulfur circuit as an optimization feature.<br />
<ol><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">Grow cultures</a> of <i>E. coli J04500-katG+LAA</i> and <i>J04500</i> overnight at 37*C in LB with appropriate antibiotics.</li><br />
<li>The following evening, innoculate 20 uL into 3 mL LB with varying concentrations of hydrogen peroxide (0 mM, 1 mM, 5 mM, 10 mM) and grow overnight.</li><br />
<li>The following morning, observe and record culture turbidity.</li><br />
</ol><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/OSCAR/DesulfurizationTeam:Calgary/Project/OSCAR/Desulfurization2012-10-26T21:50:05Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Desulfurization|<br />
<br />
CONTENT=<html><br />
<img src="https://static.igem.org/mediawiki/2012/5/5e/UCalgary2012_OSCAR_Desulfurization_Low-Res.png" style="float: right; padding: 10px;"></img><br />
<br />
<h2>Why Remove Sulfur?</h2><br />
<br />
<p align="justify"><br />
Sulfur is the third most abundant element in crude oil (Ma, 2010), and when sulfur containing hydrocarbons are burned they release S0<sub>2</sub> and S0<sub>3</sub> gasses into the atmosphere. Not only does this reduce the efficiency and value of our product, but it also contributes to global warming, acid rain, and various health issues due to the pollution (Reichmuth <i>et al</i>., 2000). Strict regulation on sulfur in fuels are now in place and low-sulfur gasoline is mandated across all of Canada (Source: Environment Canada). To upgrade the quality of our fuel we need to remove the sulfur but keep the hydrocarbon backbone.</p><br />
<br />
<h2>Our Vision</h2><p align="justify"><br />
Though a few pathways for biodesulfurization exist in the microbial world, most involve the destruction of part of the carbon skeleton (an example would be the Kodama pathway)(Soleimani <i>et al</i>., 2007). This would effectively reduce the quality of our product. With this in mind the pathway we have chosen is the 4S pathway found in <i>Rhodococcus spp</i>. It has been characterized and shown to remove sulfur from the model substrate dibenzothiophene (DBT) and convert it to 2-hydroxybiphenyl (2-HBP) in a non-destructive manner. DBT and its derivatives make up 70% of the organic sulfur compounds found in crude oil (Ma 2010), and are also some of the most difficult to remove through chemical means. By using the 4S pathway we will be able to upgrade our fuel and remove recalcitrant compounds at the same time. <br />
</p><br />
<br />
</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|750px|thumb|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD.]]<html></p><br />
<br />
<h2>4S pathway</h2><br />
<p align="justify"><br />
Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO<sub>2</sub>) through the addition of 2 oxygen atoms to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the sulfur, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-HBP. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.</p><p align="justify"><br />
The first three steps of the 4S pathway require FMNH<sub>2</sub> and subsequently reduces the reductive power of the cell. WIn order to regain this power an oxidoreductase (DszD) uses NADH to recycle the FMNH<sub>2</sub>, allowing the reaction to proceed. Without DszD the desulfurization pathway would grind to a halt.</p><p align="justify"><br />
The <i>dszA</i>,<i>B</i>, and <i>C</i> genes form an operon on the pSOX plasmid of <i>R. erythropolis</i>, while <i>dszD</i> is found in the chromosome. Naturally this pathway is slow, however using synthetic biology approaches this process can be optimized.</p><br />
<br />
<h2>Our Approach</h2><br />
<a name="Degradation"></a><h3>1) Find the genes!</h3><br />
<p align="justify">We isolated the plasmid containing the <i>dsz</i> genes from a desulfurising environmental isolate of <i>Rhodococcus</i> using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">modified miniprep procedure</a>. As the native promoter has been shown to be repressed by various sulfur-containing compounds (Li <i>et al</i>., 1996), we designed primers for just the coding sequences of the <i>A, B, </i> and <i>C</i> genes. As these genes all have some illegal cutsites in them we constructed them into the PSB1C3 vector and started our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis protocol</a>.</p><br />
<p align="justify"> We performed an experiment to measure the desulfurization rate of select organosulfur compounds by our <i>Rhodococcus</i> strain (Figures 4-6 below). These experiments monitored the degradation of the compounds by our strain over time. We discovered that the <i>dsz</i> operon is capable of desulfurizing a wider range of compounds than just the commonly studied DBT. This shows that this pathway could be a promising solution for degradation of a wide variety of sulfur containing toxins, including those that resemble naphthenic acids. </p> <br />
<br />
<p align="justify"></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 2: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through the GC/MS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GC/MS after 6 days of incubation. ]]<html></p><br />
<br />
<p align="justify"></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 3: The peak in this mass spectrum demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<p align="justify"><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound ('''A:''' dibenzothiophene, '''B:''' tetrahydro-4h-thiopyran-4-one, and '''C:''' benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdownn. Degradation is seen for DBT as well as other sulfur-containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<h3>2) Mutagenesis: Biobrick Compatability and Increasing DszB Activity </h3><br />
<p align="justify">In total the <i>dszABC</i> genes had 7 PstI sites and 1 NotI site that needed to be mutated for the biobrick standard. The primers were designed such that the site was removed without the amino acid being changed. In addition, a point mutation of Y63F in DszB increased the activity of the protein (Oshiro <i>et al</i>., 2007), and was included in the mass mutagenesis we undertook. Mutagenesis was performed as described in <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">this protocol.</a></p><br />
<br />
<a name="catalase"></a><h3>3) Replacing DszD with HpaC & Introducing Catalase </h3><br />
<p align="justify"><br />
As FMNH<sub>2</sub> is consumed in the first three steps of the pathway it needs to be regenerated or the process will grind to a halt. This usually falls to the <i>dszD</i> gene, however it has been shown that the <i>hpaC</i> gene from <i>E. coli</i> performs the same function more efficiently (Gala´n <i>et al</i>., 2000). One problem arises from this though, as high levels of FMNH<sub>2</sub> cause the production of toxic hydrogen peroxide inside the cell (Gala´n <i>et al</i>. 2000). To address this issue we have included a catalase gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"> <i>P<sub>lacI</sub>-katG-LAA</i></a>) that will remove the peroxide that would be toxic to the cell.</p><br />
<br />
<p align="justify"></html>[[File:Ucalgary_sulfur_constructs_KatandHpaC.PNG|center|250px|thumb|Figure 7: Diagrammatic representation of the full "optimization circuit", consisting of the oxidoreductase HpaC and a catalase (KatG).]]<html></p><br />
<br />
<br />
<h3>Results</h3><br />
<p align="justify">To show that catalase activity increased <i>E. coli</i> survivability in peroxide we cultured the inducible catalase against a catalase-free control with varying levels of peroxide. After growing overnight the negative didn't grow in any culture except in the absence of peroxide, while the catalase cultures could tolerate peroxide. This is shown below.</p><p align="justify"><br />
<br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 8: Catalase Assay. Overnight cultures of P<sub>lacI</sub> and P<sub>lacI</sub>-KatGLAA were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbidity was observed. It was found that at 1 mM of peroxide, cultures with just the lacI promotor perished, however when KatG-LAA was expressed, the cells survived.]]<html></p><br />
<br />
<br />
<p align="justify">To test the action of HpaC to use NADH to recycle FMN into FMNH<sub>2</sub> cell lysates were exposed to NADH and it's absorbance at 340nm (Kamali <i>et al</i>., 2010) was measured over time. Both native HpaC expression and an induced <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> system were tested as well as a negative control. The results are shown below.</p><br />
<br />
<p align="justify"> </html> <br />
[[File:Ucalgary2012 HpaC assaycumulativeforthedatapage.png|center|850px|thumb|Figure 9: HpaC Assay with '''A)''' 2 mL cell lysate and '''B)''' 100 &micro;L cell lysate. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, various amounts of cell lysate were transferred to a cuvette, and a spectrophotometer was blanked at 340 nm with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p align="justify">The assay showed that NADH does not abiotically convert into NAD+, however the native expression of HpaC did show a steady decrease in the levels of NADH. The induced overexpression of HpaC caused extremely rapid conversion into NAD+ as reflected by a sharp drop in the absorbance of NADH (see figure B). This drop was much sharper than what was seen when native levels of oxidoreductases were tested, showing that the <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> was functional and that it would effectively recycle FMN.</p><br />
<br />
<br />
<a name="UBC"></a><h3>4) Optimizing Gene Order</h3><br />
<br />
<p align="justify">Further optimization of the system was achieved through reorganization of the reconstructed operon. Natively the genes are arranged ABC, however the catalytic efficiency of the protein products are 25:1:5 for A:B:C respectively (Li <i>et al</i>., 2008). By rearranging the genes into BCA there is stronger transcription of the weaker proteins, giving a more balanced system overall. These would all be constructed with the same strong ribosomal binding site, <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>.</p><p align="justify"><br />
<br />
</html>[[File:DszOperonOptimize.png|center|400px|thumb|Figure 10: Method of optimizing gene order. The top circuit represents that found natively in the organism, with the bottom circuit representing our modified version.]]<html><br />
</p><br />
</a><h2>Final Sulfur Constructs</h2><br />
<p align="justify">After all of the above considerations are met, four final constructs for our system will be made to allow us to test desulfurization under different conditions.</p><p align="justify"><br />
<br />
</html>[[File:WikiConstructs_ucalgary_sulfur_2012_final_systems.png|center|700px|thumb|Figure 11: First set of final constructs for the desulfurization operon, with constitutive Dsz expression and inducible expression of the optimization proteins; either HpaC on its own or coexpressed with KatG]]<html></p><br />
<br />
<p align="justify"><br />
The first two constructs have the modified <i>dsz</i> operon (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902052"><i>dszB</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><i>dszC</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902050"><i>dszA</i></a>) under the control of a constitutive TetR promotor (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>) This is to allow for the testing of the optimization circuit, which is under the control of a lacI promotor inducible by IPTG (<a href="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</a>). The set-up of these two constructs will therefore allow for the expression of the <i>dsz</i> genes with the ability to test and compare their desulfurization rates <br> A) On their own <br> B) With the addition of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> <br> C) With the addition of both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K137068"><i>katG-LAA</i></a></p><br />
<br />
<p align="justify">This will allow us to determine what the optimal construct and expression levels of the additional genes must be in order to have the most effective sulfur removal system.</p><br />
<br />
</html>[[File:WikiConstructs2 sulfur ucalgary induciblesytems.PNG|center|700px||thumb|Figure 12: Second set of final constructs for the desulfurization operon, with all genes under an IPTG inducible promotor.]]<html><br />
<br />
<p align="justify"><br />
Due to the large number of proteins being expressed in this system, the possibility of forming inclusion bodies is present. As such, a backup system was built where both the optimization circuit and the <i>dsz</i> operon were under the control of the inducible lacI promoter. This system would allow us to tune the expression of the genes, and determine which expression level is optimal for desulfurization in our bioreactor.</p> <br />
<br />
<p align="justify">Currently the final steps of construction of these constructs is underway, following which functionality tests will begin.</p><br />
<br />
<br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/OSCAR/DesulfurizationTeam:Calgary/Project/OSCAR/Desulfurization2012-10-26T21:49:40Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Desulfurization|<br />
<br />
CONTENT=<html><br />
<img src="https://static.igem.org/mediawiki/2012/5/5e/UCalgary2012_OSCAR_Desulfurization_Low-Res.png" style="float: right; padding: 10px;"></img><br />
<br />
<h2>Why Remove Sulfur?</h2><br />
<br />
<p align="justify"><br />
Sulfur is the third most abundant element in crude oil (Ma, 2010), and when sulfur containing hydrocarbons are burned they release S0<sub>2</sub> and S0<sub>3</sub> gasses into the atmosphere. Not only does this reduce the efficiency and value of our product, but it also contributes to global warming, acid rain, and various health issues due to the pollution (Reichmuth <i>et al</i>., 2000). Strict regulation on sulfur in fuels are now in place and low-sulfur gasoline is mandated across all of Canada (Source: Environment Canada). To upgrade the quality of our fuel we need to remove the sulfur but keep the hydrocarbon backbone.</p><br />
<br />
<h2>Our Vision</h2><p align="justify"><br />
Though a few pathways for biodesulfurization exist in the microbial world, most involve the destruction of part of the carbon skeleton (an example would be the Kodama pathway)(Soleimani <i>et al</i>., 2007). This would effectively reduce the quality of our product. With this in mind the pathway we have chosen is the 4S pathway found in <i>Rhodococcus spp</i>. It has been characterized and shown to remove sulfur from the model substrate dibenzothiophene (DBT) and convert it to 2-hydroxybiphenyl (2-HBP) in a non-destructive manner. DBT and its derivatives make up 70% of the organic sulfur compounds found in crude oil (Ma 2010), and are also some of the most difficult to remove through chemical means. By using the 4S pathway we will be able to upgrade our fuel and remove recalcitrant compounds at the same time. <br />
</p><br />
<br />
</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|750px|thumb|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD.]]<html></p><br />
<br />
<h2>4S pathway</h2><br />
<p align="justify"><br />
Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO<sub>2</sub>) through the addition of 2 oxygen atoms to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the sulfur, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-HBP. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.</p><p align="justify"><br />
The first three steps of the 4S pathway require FMNH<sub>2</sub> and subsequently reduces the reductive power of the cell. WIn order to regain this power an oxidoreductase (DszD) uses NADH to recycle the FMNH<sub>2</sub>, allowing the reaction to proceed. Without DszD the desulfurization pathway would grind to a halt.</p><p align="justify"><br />
The <i>dszA</i>,<i>B</i>, and <i>C</i> genes form an operon on the pSOX plasmid of <i>R. erythropolis</i>, while <i>dszD</i> is found in the chromosome. Naturally this pathway is slow, however using synthetic biology approaches this process can be optimized.</p><br />
<br />
<h2>Our Approach</h2><br />
<a name="Degradation"></a><h3>1) Find the genes!</h3><br />
<p align="justify">We isolated the plasmid containing the <i>dsz</i> genes from a desulfurising environmental isolate of <i>Rhodococcus</i> using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">modified miniprep procedure</a>. As the native promoter has been shown to be repressed by various sulfur-containing compounds (Li <i>et al</i>., 1996), we designed primers for just the coding sequences of the <i>A, B, </i> and <i>C</i> genes. As these genes all have some illegal cutsites in them we constructed them into the PSB1C3 vector and started our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis protocol</a>.</p><br />
<p align="justify"> We performed an experiment to measure the desulfurization rate of select organosulfur compounds by our <i>Rhodococcus</i> strain (Figures 4-6 below). These experiments monitored the degradation of the compounds by our strain over time. We discovered that the <i>dsz</i> operon is capable of desulfurizing a wider range of compounds than just the commonly studied DBT. This shows that this pathway could be a promising solution for degradation of a wide variety of sulfur containing toxins, including those that resemble naphthenic acids. </p> <br />
<br />
<p align="justify"></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 2: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through the GC/MS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GC/MS after 6 days of incubation. ]]<html></p><br />
<br />
<p align="justify"></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 3: The peak in this mass spectrum demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<p align="justify"><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound ('''A:''' dibenzothiophene, '''B:''' tetrahydro-4h-thiopyran-4-one, and '''C:''' benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdownn. Degradation is seen for DBT as well as other sulfur-containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<h3>2) Mutagenesis: Biobrick Compatability and Increasing DszB Activity </h3><br />
<p align="justify">In total the <i>dszABC</i> genes had 7 PstI sites and 1 NotI site that needed to be mutated for the biobrick standard. The primers were designed such that the site was removed without the amino acid being changed. In addition, a point mutation of Y63F in DszB increased the activity of the protein (Oshiro <i>et al</i>., 2007), and was included in the mass mutagenesis we undertook. Mutagenesis was performed as described in <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">this protocol.</a></p><br />
<br />
<a name="catalase"></a><h3>3) Replacing DszD with HpaC & Introducing Catalase </h3><br />
<p align="justify"><br />
As FMNH<sub>2</sub> is consumed in the first three steps of the pathway it needs to be regenerated or the process will grind to a halt. This usually falls to the <i>dszD</i> gene, however it has been shown that the <i>hpaC</i> gene from <i>E. coli</i> performs the same function more efficiently (Gala´n <i>et al</i>., 2000). One problem arises from this though, as high levels of FMNH<sub>2</sub> cause the production of toxic hydrogen peroxide inside the cell (Gala´n <i>et al</i>. 2000). To address this issue we have included a catalase gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"> <i>P<sub>lacI</sub>-katG-LAA</i></a>) that will remove the peroxide that would be toxic to the cell.</p><br />
<br />
<p align="justify"></html>[[File:Ucalgary_sulfur_constructs_KatandHpaC.PNG|center|250px|thumb|Figure 7: Diagrammatic representation of the full "optimization circuit", consisting of the oxidoreductase HpaC and a catalase (KatG).]]<html></p><br />
<br />
<br />
<h3>Results</h3><br />
<p align="justify">To show that catalase activity increased <i>E. coli</i> survivability in peroxide we cultured the inducible catalase against a catalase-free control with varying levels of peroxide. After growing overnight the negative didn't grow in any culture except in the absence of peroxide, while the catalase cultures could tolerate peroxide. This is shown below.</p><p align="justify"><br />
<br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 8: Catalase Assay. Overnight cultures of P<sub>lacI</sub> and P<sub>lacI</sub>-KatGLAA were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbidity was observed. It was found that at 1 mM of peroxide, cultures with just the lacI promotor perished, however when KatG-LAA was expressed, the cells survived.]]<html></p><br />
<br />
<br />
<p align="justify">To test the action of HpaC to use NADH to recycle FMN into FMNH<sub>2</sub> cell lysates were exposed to NADH and it's absorbance at 340nm (Kamali <i>et al</i>., 2010) was measured over time. Both native HpaC expression and an induced <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> system were tested as well as a negative control. The results are shown below.</p><br />
<br />
<p align="justify"> </html> <br />
[[File:Ucalgary2012 HpaC assaycumulativeforthedatapage.png|center|850px|thumb|Figure 9: HpaC Assay with '''A)''' 2 mL cell lysate and '''B)''' 100 &micro;L cell lysate. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, various amounts of cell lysate were transferred to a cuvette, and a spectrophotometer was blanked at 340 nm with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p align="justify">The assay showed that NADH does not abiotically convert into NAD+, however the native expression of HpaC did show a steady decrease in the levels of NADH. The induced overexpression of HpaC caused extremely rapid conversion into NAD+ as reflected by a sharp drop in the absorbance of NADH (see figure B). This drop was much sharper than what was seen when native levels of oxidoreductases were tested, showing that the <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> was functional and that it would effectively recycle FMN.</p><br />
<br />
<br />
<a name="UBC"></a><h3>4) Optimizing Gene Order</h3><br />
<br />
<p align="justify">Further optimization of the system was achieved through reorganization of the reconstructed operon. Natively the genes are arranged ABC, however the catalytic efficiency of the protein products are 25:1:5 for A:B:C respectively (Li <i>et al</i>., 2008). By rearranging the genes into BCA there is stronger transcription of the weaker proteins, giving a more balanced system overall. These would all be constructed with the same strong ribosomal binding site, <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>.</p><p align="justify"><br />
<br />
</html>[[File:DszOperonOptimize.png|center|400px|thumb|Figure 10: Method of optimizing gene order. The top circuit represents that found natively in the organism, with the bottom circuit representing our modified version.]]<html><br />
</p><br />
<br />
<a name="sulfur"></a><h2>Final Constructs</h2><br />
<p align="justify">After all of the above considerations are met, four final constructs for our system will be made to allow us to test desulfurization under different conditions.</p><p align="justify"><br />
<br />
</html>[[File:WikiConstructs_ucalgary_sulfur_2012_final_systems.png|center|700px|thumb|Figure 11: First set of final constructs for the desulfurization operon, with constitutive Dsz expression and inducible expression of the optimization proteins; either HpaC on its own or coexpressed with KatG]]<html></p><br />
<br />
<p align="justify"><br />
The first two constructs have the modified <i>dsz</i> operon (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902052"><i>dszB</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><i>dszC</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902050"><i>dszA</i></a>) under the control of a constitutive TetR promotor (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>) This is to allow for the testing of the optimization circuit, which is under the control of a lacI promotor inducible by IPTG (<a href="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</a>). The set-up of these two constructs will therefore allow for the expression of the <i>dsz</i> genes with the ability to test and compare their desulfurization rates <br> A) On their own <br> B) With the addition of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> <br> C) With the addition of both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K137068"><i>katG-LAA</i></a></p><br />
<br />
<p align="justify">This will allow us to determine what the optimal construct and expression levels of the additional genes must be in order to have the most effective sulfur removal system.</p><br />
<br />
</html>[[File:WikiConstructs2 sulfur ucalgary induciblesytems.PNG|center|700px||thumb|Figure 12: Second set of final constructs for the desulfurization operon, with all genes under an IPTG inducible promotor.]]<html><br />
<br />
<p align="justify"><br />
Due to the large number of proteins being expressed in this system, the possibility of forming inclusion bodies is present. As such, a backup system was built where both the optimization circuit and the <i>dsz</i> operon were under the control of the inducible lacI promoter. This system would allow us to tune the expression of the genes, and determine which expression level is optimal for desulfurization in our bioreactor.</p> <br />
<br />
<p align="justify">Currently the final steps of construction of these constructs is underway, following which functionality tests will begin.</p><br />
<br />
<br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/OSCAR/DesulfurizationTeam:Calgary/Project/OSCAR/Desulfurization2012-10-26T21:48:52Z<p>Lisa.O: </p>
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<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Desulfurization|<br />
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CONTENT=<html><br />
<img src="https://static.igem.org/mediawiki/2012/5/5e/UCalgary2012_OSCAR_Desulfurization_Low-Res.png" style="float: right; padding: 10px;"></img><br />
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<h2>Why Remove Sulfur?</h2><br />
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<p align="justify"><br />
Sulfur is the third most abundant element in crude oil (Ma, 2010), and when sulfur containing hydrocarbons are burned they release S0<sub>2</sub> and S0<sub>3</sub> gasses into the atmosphere. Not only does this reduce the efficiency and value of our product, but it also contributes to global warming, acid rain, and various health issues due to the pollution (Reichmuth <i>et al</i>., 2000). Strict regulation on sulfur in fuels are now in place and low-sulfur gasoline is mandated across all of Canada (Source: Environment Canada). To upgrade the quality of our fuel we need to remove the sulfur but keep the hydrocarbon backbone.</p><br />
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<h2>Our Vision</h2><p align="justify"><br />
Though a few pathways for biodesulfurization exist in the microbial world, most involve the destruction of part of the carbon skeleton (an example would be the Kodama pathway)(Soleimani <i>et al</i>., 2007). This would effectively reduce the quality of our product. With this in mind the pathway we have chosen is the 4S pathway found in <i>Rhodococcus spp</i>. It has been characterized and shown to remove sulfur from the model substrate dibenzothiophene (DBT) and convert it to 2-hydroxybiphenyl (2-HBP) in a non-destructive manner. DBT and its derivatives make up 70% of the organic sulfur compounds found in crude oil (Ma 2010), and are also some of the most difficult to remove through chemical means. By using the 4S pathway we will be able to upgrade our fuel and remove recalcitrant compounds at the same time. <br />
</p><br />
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</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|750px|thumb|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD.]]<html></p><br />
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<h2>4S pathway</h2><br />
<p align="justify"><br />
Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO<sub>2</sub>) through the addition of 2 oxygen atoms to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the sulfur, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-HBP. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.</p><p align="justify"><br />
The first three steps of the 4S pathway require FMNH<sub>2</sub> and subsequently reduces the reductive power of the cell. WIn order to regain this power an oxidoreductase (DszD) uses NADH to recycle the FMNH<sub>2</sub>, allowing the reaction to proceed. Without DszD the desulfurization pathway would grind to a halt.</p><p align="justify"><br />
The <i>dszA</i>,<i>B</i>, and <i>C</i> genes form an operon on the pSOX plasmid of <i>R. erythropolis</i>, while <i>dszD</i> is found in the chromosome. Naturally this pathway is slow, however using synthetic biology approaches this process can be optimized.</p><br />
<br />
<h2>Our Approach</h2><br />
<a name="Degradation"></a><h3>1) Find the genes!</h3><br />
<p align="justify">We isolated the plasmid containing the <i>dsz</i> genes from a desulfurising environmental isolate of <i>Rhodococcus</i> using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">modified miniprep procedure</a>. As the native promoter has been shown to be repressed by various sulfur-containing compounds (Li <i>et al</i>., 1996), we designed primers for just the coding sequences of the <i>A, B, </i> and <i>C</i> genes. As these genes all have some illegal cutsites in them we constructed them into the PSB1C3 vector and started our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis protocol</a>.</p><br />
<p align="justify"> We performed an experiment to measure the desulfurization rate of select organosulfur compounds by our <i>Rhodococcus</i> strain (Figures 4-6 below). These experiments monitored the degradation of the compounds by our strain over time. We discovered that the <i>dsz</i> operon is capable of desulfurizing a wider range of compounds than just the commonly studied DBT. This shows that this pathway could be a promising solution for degradation of a wide variety of sulfur containing toxins, including those that resemble naphthenic acids. </p> <br />
<br />
<p align="justify"></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 2: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through the GC/MS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GC/MS after 6 days of incubation. ]]<html></p><br />
<br />
<p align="justify"></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 3: The peak in this mass spectrum demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<p align="justify"><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound ('''A:''' dibenzothiophene, '''B:''' tetrahydro-4h-thiopyran-4-one, and '''C:''' benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdownn. Degradation is seen for DBT (the model compound) as well as other sulfur containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<h3>2) Mutagenesis: Biobrick Compatability and Increasing DszB Activity </h3><br />
<p align="justify">In total the <i>dszABC</i> genes had 7 PstI sites and 1 NotI site that needed to be mutated for the biobrick standard. The primers were designed such that the site was removed without the amino acid being changed. In addition, a point mutation of Y63F in DszB increased the activity of the protein (Oshiro <i>et al</i>., 2007), and was included in the mass mutagenesis we undertook. Mutagenesis was performed as described in <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">this protocol.</a></p><br />
<br />
<a name="catalase"></a><h3>3) Replacing DszD with HpaC & Introducing Catalase </h3><br />
<p align="justify"><br />
As FMNH<sub>2</sub> is consumed in the first three steps of the pathway it needs to be regenerated or the process will grind to a halt. This usually falls to the <i>dszD</i> gene, however it has been shown that the <i>hpaC</i> gene from <i>E. coli</i> performs the same function more efficiently (Gala´n <i>et al</i>., 2000). One problem arises from this though, as high levels of FMNH<sub>2</sub> cause the production of toxic hydrogen peroxide inside the cell (Gala´n <i>et al</i>. 2000). To address this issue we have included a catalase gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"> <i>P<sub>lacI</sub>-katG-LAA</i></a>) that will remove the peroxide that would be toxic to the cell.</p><br />
<br />
<p align="justify"></html>[[File:Ucalgary_sulfur_constructs_KatandHpaC.PNG|center|250px|thumb|Figure 7: Diagrammatic representation of the full "optimization circuit", consisting of the oxidoreductase HpaC and a catalase (KatG).]]<html></p><br />
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<br />
<h3>Results</h3><br />
<p align="justify">To show that catalase activity increased <i>E. coli</i> survivability in peroxide we cultured the inducible catalase against a catalase-free control with varying levels of peroxide. After growing overnight the negative didn't grow in any culture except in the absence of peroxide, while the catalase cultures could tolerate peroxide. This is shown below.</p><p align="justify"><br />
<br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 8: Catalase Assay. Overnight cultures of P<sub>lacI</sub> and P<sub>lacI</sub>-KatGLAA were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbidity was observed. It was found that at 1 mM of peroxide, cultures with just the lacI promotor perished, however when KatG-LAA was expressed, the cells survived.]]<html></p><br />
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<br />
<p align="justify">To test the action of HpaC to use NADH to recycle FMN into FMNH<sub>2</sub> cell lysates were exposed to NADH and it's absorbance at 340nm (Kamali <i>et al</i>., 2010) was measured over time. Both native HpaC expression and an induced <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> system were tested as well as a negative control. The results are shown below.</p><br />
<br />
<p align="justify"> </html> <br />
[[File:Ucalgary2012 HpaC assaycumulativeforthedatapage.png|center|850px|thumb|Figure 9: HpaC Assay with '''A)''' 2 mL cell lysate and '''B)''' 100 &micro;L cell lysate. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, various amounts of cell lysate were transferred to a cuvette, and a spectrophotometer was blanked at 340 nm with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p align="justify">The assay showed that NADH does not abiotically convert into NAD+, however the native expression of HpaC did show a steady decrease in the levels of NADH. The induced overexpression of HpaC caused extremely rapid conversion into NAD+ as reflected by a sharp drop in the absorbance of NADH (see figure B). This drop was much sharper than what was seen when native levels of oxidoreductases were tested, showing that the <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> was functional and that it would effectively recycle FMN.</p><br />
<br />
<br />
<a name="UBC"></a><h3>4) Optimizing Gene Order</h3><br />
<br />
<p align="justify">Further optimization of the system was achieved through reorganization of the reconstructed operon. Natively the genes are arranged ABC, however the catalytic efficiency of the protein products are 25:1:5 for A:B:C respectively (Li <i>et al</i>., 2008). By rearranging the genes into BCA there is stronger transcription of the weaker proteins, giving a more balanced system overall. These would all be constructed with the same strong ribosomal binding site, <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>.</p><p align="justify"><br />
<br />
</html>[[File:DszOperonOptimize.png|center|400px|thumb|Figure 10: Method of optimizing gene order. The top circuit represents that found natively in the organism, with the bottom circuit representing our modified version.]]<html><br />
</p><br />
<br />
<a name="sulfur"></a><h2>Final Constructs</h2><br />
<p align="justify">After all of the above considerations are met, four final constructs for our system will be made to allow us to test desulfurization under different conditions.</p><p align="justify"><br />
<br />
</html>[[File:WikiConstructs_ucalgary_sulfur_2012_final_systems.png|center|700px|thumb|Figure 11: First set of final constructs for the desulfurization operon, with constitutive Dsz expression and inducible expression of the optimization proteins; either HpaC on its own or coexpressed with KatG]]<html></p><br />
<br />
<p align="justify"><br />
The first two constructs have the modified <i>dsz</i> operon (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902052"><i>dszB</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><i>dszC</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902050"><i>dszA</i></a>) under the control of a constitutive TetR promotor (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>) This is to allow for the testing of the optimization circuit, which is under the control of a lacI promotor inducible by IPTG (<a href="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</a>). The set-up of these two constructs will therefore allow for the expression of the <i>dsz</i> genes with the ability to test and compare their desulfurization rates <br> A) On their own <br> B) With the addition of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> <br> C) With the addition of both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K137068"><i>katG-LAA</i></a></p><br />
<br />
<p align="justify">This will allow us to determine what the optimal construct and expression levels of the additional genes must be in order to have the most effective sulfur removal system.</p><br />
<br />
</html>[[File:WikiConstructs2 sulfur ucalgary induciblesytems.PNG|center|700px||thumb|Figure 12: Second set of final constructs for the desulfurization operon, with all genes under an IPTG inducible promotor.]]<html><br />
<br />
<p align="justify"><br />
Due to the large number of proteins being expressed in this system, the possibility of forming inclusion bodies is present. As such, a backup system was built where both the optimization circuit and the <i>dsz</i> operon were under the control of the inducible lacI promoter. This system would allow us to tune the expression of the genes, and determine which expression level is optimal for desulfurization in our bioreactor.</p> <br />
<br />
<p align="justify">Currently the final steps of construction of these constructs is underway, following which functionality tests will begin.</p><br />
<br />
<br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/DesulfurizationTeam:Calgary/Notebook/Desulfurization2012-10-26T21:47:54Z<p>Lisa.O: </p>
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<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Desulfurization Journal|<br />
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CONTENT=<br />
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<h2>Week 1 (May 1-4)</h2><br />
<p>During this week, literature search was performed.</p><br />
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<h2>Week 2 (May 7-11)</h2><br />
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<p>Along with the rest of the team, this week was dedicated to familiarizing ourselves on the protocols that will be utilized during this years project; specifically the polymerase chain reaction, gel verification, preparation of overnight cultures, as well as developing a procedural flowchart to transform competent cells with registry biobricks. With regards to our sub-group specific goals, we reviewed the current available literature around various industrial and laboratory approaches to desulfurization of organic groups, especially in the petroleum industry. This included a comparison of non-biological processes such as conventional hydrodesulfurization, which is currently employed in petroleum product refinery stages, and how a biological approach would supplement and perhaps even offer several advantages over these methods. Current limitations to biological desulfurization, however, include such factors as biocatalyst stability, enzyme specificity and desulfurization rate. We also identified the enzyme desulfinase (DszB) as being one of the bottlenecks in the desulfurization 4S pathway. Overall, our goals moving forward involve determining the specific pathways involved in the desulfurization process as well as the reaction conditions we would want to employ, and identifying specific model compounds in addition to dibenzothiophene (DBT) that we could use to test the effectivity of our biosystem in order to determine its functionality in the conversion of naphthenic acids to economically valuable hydrocarbons.</p><br />
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<h2>Week 3 (May 14-18)</h2><br />
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<br />
<p> Building on the previous week's literature review, the 4S pathway was recognized as the preferred biological mechanism that we would explore in devising a desulfurization biosystem. Of specific interest is the dsz operon consisting of the genes for <i> dszA, dszB, </i>and<i> dszC</i> which selectively and non-destructively remove the sulfur from the hydrocarbon structure, and therefore preserves the carbon skeleton. In addition to these, another dsz gene exists.<i> dszD</i>, which codes for a FMN:NADH reductase, is an essential component of the pathway, but not part of the operon. Instead, it is on the genome. The enzyme produced by this gene is required to regenerate the FMNH2 consumed by the reactions carried out by DszA and DszC. <i>Rhodococcus erythropolis</i> IGTS8 is the most studied model organism in investigations of the 4S pathway, and has been shown in many different research endeavors to be capable of converting DBT to 2-HBP.</p><p><br />
</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|thumb|750px|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD. Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO2) through the addition of oxygen to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the heteroatom, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-hydroxybiphenyl. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.]]<html></p><p><br />
An alternative to the DszD gene is HpaC, an oxidoreductase encoded in the <i>E. coli</i> W genome. This enzyme has been shown to increase the rate of desulfurization. Following this, other protocols added to our growing lab methods 'toolkit' were a restriction digest protocol, PCR purification, and finally, DNA construction digest. Aims moving forward include obtaining strains of the <i> R. erythropolis </i>, while also executing a timeline devised to biobrick, test, and incorporate the genes necessary in the above processes in a biobrick circuit.</p><br />
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<h2>Week 4 (May 22-25)</h2><br />
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<br />
<p>This week was kicked off with a project development meeting with Emily and David, and we devised a protocol for biobricking the <i> hpaC </i> gene. Additionally, methods to place the genes coding for the 4 enzymes, DszA,B,C and HpaC into a single construct were explored. Within the lab, the PCR performed on the resuspended pUC18-hpaC was not successful initially. <br />
Furthermore, we ordered the substrates/compounds that we intend to use for desulfurization tests. Once the substrates and the <i>Rhodococcus</i> strain arrive we are going to test how effectively the bacteria can desulfurize different sulphur-containing compounds that resemble naphthenic acids. Finally, we came across a paper where a team had developed an improved efficiency DszB through site-directed mutagenesis in 2007. This was through a point mutation to the gene, converting a tyrosine at position 63 to a phenylalanine residue. A member of this team was contacted to request the plasmid that contains the mutated gene. The conversion step carried out by DszB is the major bottleneck in the 4S pathway and if a strain or sample containing this mutation was obtained, it would significantly bolster our later testing efforts on DBT, as well as other compounds such as thiophane.<br />
</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<br />
<br />
<p>Since we wanted to make sure we would not run out of pUC18(plasmid containing the hpaC gene), we transformed some E.coli cells with it. We grew them on plates containing ampicillin (A), kanamycin (K), tetracycline (T) and chloramphenicol (C) antibiotics and they only grew on A. Therefore pUC18 has A resistance. We did a three sets of PCR with primers designed against <i>hpaC</i>, one using 1/10 dilution of pUC18, the other using 1/100 dilution of pUC18 and one with the colonies we had just obtained by transforming the <i>E.coli</i> cells. </p><br />
<p>The PCR worked and we saw bands of the same size for all three sets of PCR. (Unfortunately, the picture we saved is not a good one since some of the bands faded away under UV due to prolonged exposure. Following this, PCR purification was performed to obtain the pure <i>hpaC</i> with biobrick prefix and suffix attatched to gene, which would allow us to insert the sequence into a biobrick standard backbone. 3 sets of digestion, ligation, and transformation (using pairs of X&P enzymes, E&S enzymes and E&P enzymes) were carried out in order to insert the <i>hpaC </i> gene into the <a href=" http://partsregistry.org/Part:pSB1C3">pSB1C3</a><br />
vector. All the sets grew successfully.<br />
Following the above successes with<i> hpaC</i>, the arrival of our <i>Rhodococcus</i> strain afforded us the opportunity to begin investigation of the Dsz operon using the primers currently in our possession. This strain is an environmental isolate that has been shown to be an active desulfurizer. The gram-positive nature of the strain also dictated we explore various lysing strategies before the genes encoding the Dsz enzymes could be amplified for further purification and biobrick construction steps. PCR was carried out using <i>dszA</i> primers on three different treatments {microwave, lysate buffer, and a control} which yielded banding pattern around 1200 base pairs for the lysate treatment (2%SDS and 10% tritonX-100, plus heat for 5mins at 98C).<br />
</p> <br />
<br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<br />
<br />
<p> In order to confirm the <i>hpaC</i> biobrick construction, two sets of colony PCR were performed, choosing white colonies from the 3 plates we grew last week (white colonies indicate a loss of the RFP generator in the <a href=" http://partsregistry.org/Part:pSB1C3">pSB1C3</a><br />
backbone, and therefore allow for weeding out of the colonies which are simply the original plasmid vector). These reactions were carried out both with <i>hpaC</i> primers and with standard biobrick primers designed against the plasmid backbone. After running them on the gel we saw equal bands for the PCR reactions performed using <i>hpaC</i> primers (However, a PCR using biobrick primers was performed later and the same results were obtained). Colonies 1(-) and 5(-) were used to make overnight cultures, which were then miniprepped the following day to obtain the plasmid DNA of the putative <i>hpaC</i> biobrick. Digestions were performed on the miniprep products using EcoRI and PstI to look for part size as further verification for the genes presence in the plasmid. The results were good and two bands were observed on each column (one for vector and the other for <i>hpaC</i>)). <i>hpaC</i> was sent in for sequencing. </p><br />
<p><br />
<br />
<br />
<br />
</html>[[File:UCalgary2012_04.06.2012-desulfurisation_hpacverification.jpg|thumb|700px|center|Figure 2: HpaC verification cPCR. HpaC gene was inserted into the pSB1C33 vector and E. coli cells were transformed. In order to confirm that pSB1C3 contains the hpaC gene, two sets of colony PCR's were conducted. One with biobrick primers, and the other with hpaC primers. Bands indicate successful amplification at the approximate size of the hpaC gene (517 bp)]]<br />
[[File:Ucalgary2012 06.06.2012-digestion of hpaC with E and P.jpg|thumb|500px|center|Figure 3: HpaC confirmation digest. Plasmid was miniprepped and digested for 2h with EcoRI and SpeI before being run on a 1% gel for 1h and 120V. Bands between 500 and 700 bp indicate the hpaC part is present as an insert. Bands of about 2000bp show the size of the psb1c3 vector.]]<html></p><br />
<br />
<br />
<br />
<p> PCR reagents were prepared to re-test/confirm previous results of <i>dszA</i> amplification following two different lysing treatments (microwave + lysate buffer). This time, all three genes were amplified and gel verification showed clear banding patterns around 500bp range for all three genes for the microwave treatment. Remaining PCR products were run on a gel and extracted for further purification steps; however, presence of any genetic material were not confirmed through nanodropping which raised concerns about the composition of the purified products, the success of the initial amplification step, or perhaps even the lysis treatment. Further experimentation will have to be carried out to troubleshoot.</p><br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<br />
<br />
<p> This week, we focused on amplifying <i>dsz</i> genes from our <i>Rhodococcus</i> strain for construction into biobricks. We also wanted to purify the <a href=" http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"> BBa_K902057 <i>hpaC</i></a> and pUC18-<i>hpaC</i> plasmids to replenish our current stocks. For the <i>dsz</i> aspect, we were able to successfully grow extra plates of <i>Rhodococcus</i> strain which was used to inoculate PCR tubes. The PCR did not go well, with significant streaking and false positives with similar banding pattern to previous gels run in the previous week. A final gel verification of a random sample of a tube of PCR products from dszA,B,C respectively and two negative control treatments involving master mix only and the lysed cells only illustrated the lack of discrepancy between the supposed successful amplification and the lysed cells (with lysate buffer) alone. Because of this we decided to take a different approach involving plasmid isolation carried out before PCR, rather than applying the PCR reagents directly to a lysed culture sample.</p><br />
<p> <a href=" http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> verification through sequencing was successful, confirming the construction of our first biobrick. Subsequently, O/N cultures of the plasmid containing cultures were prepared and stored in glycerol at -80C. Furthermore, verification of catalase gene part (<a href=" http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a>), which was sent as a culture stab from the parts registry was initiated. Our newly identified biobricked-hpaC was used as a positive control, but the banding pattern was not very conclusive. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<br />
<br />
<p>PCR was reattempted on <i>Rhodoccocus</i> that was lysed using two different dilutions of the lysate buffer, but the gel verification confirmed the previous failure in using this approach. An alternative that involved preparation of an overnight culture of the <i>Rhodococcus</i> cells followed by a plasmid purification was followed. The plasmid purification eventually yielded plasmid samples with concentrations of 98.6ng/μL to 182.7ng/μL (4 samples obtained overall). Additionally, the catalase biobrick was used to transform some stock competent cells, and samples of some colonies were subsequently PCR'ed. Although, the gel verification showed some potential contamination, and the required banding patterns at around 2200bp was not obtained. </p><br />
<br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<br />
<br />
<p> PCR was attempted to amplify the genes of the <I>dsz</i> operon utilising an adapted PCR protocol with purified <i>Taq</i> polymerase that had been isolated from the host organism. Eventually, some banding pattern was obtained between 1200 and 1500 base pairs when a gradient thermocycler was used with melting temperatures ranging betweeen 55°C to 65°C. This was assumed to be indicative of successful amplification of <i>dszB</i>; however, further purification and gel verification results were inconclusive and no yield was obtained when placed tested using a nanodrop machine. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<br />
<br />
<p>Top 10 E.coli cells were transformed with <a href="http://partsregistry.org/Part:BBa_R0011">BBa_R0011</a><br />
(IPTG inducible promoter in psb1C3 backbone), and resulting colonies were tested using cPCR. <br />
Colony PCR was performed on cells containing the catalase biobrick. Catalase is 2217bp long but since biobrick primers add about 200bp, bands of 2400 bp were expected if the part was present in the biobrick. These bands were observed, indicating that the <a href=" http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a> gene was most likely present.</p><br />
<p></html>[[File:Ucalgary2012 4.7.2012 catalase colony pcr 2.jpg|thumb|500px|center|Figure 4: Colony PCR on potential colonies containing katG-LAA. Biobrick primers were used. Bands at 2200 indicate successful amplification of the catalase part within the biobrick backbone. Smaller bands are indicative of primer degradation and are nonspecific amplification. +C indicates the positive control, and RFP generator, which should amplify at around 1000 bp.]]<html></p> <p><br />
<br />
PCR using Phusion high fidelity polymerase was carried out on <i>dszA</i>, <i>dszB</i>, and <i>dszC</i> in a gradient thermocycler. Amplification of non-specific bands was present for <i>dszA</i> and <i>dszB</i>, however strong banding for the desired size of the gene was observed for both (around 1500 for <i>dszA</i>, 1100 for <i>dszB</i> </p><br />
<br />
<p></html>[[File:Ucalgary2012 6.7.2012.dszABphusionPCR.jpg|thumb|500px|center|Figure 5:DszA and DszB PCR from <i>Rhodococcus</i>. DszA amplicon is around 1300 bp, and is observed to run higher on the gel. DszB amplicon is expected to be 1098 bp, which is observed in addition to multiple nonspecific banding. +C indicates the positive control, and RFP generator, which when PCRed with biobrick primers should be around 1000 bp. As this control is running high as well, it is believed that both amplicons for DszA and DszB have been obtained. No contamination is observed in the NTC (no template control).]]<html><br />
</p> <br />
<p></html>[[File:Ucalgary2012 7.5.2012 dszC.png|thumb|500px|center|Figure 6:DszC PCR from <i>Rhodococcus</i>. The numbers above the figure show the colony number. DszC amplicon is around 1450 bp, and is observed to run at almost the right size on the gel. No contamination is observed in the NTC (no template control).]]<html><br />
<p><br />
Examining the sequences of the <i>dszABC</i> genes led to the discovery that all 4 had multiple illegal enzyme cut-sites in them that we have to eliminate before biobrick composite part construction can occur. <i>dszA</i> has four PstI cut sites, <i>dszB</i> has a PstI and a NotI and <i>dszC</i> has a PstI cut site. The Stratagene QuickChange mutagenesis procedure is going to be used to eliminate illegal cut sites with the only alteration being that Kapa HiFi polymerase would be used during the process. Primers needed for the mutagenesis were designed based on the procedure mentioned above. </p><br />
<br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<br />
<p> Following successful amplification of the <i>dsz</i> operon genes in the previous week, the genes were constructed into the <a href="http://partsregistry.org/Part:pSB1C3"> pSB1C3</a> vector. Colony PCR verifications were observed to be positive. Furthermore, the insertion of part <a href="http://partsregistry.org/Part:BBa_J13002"> BBa_J13002, (P<sub>tetR</sub>-RBS)</a><br />
in front of the previously biobricked <i>hpaC</i> was attempted. Overnight cultures were also prepared using two colonies each for <a href="http://partsregistry.org/Part:BBa_J13002"> <i>P<sub>tetR</sub>-RBS</i></a> and <a href="http://partsregistry.org/Part:BBa_R0011"> <i>P<sub>lacI</sub></i> </a> (an IPTG inducible promoter that we hope to build in front of <ahref="http://partsregistry.org/Part:BBa_B0034">an RBS site, BBa_B0034</a>). These cultures were then miniprepped to yield the respective parts.</p> <br />
<br />
<p>Additionally, <a href="http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a> was built into a <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> backbone. The construction and availability of all these parts will be critical in the construction of our overall circuit for biodesulfurization. Colonies which looked good on cPCR were used to prepare overnight cultures, and were miniprepped and sent in for sequencing verification the following day.<br />
On the side, M9 minimal media was also prepared to carry out growth experimentation and overall desulfurization capability of <i>Rhodococcus</i> when exposed to DBT. The various growth treatments were M9 Media and glucose only, M9+glucose+DBT, M9+glucose+MgSO4+/-DBT, M9+glucose+MgCl2+/-DBT. 0.008g of FeCl2.4H2O was also added to each of the tubes. Samples were then inoculated with colonies of the <i>Rhodococcus</i>.</p><br />
<br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<br />
<p>This week, while awaiting sequencing verification results which were required before we could begin the construction process, the desulfurization team initially aided in some of the tasks related to the other hydrocarbon groups. The success of the construction of <a href="http://partsregistry.org/Part:BBa_J13002"><i>P<sub>tetR</sub></i>-RBS</a><br />
with <a href="http://partsregistry.org/Part:BBa_K902057"><i>hpaC</i></a> was also explored by using forward and reverse primers of <a href="http://partsregistry.org/Part:BBa_R0040">BBa_R0040</a> (the promoter component of the composite part BBa_J13002). However, the eventual gel verification was inconclusive and sequencing results finally indicated an unsuccessful ligation. Additionally, the minimal media M9 preparation had been contaminated in the previous effort so this process was repeated to create tubes of each of the growth condition treatments detailed previously, and two repeats, one with an extra filtration step and one without was used to prepare the cultures.</p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<br />
<p> Mutagenic primers were redesigned after the initial ones were found to have premature stop codons. As part of the redesign process in constructing our overall gene circuits for desulfurization, a backbone switch of <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> into a chloramphenicol (Chlor) resistant vector was necessary. The subsequent transformed products were plated on a Chlor plate and selected colonies were used to prepare O/N cultures, then minipreped before finally being digested with enzymes EcoRI and PstI. The resulting gel verification images were inconclusive as they did not show the required banding pattern around 50bp. Meanwhile, colony PCR was run on colonies transformed with <i>katG-LAA</i> constructed into a <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> backbone, as well as the <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> +<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a> construct. <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></a> was shown to have been successfully amplified, so overnight cultures were prepared and subsequently miniprepped. On the other hand, the construct was not successful so a third attempt was carried out. Colony PCR treatments that used either <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> forward primers or <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
primers were used and the overall constructs were made either on a chlor-resistant, or ampicillin-resistant vectors. Preliminary images of the gel verification appeared to have confirmed the construct, although sequencing verification will be the final indicator of overall success. </p><br />
<br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<br />
<br />
<p>Sequencing results from the previous week's constructs were available confirming that we constructed KatGLAA in a chlor-resistant backbone. However, switching the plasmid backbone of <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> to <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> was not successful. The construction of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>+<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> was finally sent in for sequencing. Site-directed mutagenesis of the <i>dsz</i> operon was also initiated: <i>dszA</i> has four PstI cut sites; <i>dszB</i> has a PstI and a NotI site; <i>dszC</i> has two PstI cut sites. Site directed mutagenesis was started this week to change a single base pair in these genes in a way that eliminates the cut site but preserves the amino acid codons, so as to not mutate the protein coding sequence. Ohshiro 2007 demonstrated that replacing the Tyr residue at position 63 of <i>dszB</i> gene with a Phe increases the activity of the enzyme. Therefore we want to introduce the same mutation into our <i>dszB</i>.</p><p><br />
For the first attempt at mutagenesis we chose to mutate the second PstI site in <i>dszC</i> (PstI2). As a positive control for the procedure, we also performed the mutagenic PCR on a plasmid containing the β-galactosidase gene with a point mutation where the PCR would cause it to regain its function. For both mutagenesis protocols we used the Kappa Hifi kit. After confirming that the PCR worked by running some produce on a gel, the PCR products were DpnI digested, the purpose of which is to degrade the unmodified parental DNA (DpnI degrades methylated DNA only). Control PCR products were plated on an ampicillin plate containing IPTG and X-gal. The colonies that grew on the control plates were blue indicating that the mutagenesis had worked for the β-galactosidase gene. Minipreps of the O/N culture of <i>dszC</i> mutants were digested with PstI enzyme and the results indicated that the mutagenesis was successful.</p><p> <br />
Attempts to simultaneously perform all the mutations in <i>dszC</i> genes in one step using the Knight procedure failed (<a href="http://openwetware.org/wiki/Knight:Site-directed_mutagenesis/Multi_site">Knight Multi-site Mutagenesis Procedure</a>). What enables simultaneous mutations is that Taq ligase closes the gaps in PCR products after each cycle. In the protocol it instructs to use Taq ligase buffer only for the PCR/ligation protocol. We suspected that the reason this procedure did not work might be that the Kappa polymerase is not functional in Taq ligase buffer. Therefore we did some experiments on the controls in Taq ligase kit and kappa polymerase kit to find out which buffer that Kappa polymerase and Taq ligase both work best in. The result was that both enzymes work best in a buffer made of half Taq ligase buffer and half Kappa polymerase buffer. </p><br />
<br />
<br />
<br />
</html>[[File:Ucalgary2012 31.7.2012 dszc mutagenesis 5 20 and 50ng.jpg|thumb|500px|center|Figure 7: DszC PstI2 mutagenesis PCR with varying concentrations of template plasmid was performed. The gel shows the PCR products that were run on a gel. +C lanes show the PCR products of the control. The control was pWhitescript™ 4.5-kb control plasmid from Stratagene mutagenesis kit which was mutated with primers also from the kit. Bands indicate successful amplification of the plasmid.]]<br />
[[File:UCalgary 02.08.12 dszC psti digest mutagenesis.jpg|thumb|700px|center|Figure 8: The control and the mutated plasmids were digested with PstI restriction enzyme. The control is dszC biobrick. Other lanes show the plasmids purified from the cells transformed with PstI2 mutagenesis PCR products followed by digestion. The control shows three bands since it has three PstI cut sites. The other lanes show two bands which indicates one of the cut sites has been eliminated through mutagenesis. ]]<html><br />
<br />
</html>[[File:Ucalgary2012 2.8.2012 finding the right buffer for multisite mutagenesis.png|thumb|800px|center|Figure 9: The multisite mutagenesis using the Knight procedure was tried in different buffers to find out the buffer that Kappa works optimally at. All the PCRs were performed on the pWhitescript™ 4.5-kb control plasmid from Stratagene mutagenesis kit which contains the beta-galactosidase gene. Based on this gel, the optimal buffer is composed of 50% kappa buffer and 50% Taq ligase buffer.]]<html><br />
<br />
</html>[[File:Ucalgary 3.8.2012 optimal buffer for Taq ligase.png|thumb|800px|center|Figure 10: Using the control provided in the NEB Taq ligase buffer (BsteII digested lambda DNA), we tried to find if the Taq ligase enzyme can function in combinations of Kappa Hifi buffer and Taq ligase buffer. Lane two is the control which is only the digested lambda DNA. Lanes 1 and 3 show that some of the bands compared to control have been ligated together. Therefore, Taq ligase functions just as good in the buffer composed of 50% kappa hifi buffer and 50% Taq ligase as it would in its own buffer.]]<html><br />
<br />
<h2>Week 15 (August 6 - August 11)</h2><br />
<br />
<p>Sequencing results for <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A> <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> returned negative, so a 3-part ligation method was used to retry this construction. The following parts were ligated with the restriction enzymes indicated in brackets after each: <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>(EcoRI/SpeI) + <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> (XbaI/PstI) + <A HREF=" http://partsregistry.org/Part:PSB1K3">BBa_PSB1K3</A> (EcoRI/PstI). Also, the more conventional construction (only 1 insert) of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>(SpeI/PstI) + <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> (XbaI/PstI) was reattempted. Furthermore, 3-way ligations were also attempted for <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
+<A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A>+<A HREF="http://partsregistry.org/Part:PSB1K3">BBa_PSB1K3</A>, and <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a>+<a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
+<A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A>, as well as the two-way contruction of just <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> after the <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a>. After plating these transformations, colony PCRs were carried out and samples that gave an indication of being successful on the gels were used to prepare O/N cultures followed by miniprep. With regards to the site-directed mutagenesis side of the experimentation, <i>dszA</i>-PstI1 (the first PstI cut site in <i>dszA</i>) ,<i> dszB</i>-PstI and <i>dszC</i>(PstI2 mutated)-PstI1 mutagenesis were performed following the procedure explained in the previous week. The gel below shows the successful result of digest confirmation (Fig. 8). <br />
Multisite mutagenesis (Knight method) was repeated using the modified buffer (half Taq ligase buffer and half Kappa buffer). However it was not successful again. We also tried doing multisite mutagenesis using Pfu Turbo polymerase and following the Knight procedure without any buffer modifications. No successful results were observed. </p><br />
<br />
<br />
<br />
</html>[[File:Ucalgary2012 Digestion confirmation of mutagenesis in dszAPstI1, dszBPstI and dszCPstI1 (PstI2 site mutated)..jpg|thumb|800px|center|Figure 11: Confirmation of site-directed mutagensis to eliminate PstI cut sites in: <i>dszA</i>PstI1 cut site, <i>dszB</i>PstI cut site and <i>dszC</i>PstI1 (PstI2 site mutated). Note that in each case, the unmutated controls for each gene are to the left of each set of plasmids derived from mutagenic PCRs. Plasmids from cloned <i>dsz</i> genes are smaller than the bands to the right of them (which have undergone mutagenesis), indicating that in the plasmids on the right, the PstI cut site was lost. ]]<html><br />
<br />
<br />
<h2>Week 16 (August 12 - August 18)</h2><br />
<br />
<br />
<p>The progress in mutagenesis of <i>dsz</i> genes was continued from the previous week: <i>dszB</i>(PstI mutated)-Y63F and <i>dszA</i>(PstI1 mutated)-PstI3 mutagenesis. The gel below shows the digest confirmation.</p><br />
<br />
<br />
</html><br />
[[File:Ucalgary2012 15.08.2012 dszAPstI1&amp;3 dszB Psti y63f muta diges-1.jpg|thumb|800px|center|Figure 12: Digestion confirmation of biobricks of a) dszA (PstI1 mutated) PstI3 mutagenesis and b) dszB (PstI mutated) Y63F mutagenesis. Cutting dszA (PstI1 mutated) with PstI is expected to produce fragments of 2790, 333, 255, and 114bp. Cutting dszA (PstI1 and PstI3 mutated) is expected to produce fragments of 2730, 588, and 114bp. The primers for dszB-Y63F mutagenesis introduce an HpyAV cut site. pSB1C3 inteslf has two more HpyAV cut sites. Before the mutation bands of 2858 abd 310bp are expected, and after successful mutation bands of 1502, 1356, 310 are expected. Lane legend: 1- dszA (PstI1 mutated). 2-dszA (PstI1 mutated) digested with PstI. 3-dszA (PstI1 mutated) PstI3 mutagenesis c1 Digested with PstI. 4- dszA (PstI1 mutated) PstI3 mutagenesis c2 Digested with PstI. 5-dszA (PstI1 mutated) PstI3 mutagenesis c3 Digested with PstI. 6- dszA (PstI1 mutated) PstI3 mutagenesis c4 Digested with PstI. 7- Fermentas 1kb Plus Ladder. 8- Empty. 9- dszB (PstI mutated). 10-dszB (PstI mutated) digested with HpyAV. 11- dszB (PstI mutated) Y63F mutagenesis c1 digested with HpyAV. 12- dszB (PstI mutated) Y63F mutagenesis c2 digested with HpyAV. 13- dszB (PstI mutated) Y63F mutagenesis c3 digested with HpyAV. 14-dszB (PstI mutated) Y63F mutagenesis c4 digested with HpyAV.]]<html></p><br />
<br />
<p>We attempted a different approach to speed up the turnover time of the mutagenesis PCR. Briefly, after the PCR mutagenesis the PCR products were purified and then incubated with T4 polynucleotide kinase (PNK) and ligase. After heat inactivating the ligase and T4 PNK, the products were DpnI digested. Subsequently another round of DNA purification was performed. However, the results were unsatisfactory after the digest confirmation.</p><p><br />
Sequencing results came back. <i>dszA</i> (PstI1 and PstI3 mutated) and <i>dszB</i>(PstI and Y63F mutated) were good. However <i>dszC</i> (PstI1 and PstI2 mutated) had an insertion next to the PstI1 cut site. Mutagenesis was repeated on the <i>dszC</i>(PstI2 mutated).<br />
<i>dszB</i>(PstI and Y63F mutated)-NotI and <i>dszA</i>(PstI1 and PstI3 mutated)-PstI4 mutagenesis were also performed.</p><br />
<p> To investigate the desulfurisation capability of the <i>Rhodococcus</i> sp. from which we cloned the <i>dsz</i> operon, a desulfurization assay was prepared by inoculating different treatments of M9 media. We also prepared some solutions that will be needed for analysis in the following week: a conditioning agent composed of 100ml of 95% ethanol, 50ml glycerol, 30ml of 12M HCl (aq) and 70g of NaCl(s) was prepared. The assay relies on the turbidity of a sample containing sulphate ions which are precipitated (hence the turbidometric nature of the assay) upon adding BaCl2(s), therefore if the <i>dsz</i> pathway is active, we expect a more turbid solution to form than in control samples. </p><br />
<br />
<br />
<br />
<h2>Week 17 (August 19 - August 25)</h2><br />
<br />
<br />
<p>This week, progress was made in determining the desulfurization activity of our <i>Rhodococcus</i> strain as measured by the sulfate release using a turbidometric assay. We encountered several challenges in our prescribed protocol as the concentrations that we used to prepare the standard curve may have been too dilute, or the composition of out conditioning agent may have been flawed. Additionally, steps were taken to determine the decomposition of DBT to 2-HBP through Gas Chromatograph-Mass Spectroscopy (GC-MS) analysis, but due to a preparation error, the DBT was added to a growth solution of M9 media prematurely and the autoclaving process decomposed the DBT releasing a yellow colouration into the solution. These two approaches in determining the desulfurization capability of the <i>dsz</i> operon will be further investigated. </p><br />
<br />
<p>Since the <i>dszC</i> second mutagenesis had proven to be unsuccessful last week, the <i>dszC</i>(PstI2 mutated)PstI1 mutagenesis was repeated. Also <i>dszA</i>(PstI1,3,4 mutated) PstI2 mutagenesis was performed. <i>dszA</i> and <i>dszC</i> were sent for sequencing on Wednesday. <i>dszB</i> was sent for sequencing on Friday. Sequencing results of <i>dszA</i> and <i>dszC</i> were back by Friday. <i>dszC</i> was successful. However, <i>dszA</i> contained an insertion next to the binding site of PstI4 cut sit, so the last two mutations must be redone. <i>dszB</i>(PstI and Y63F mutated)-NotI-mutagenesis was also repeated in case the result of the sequencing was not successful. These constructions were repeated. <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>-<A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
-<i>dszC</i> constructions were attempted, however they were not successful as indicated by colony PCR. Constructions of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>/<i>hpaC</i> were carried out and also came back negative in sequencing, however <a href="http://partsregistry.org/Part:BBa_K902059">BBa_B0034<br />
/<i>katG-LAA</i> (BBa_K902059)</a> was sequence confirmed.</p><br />
</html>[[File:Ucalgary2012 20.8.2012 dszC(psti2)psti1 mutagenesis.png|thumb|700px|center|Figure 13: Another attempt to mutagenize dszC(PstI2 mutated)at PstI1 site. The gel shows the confirmation digestion. C is the control which is dszC(PstI2 mutated). Since the control has two PstI cut sites, two bands are observed on the gel. The mutagenesis has been successful since only one band is observed on the other lanes indicating that one of the cut sites has been eliminated during mutagenesis.]]<html><br />
<br />
</html>[[File:Ucalgary2012 21.8.2012 dszA final mutagenesis digest.png|thumb|700px|center|Figure 14: DszA (PstI1, PstI2, PstI3 mutated)PstI4 mutagenesis digestion confirmation gel. All the lanes show the results of the plasmids being cut with PstI enzyme. C indicates the lane containing the control which is dszA(PstI1, PstI3 and PstI4 mutated). The other lanes are the digestions of the plasmids that are mutagenized, from different colonies. Control plasmid has two PstI cut sites and therefore the two bands observed on the gel were expected. The other lanes show only one band of about 3500bp which is about the right size for dszA in a psb1c3 vector. Therefore one of the cutsites has been eliminated during mutagenesis.]]<html><br />
<br />
<br />
<br />
<br />
<h2>Week 18 (August 26 - September 1)</h2><br />
<br />
<p> <A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i> (BBa_K902052)</i></A> sequencing results came back as successful. <i>dszA</i>(PstI1,3 mutated)-PstI2-mutagenesis was performed and sent for sequencing. Also <i>dszA</i>(PstI1,2,3 mutated)-PstI4-mutagenesis was performed, and this was also sent for sequencing. </p><br />
<p>Constructions of <A HREF="http://partsregistry.org/Part:BBa_K902053">P<sub>tetR</sub>-RBS/<i>dszB</i> (BBa_K902053)</a> and <a href="http://partsregistry.org/Part:BBa_K902056">BBa_B0034<br />
/<i>dszC</i> (BBa_K902056)</a> were attempted, verification digested, and sent for sequencing. Sequencing results for these constructs came back as positive, along with successful mutagenesis of <A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i> (BBa_K902050)</A>.</p><br />
<p>At this point, all of the <i>dsz</i> genes have been successfully made biobrick compatible, and <i>hpaC</i> has been biobricked. We have also successfully constructed <a href="http://partsregistry.org/Part:BBa_K902059">BBa_B0034 with <i>katG-LAA</i></a> to be used in the optimization circuit, as well as <A HREF="http://partsregistry.org/Part:BBa_K902053"><i>P<sub>tetR</sub>-RBS</i>/<i>dszB</i> (BBa_K902053) </a>and <a href="http://partsregistry.org/Part:BBa_K902056">BBa_B0034/<i>dszC</i> (BBa_K902056)</a>.<br />
<p> Constructions of <A HREF="http://partsregistry.org/Part:BBa_J04500">P<sub>lacI</sub>-RBS</A>with <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> were performed. As well, attempts to construct <A HREF="http://partsregistry.org/Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></a>/<a href="http://partsregistry.org/Part:BBa_K902056">RBS-<i>dszC</i></a> as well as <A HREF="http://partsregistry.org/Part:BBa_J13002">P<sub>tetR</sub>-RBS</A>/<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
/<A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i></A>, and <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>/<A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> were also carried out. These parts are intended as construction intermediates towards building the final systems, as well as providing a way of testing the genes functionality (namely, to test HpaC for oxidoreductase activity and to test if over-expression of KatG in the cell will increase its ability to survive H<sub>2</sub>O<sub>2</sub> stress). Transformations of all these constructions were carried out at the end of the week.</p><br />
<br />
<h2>Week 19 (September 2- September 8)</h2><br />
<p>Confirmation digests on colonies of the previous constructions that gave bands of the expected size with cPCR were performed. Positive results were found for colonies of <A HREF="http://partsregistry.org/Part:BBa_J04500"><i>P<sub>lacI</sub>-RBS</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <A HREF="http://partsregistry.org/Part:BBa_J04500"><i>P<sub>lacI</sub>-RBS</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
/<A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i></A>. Sequencing was sent, and results indicated that the constructions of <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS/hpaC</i> (BBa_K902058)</a> were successful, meaning that after many months of trying we FINALLY have a promoter in front of the <i>hpaC</i> gene and can proceed to test the parts functionality. Attempts to construct <i>hpaC</i> with the (<A HREF="http://partsregistry.org/Part:BBa_J13002">evil TetR promotor, BBa_J13002</A>) were abandoned, as it was believed that this construction was failing due to toxicity of over-expressing the protein, and it was determined that this part was not necessary after all. <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub>-RBS/dszB</i>, (BBa_K902054)</a> also worked, though this was less exciting. <a href="http://partsregistry.org/Part:BBa_K902051">BBa_B0034<br />
/<i>dszA</i></a> came back as a bad read despite looking very good on the confirmation digest gel, so this part will be resent for sequencing. Constructions of <A HREF="http://partsregistry.org/Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> were reattempted, and confirmation digests for this part looked good, and so samples were sent for sequencing.</p><br />
<br />
<br />
<h2>Week 20 (September 9- September 15)</h2><br />
<p>Construction attempts on <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> were performed. Colonies grew for the constructions, however further confirmation results were dissapointing (only 2 clones of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a> appeared to have been successful). These clones were sent for sequencing, and constructions were reattempted.</p><p> However, when sequencing came back, somehow reads indicated that these clones were in fact a gene from the Denitrogenation project (which is 990bp and a completely different band then what we saw on the gel). We believe, somewhere, something has gone very wrong- further investigation into this will be carried out. In the meantime, the above constructions were reattempted, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902051">BBa_B0034/<i>dszA</i></A> was re-prepped in case a contaminant in the plasmid stock was to blame for the bad reads found in this batch of sequencing as well as the last. In addition, plasmid switches of multiple sequence confirmed parts into a <a href=" http://partsregistry.org/Part:pSB1C3"> pSB1C3</a> backbone were carried out. </p><br />
<br />
<h2>Week 21 (September 16- September 22)</h2><br />
<p>Colonies for the transforms of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> have been few and far between, and cPCR results are always discouraging. Sequencing results for other sections of the project have once again come back very confusing, and further research continues into the source of this madness. </p><br />
<p> The sulfur compound degradation assay was set up to test the desulfurization rate of the original <i>Rhodococcus baikonurensis </i> (refer to the protocol page).</p><br />
<br />
<br />
<h2>Week 22 (September 23- September 29)</h2><br />
<p>Attempts to construct <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, , and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> continue. In the meantime, <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> was tested for functionality. In order to do this, cultures of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> were grown up overnight in LB media. A strain carrying <A HREF="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</A> only was used as a negative control. The following morning, 20 &micro;L of each culture was inoculated into 3 mL of LB with various concentrations of hydrogen peroxide; 0 mM, 1 mM, 5 mM, and 10 mM. These cultures were then allowed to grow overnight, and culture turbidity was observed. It was found that the negative control exhibited no growth after 12h at 1 mM peroxide, however cultures with induced expression of catalase were turbid after 12 h of growth at this concentration (Fig. 10). This demonstrated the ability of the catalase to protect the cells from excessive peroxide concentrations.</p><p><br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 15: Catalase Assay. Overnight cultures of J04500 and J04500-<i>KatGLAA</i> were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbitity was observed.]]<html></p><br />
</p><br />
<p>In addition to this, activity of <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-hpaC</i></a> was tested. In order to do this, cultures of <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-hpaC</i></a> and <a href="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub>-dszB</i></a> were grown up overnight in LB with appropriate antibiotics. Following this, protein expression was induced with IPTG, after which the assay was carried out as described in the following figure and on the protocols page.</p><br />
<p> </html> <br />
[[File:Ucalgary2012 DesulfurizationGroup HpaC assayTake1.png|center|600px|thumb|Figure 16: HpaC Assay. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 2 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, 1 mL of cell lysate was transferred to a cuvette, and a spectrophotometer was blanked at 340 nm (maximal absorbance of NADH) with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p>When the assay was run, it was found that NADH does not convert readily to NAD+ on its own. When cell lysate containing the naturally expressed amounts of oxidoreductase was added, a decrease in absorbance could quickly be observed as the NADH was converted to NAD+. When cultures over-expressing HpaC were tested, the absorbance levels were found to start much lower than the control. We believe that this is because with the amount of cell lysate tested, when the HpaC protein is overexpressed the NADH is consumed almost immediately and therefore the data reflecting the drop in absorbance is missed. Further tests will use differing amounts of cell lysate in order to try to capture data that shows the drop in absorbance for HpaC cultures.</p><br />
<br />
<h2>Week 23 (September 30-October 3): Wiki-Freeze PANIC!!</h2><br />
<br />
<p>The GCMS results of the sulfur assay were received. DBT desulfurization was successful (figures below). Some compounds were not detected by GCMS since they were too polar. However, 2 additional compounds showed degradation in addition to DBT, indicating that the pathway has a wider substrate specificity!</p><br />
<p></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 17: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol in the protocols section for details). Samples were taken out at different time points and were run through GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria, run through the GCMS after 6 days of being in the incubator to account for abiotic degradation. ]]<html></p<br />
<br />
<p></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 18: The peak in this mass spectra demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound ('''A:''' dibenzothiophene, '''B:''' tetrahydro-4h-thiopyran-4-one, and '''C''': benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdownn. Degradation is seen for DBT (the model studied compound) as well as other sulfur containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<p>The HpaC assay was repeated using 100 &micro;L of cell lysate instead of 1 mL of 2x concentrated cell lysate, following the previously used protocol. With this smaller amount, a clear sharp decrease in the absorbance of NADH can be observed, indicating a very fast conversion by HpaC, and further confirming that the part was functional.</p><br />
<br />
<p></html>[[File:Ucalgary2012Desulfurization-Hpacasay2.PNG|center|550px|thumb|Figure 20: HpaC Assay. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, 100 &micro;L of cell lysate was transferred to a cuvette, and a spectrophotometer was blanked at 340 nm (maximal absorbance of NADH) with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<h2>Week 24 (October 4- October 11)</h2><br />
<p>This week, before the Americas West Regional Competition, attempts to construct the previously mentioned parts continued. Unfortunately, they were unsuccessful.<br />
<br />
<h2>Week 25 (October 16- October 20)</h2><br />
<p>Traditional attempts to build the sulfur operon and its sub-parts were repeated, however attempts failed once more. During this week, it was decided that due to time consstraints, alternative approaches to construct the operon would be undertaken. Due to this, splice-overlap-extention (SOE) PCR primers (containing scar-sites where appropriate) were designed according to <b>protocol</b> and ordered.</p><br />
<p><br><br />
<br />
<br />
Primer: 1a (BBK)-J13/DszB F - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGTCCCTATCAGTGATAGAGATTGACATCCC-3'<br />
<br><br><br />
Primer: 1b (BBK)-J04/DszB F - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 2 Jxx/B-(RBS/C) R - 5'-GGTGACAGTGTCATCTAGTATTTCTCCTCTTTCTAGTACTATCGGTGGCGATTGAGGCTGTTG-3'<br />
<br><br><br />
Primer: 3 (Jxx/B)-RBS/C F - 5'-CAACAGCCTCAATCGCCACCGATAGTACTAGAAAGAGGAGAAATACTAGATGACACTGTCACC-3'<br />
<br><br><br />
Primer: 4 RBC/C-(RBS/A) R - 5'-GCCAGATGCATTTGTCGTTGTTGAGTCATCTAGTATTTCTCCTCTTTCTAGTATCAGGAGGTGAAGCCGGGAATCG-3'<br />
<br><br><br />
Primer: 5 (RBS/C)-RBS/A F - 5'-CGATTCCCGGCTTCACCTCCTGATACTAGAAAGAGGAGAAATACTAGATGACTCAACAACGACAAATGCATCTGGC-3'<br />
<br><br><br />
Primer: 6 RBS/A-(J04/HpaC) R - 5'-GGGGAGAGGCGGTTTGCGTATTGCTAGTATCATGAAGGTTGTCCTTGCAGTTGTGG-3'<br />
<br><br><br />
Primer: 7 RBS/A-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATCATGAAGGTTGTCCTTGCAGTTGTGG-3'<br />
<br><br><br />
Primer: 8 (BBK)-J04/HpaC F - 5'-CGAATTCGCGGCCGCTTCTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 9 (RBS/A)-J04/HpaC F - 5'-CCACAACTGCAAGGACAACCTTCATGATACTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 10 J04/HpaC-(RBS/Kat) R - 5'-CTGACGTGCTCATCTAGTATTTCTCCTCTTTCTAGTATTAAATCGCAGCTTCCATTTCCAGCATCAC-3'<br />
<br><br><br />
Primer: 11 J04/HpaC-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTAAATCGCAGCTTCCATTTCCAGCATCAC-3'<br />
<br><br><br />
Primer: 12 (J04/HpaC)-RBS/Kat F - 5'-GTGATGCTGGAAATGGAAGCTGCGATTTAATACTAGAAAGAGGAGAAATACTAGATGAGCACGTCAG-3'<br />
<br><br><br />
Primer: 13 RBS/Kat-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTATTAAGCAGCCAGAGCGTAGTTTTCGTC-3'<br />
</p><br><br />
<p><br />
Colony PCR was done on potential colonies of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, , and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A>. Faint bands around the expected size were seen, and these were stabbed with a pipette tip and stirred into water in order to be used as template in a further round of PCR to get amplification of the part. The plan was that these amplicons could then be used as an insert in further constructions, however no amplification was seen. Further optimization of this procedure could possibly result in a short-cut to construction, however the time needed to do this is a luxury that we do not have.</p><p>Additional attempts to create inserts by doing PCR on ligation reactions in addition to gel extraction of colony PCR bands were attempted as well, however these attempts were unsuccessful as well. SOE PCR and possible Gibson Assembly appear to be the Sulfur Teams last hope at this point, and we are eagerly awaiting our primers.<br />
</p><br />
<br />
<br />
<h2>Week 26 (October 21- October 26)</h2><br />
<p><br />
Much to our delight, SOE PCR primers were recieved this week. Therefore, alongside traditional construction of the aforementioned parts, SOE PCR was carried out according to <b>protocol</b> and these parameters:</p><br />
<br><br />
<h4>Round 1:</h4><p><br />
<ul><br />
<li>Primers 1a and 2 with <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A> as a template = Product L1 (J13002/DszB with BBk prefix and B0034/DszC overlap)<br />
<br></li><li><br />
Primers 1b and 2 with <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A> as a template = Product L2 (J04500/<i>dszB</i> with BBk prefix and B0034/<i>dszC</i> overlap)<br />
<br></li><li><br />
Primers 3 and 4 with <A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a> as a template = Product L3 (B0034/<i>dszC</i> with <i>dszB</i> and B0034/<i>dszA</i> overlap)<br />
<br></li><li><br />
Primers 5 and 6 with <A HREF="http://partsregistry.org/Part:BBa_K902050"><i>dszA</i></a> as a template = Product L4 (B0034/<i>dszA</i> with <i>dszC</i> and J04500/<i>hpaC</i> overlap)<br />
<br></li><li><br />
Primers 5 and 7 with <A HREF="http://partsregistry.org/Part:BBa_K902050"><i>dszA</i></a> as a template = Product L5 (B0034/<i>dszA</i> with <i>dszC</i> overlap and BBk suffix)<br />
<br></li><li><br />
Primers 8 and 10 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L6 (J04500/<i>hpaC</i> with BBk prefix and B0034/<i>katG-LAA</i> overlap)<br />
<br></li><li><br />
Primers 9 and 10 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L7 (J04500/<i>hpaC</i> with <i>dszA</i> and B0034/<i>katG-LAA</i> overlap)<br />
<br></li><li><br />
Primers 9 and 11 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L8 (J04500/<i>hpaC</i> with <i>dszA</i> overlap and BBk suffix)<br />
<br></li><li><br />
Primers 12 and 13 with <A HREF="http://partsregistry.org/Part:BBa_K902059"><i>RBS-katG-LAA</i></a> as a template = Product L9 (B0034/<i>katG-LAA</i> with J04500/<i>hpaC</i> overlap and BBk suffix)<br />
</li></ul><br />
</p><br />
<p><br />
</html>[[File:Ucalgary2012 Spliceoverlapextentionpcr1.png|center|thumb|800px|Figure 21: Round 1 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with varying amounts of DNA template as indicated above the wells. Cycling conditions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 55%deg;C for 1 min, 72%deg;C for 2 min. 30 s.)x 31, Stage 3 (72%deg;C for 10 min.). Bands seen at ~1500 bp for L1 through L5, ~800 bp for L6-L8, and ~2200 bp for L9 indicate successful amplification of the desired product.]]<html><br />
</p><br />
<h4>Round 2</h4><br />
<p><br />
<ul><br />
<li>Primers 1a and 4 with templates L1 and L3 = Product X1 (J13002/<i>dszB</i>/B0034/<i>dszC</i> with BBk prefix and B0034/<i>dszA</i> overlap)</li><br />
<li>Primers 1b and 4 with templates L2 and L3 = Product X2 (J04500/<i>dszB</i>/B0034/<i>dszC</i> with BBk prefix and B0034/<i>dszA</i> overlap)</li><br />
<li>Primers 3 and 6 with templates L3 and L4 = Product X3 (B0034/<i>dszC</i>/B0034/<i>dszA</i> with <i>dszB</i> and J04500/<i>hpaC</i> overlap)</li><br />
<li>Primers 3 and 7 with templates L3 and L5 = Product X4 (B0034/<i>dszC</i>/B0034/<i>dszA</i> with <i>dszB</i> overlap and BBk suffix)</li><br />
<li>Primers 5 and 10 with templates L4 and L7 = Product X5 (B0034/<i>dszA</i>/J04500/<i>hpaC</i> with <i>dszC</i> and B0034/<i>katG-LAA</i> overlap)</li><br />
<li>Primers 5 and 11 with templates L4 and L8 = Product X6 (B0034/<i>dszA</i>/J04500/<i>hpaC</i> with <i>dszC</i> overlap and BBk suffix)</li><br />
<li>Primers 8 and 13 with templates L6 and L9 = Product X7 (J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with BBk prefix and suffix)</li><br />
<li>Primers 9 and 13 with templates L7 and L9 = Product X8 (J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with <i>dszA</i> overlap and BBk suffix)</li><br />
</ul></p><p><br />
</html>[[File:Ucalgary2012 Spliceoverlapextentionpcr2.png|center|thumb|800px|Figure 22: Round 2 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with varying amounts of gel-extracted DNA template from the previous PCR round as indicated above the wells. Bands were extracted using Qiagen QIAquick Gel Extraction Kit. Cycling conditions for the PCR reactions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 65%deg;C for 1 min, 72%deg;C for 3 min. 30 s.)x 31, Stage 3 (72%deg;C for 10 min.). Faint bands seen at ~3000bp indicate amplification of the desired product.]]<html> <br />
</p><br />
<h4>Round 3</h4><br />
<p><br />
<ul><br />
<li>Primers 1a and 10 with templates X1 and X5 = Product Z1 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with B0034/<i>katG-LAA</i> overlap and BBk prefix)</li><br />
<li>Primers 1a and 11 with templates X1 and X6 = Product Z2 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with BBk prefix and suffix)</li><br />
<li>Primers 1a and 7 with templates X1 and L5 = Product Z3 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 10 with templates X2 and X5 = Product Z4 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with B0034/<i>katG-LAA</i> overlap and BBk prefix)</li><br />
<li>Primers 1b and 7 with templates X2 and L5 = Product Z5 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 11 with templates X2 and X6 = Product Z6 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 6 with templates L2 and X3 = Product Z7(J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with J04500/<i>hpaC</i> overlap and BBk prefix)</li><br />
<li>Primers 1b and 7 with templates L2 and X4 = Product Z8 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1a and 6 with templates L1 and X3 = Product Z9 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with J04500/<i>hpaC</i> overlap and BBk prefix)</li><br />
<li>Primers 1a and 7 with templates L1 and X4 = Product Z10 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 5 and 13 with templates X5 and X8 = Product Z11 (B0034/<i>dszA</i>/J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with <i>dszC</i> overlap and BBk suffix)</li><br />
</ul></p><p><br />
</html>[[File:Ucalgary2012 sulfurSpliceoverlapextentionpcr3.png|center|thumb|800px|Figure 23: Round 3 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with 50 ng of gel-extracted DNA template from the previous PCR round. Bands were extracted using Qiagen QIAquick Gel Extraction Kit. Cycling conditions for the PCR reactions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 65%deg;C for 1 min, 72%deg;C for 6 min.)x 31, Stage 3 (72%deg;C for 10 min.). A faint band (~6000bp) can be seen in lane 4, indicating possible amplification of the desired product. Other reactions appear to have failed, as this bands are not present elsewhere.]]<html> <br />
</p><br />
<p><br />
Though it appears that S.O.E. PCR consists of crushed dreams and lies (or that extensive optimization would have to be performed in order to amplify the correct product), extraction was carried out on the single band seen, in the hopes that a subsequent round of PCR with the terminal primers for this product would possibly amplify it to a concentration that can be used in construction in the next week to assemble and test the sulfur operon constructs before competition. Conveniently, the first round of PCR creates <b>Gibson Assembly</b> compatible products. Because a kit is available in the lab, this will be attempted in the following days as well. Meanwhile, a round of traditional construction appears to have finally created the parts <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902079"><i>P<sub>tetR</sub></i>-<i>dszB</i>-B0034-<i>dszC</i></A>, <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902080"><i>P<sub>lacI</sub></i>-<i>dszB</i>-B0034-<i>dszC</i></A>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902081"><i>P<sub>lacI</sub></i>-<i>hpaC</i>-B0034-<i>katG</i></A>. Though sequence confirmation could not be obtained before Wiki-Freeze, we are confident that these parts are correct due to the clear size difference seen between these parts and their sub-parts. A plasmid switch was attempted the night before DNA submission had to be sent, however due to technical issues the incubator stayed at a low temperature overnight, and cultures did not grow enough to obtain the first two parts in a pSB1C3 backbone. Because of the difficulty assembling these parts, the deadline for DNA submission, and the fact that having them in an alternate backbone in the registry would allow for easier and quicker assembly of the sulfur operon by others (no plasmid switches of large parts would have to be performed, as we have found that plasmid switching the larger constructs is quite difficult.) we decided to submit these parts in an ampicillin backbone. Further attempts to switch the backbone for these parts will continue, and hopefully we will be able to submit them in the standard backbone at a later date before the competition. In addition, a reconstruction of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902051">B0034-<i>dszA</i></A> was obtained and finally, at long last, sequence confirmed. Because of the verification of this part and the excellent sequence read when compared to those obtained for the previously submitted version, we decided to also resubmit this part to the registry.</p><p>Construction attempts of the operon will continue into the following week, as only 1 additional construction is needed to assemble a testable version of the operon. Hopefully, this data will be obtained before the competition, and documented on the respective parts pages.</p><br />
<p><br><br>This is the Desulfurization Team, signing off.</p><br />
</h><br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/DesulfurizationTeam:Calgary/Notebook/Desulfurization2012-10-26T21:47:11Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Desulfurization Journal|<br />
<br />
CONTENT=<br />
<html><br />
<h2>Week 1 (May 1-4)</h2><br />
<p>During this week, literature search was performed.</p><br />
<br />
<br />
<br />
<br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<br />
<br />
<p>Along with the rest of the team, this week was dedicated to familiarizing ourselves on the protocols that will be utilized during this years project; specifically the polymerase chain reaction, gel verification, preparation of overnight cultures, as well as developing a procedural flowchart to transform competent cells with registry biobricks. With regards to our sub-group specific goals, we reviewed the current available literature around various industrial and laboratory approaches to desulfurization of organic groups, especially in the petroleum industry. This included a comparison of non-biological processes such as conventional hydrodesulfurization, which is currently employed in petroleum product refinery stages, and how a biological approach would supplement and perhaps even offer several advantages over these methods. Current limitations to biological desulfurization, however, include such factors as biocatalyst stability, enzyme specificity and desulfurization rate. We also identified the enzyme desulfinase (DszB) as being one of the bottlenecks in the desulfurization 4S pathway. Overall, our goals moving forward involve determining the specific pathways involved in the desulfurization process as well as the reaction conditions we would want to employ, and identifying specific model compounds in addition to dibenzothiophene (DBT) that we could use to test the effectivity of our biosystem in order to determine its functionality in the conversion of naphthenic acids to economically valuable hydrocarbons.</p><br />
<br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<br />
<br />
<p> Building on the previous week's literature review, the 4S pathway was recognized as the preferred biological mechanism that we would explore in devising a desulfurization biosystem. Of specific interest is the dsz operon consisting of the genes for <i> dszA, dszB, </i>and<i> dszC</i> which selectively and non-destructively remove the sulfur from the hydrocarbon structure, and therefore preserves the carbon skeleton. In addition to these, another dsz gene exists.<i> dszD</i>, which codes for a FMN:NADH reductase, is an essential component of the pathway, but not part of the operon. Instead, it is on the genome. The enzyme produced by this gene is required to regenerate the FMNH2 consumed by the reactions carried out by DszA and DszC. <i>Rhodococcus erythropolis</i> IGTS8 is the most studied model organism in investigations of the 4S pathway, and has been shown in many different research endeavors to be capable of converting DBT to 2-HBP.</p><p><br />
</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|thumb|700px|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD. Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO2) through the addition of oxygen to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the heteroatom, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-hydroxybiphenyl. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.]]<html></p><p><br />
An alternative to the DszD gene is HpaC, an oxidoreductase encoded in the <i>E. coli</i> W genome. This enzyme has been shown to increase the rate of desulfurization. Following this, other protocols added to our growing lab methods 'toolkit' were a restriction digest protocol, PCR purification, and finally, DNA construction digest. Aims moving forward include obtaining strains of the <i> R. erythropolis </i>, while also executing a timeline devised to biobrick, test, and incorporate the genes necessary in the above processes in a biobrick circuit.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<br />
<br />
<p>This week was kicked off with a project development meeting with Emily and David, and we devised a protocol for biobricking the <i> hpaC </i> gene. Additionally, methods to place the genes coding for the 4 enzymes, DszA,B,C and HpaC into a single construct were explored. Within the lab, the PCR performed on the resuspended pUC18-hpaC was not successful initially. <br />
Furthermore, we ordered the substrates/compounds that we intend to use for desulfurization tests. Once the substrates and the <i>Rhodococcus</i> strain arrive we are going to test how effectively the bacteria can desulfurize different sulphur-containing compounds that resemble naphthenic acids. Finally, we came across a paper where a team had developed an improved efficiency DszB through site-directed mutagenesis in 2007. This was through a point mutation to the gene, converting a tyrosine at position 63 to a phenylalanine residue. A member of this team was contacted to request the plasmid that contains the mutated gene. The conversion step carried out by DszB is the major bottleneck in the 4S pathway and if a strain or sample containing this mutation was obtained, it would significantly bolster our later testing efforts on DBT, as well as other compounds such as thiophane.<br />
</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<br />
<br />
<p>Since we wanted to make sure we would not run out of pUC18(plasmid containing the hpaC gene), we transformed some E.coli cells with it. We grew them on plates containing ampicillin (A), kanamycin (K), tetracycline (T) and chloramphenicol (C) antibiotics and they only grew on A. Therefore pUC18 has A resistance. We did a three sets of PCR with primers designed against <i>hpaC</i>, one using 1/10 dilution of pUC18, the other using 1/100 dilution of pUC18 and one with the colonies we had just obtained by transforming the <i>E.coli</i> cells. </p><br />
<p>The PCR worked and we saw bands of the same size for all three sets of PCR. (Unfortunately, the picture we saved is not a good one since some of the bands faded away under UV due to prolonged exposure. Following this, PCR purification was performed to obtain the pure <i>hpaC</i> with biobrick prefix and suffix attatched to gene, which would allow us to insert the sequence into a biobrick standard backbone. 3 sets of digestion, ligation, and transformation (using pairs of X&P enzymes, E&S enzymes and E&P enzymes) were carried out in order to insert the <i>hpaC </i> gene into the <a href=" http://partsregistry.org/Part:pSB1C3">pSB1C3</a><br />
vector. All the sets grew successfully.<br />
Following the above successes with<i> hpaC</i>, the arrival of our <i>Rhodococcus</i> strain afforded us the opportunity to begin investigation of the Dsz operon using the primers currently in our possession. This strain is an environmental isolate that has been shown to be an active desulfurizer. The gram-positive nature of the strain also dictated we explore various lysing strategies before the genes encoding the Dsz enzymes could be amplified for further purification and biobrick construction steps. PCR was carried out using <i>dszA</i> primers on three different treatments {microwave, lysate buffer, and a control} which yielded banding pattern around 1200 base pairs for the lysate treatment (2%SDS and 10% tritonX-100, plus heat for 5mins at 98C).<br />
</p> <br />
<br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<br />
<br />
<p> In order to confirm the <i>hpaC</i> biobrick construction, two sets of colony PCR were performed, choosing white colonies from the 3 plates we grew last week (white colonies indicate a loss of the RFP generator in the <a href=" http://partsregistry.org/Part:pSB1C3">pSB1C3</a><br />
backbone, and therefore allow for weeding out of the colonies which are simply the original plasmid vector). These reactions were carried out both with <i>hpaC</i> primers and with standard biobrick primers designed against the plasmid backbone. After running them on the gel we saw equal bands for the PCR reactions performed using <i>hpaC</i> primers (However, a PCR using biobrick primers was performed later and the same results were obtained). Colonies 1(-) and 5(-) were used to make overnight cultures, which were then miniprepped the following day to obtain the plasmid DNA of the putative <i>hpaC</i> biobrick. Digestions were performed on the miniprep products using EcoRI and PstI to look for part size as further verification for the genes presence in the plasmid. The results were good and two bands were observed on each column (one for vector and the other for <i>hpaC</i>)). <i>hpaC</i> was sent in for sequencing. </p><br />
<p><br />
<br />
<br />
<br />
</html>[[File:UCalgary2012_04.06.2012-desulfurisation_hpacverification.jpg|thumb|700px|center|Figure 2: HpaC verification cPCR. HpaC gene was inserted into the pSB1C33 vector and E. coli cells were transformed. In order to confirm that pSB1C3 contains the hpaC gene, two sets of colony PCR's were conducted. One with biobrick primers, and the other with hpaC primers. Bands indicate successful amplification at the approximate size of the hpaC gene (517 bp)]]<br />
[[File:Ucalgary2012 06.06.2012-digestion of hpaC with E and P.jpg|thumb|500px|center|Figure 3: HpaC confirmation digest. Plasmid was miniprepped and digested for 2h with EcoRI and SpeI before being run on a 1% gel for 1h and 120V. Bands between 500 and 700 bp indicate the hpaC part is present as an insert. Bands of about 2000bp show the size of the psb1c3 vector.]]<html></p><br />
<br />
<br />
<br />
<p> PCR reagents were prepared to re-test/confirm previous results of <i>dszA</i> amplification following two different lysing treatments (microwave + lysate buffer). This time, all three genes were amplified and gel verification showed clear banding patterns around 500bp range for all three genes for the microwave treatment. Remaining PCR products were run on a gel and extracted for further purification steps; however, presence of any genetic material were not confirmed through nanodropping which raised concerns about the composition of the purified products, the success of the initial amplification step, or perhaps even the lysis treatment. Further experimentation will have to be carried out to troubleshoot.</p><br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<br />
<br />
<p> This week, we focused on amplifying <i>dsz</i> genes from our <i>Rhodococcus</i> strain for construction into biobricks. We also wanted to purify the <a href=" http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"> BBa_K902057 <i>hpaC</i></a> and pUC18-<i>hpaC</i> plasmids to replenish our current stocks. For the <i>dsz</i> aspect, we were able to successfully grow extra plates of <i>Rhodococcus</i> strain which was used to inoculate PCR tubes. The PCR did not go well, with significant streaking and false positives with similar banding pattern to previous gels run in the previous week. A final gel verification of a random sample of a tube of PCR products from dszA,B,C respectively and two negative control treatments involving master mix only and the lysed cells only illustrated the lack of discrepancy between the supposed successful amplification and the lysed cells (with lysate buffer) alone. Because of this we decided to take a different approach involving plasmid isolation carried out before PCR, rather than applying the PCR reagents directly to a lysed culture sample.</p><br />
<p> <a href=" http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> verification through sequencing was successful, confirming the construction of our first biobrick. Subsequently, O/N cultures of the plasmid containing cultures were prepared and stored in glycerol at -80C. Furthermore, verification of catalase gene part (<a href=" http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a>), which was sent as a culture stab from the parts registry was initiated. Our newly identified biobricked-hpaC was used as a positive control, but the banding pattern was not very conclusive. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<br />
<br />
<p>PCR was reattempted on <i>Rhodoccocus</i> that was lysed using two different dilutions of the lysate buffer, but the gel verification confirmed the previous failure in using this approach. An alternative that involved preparation of an overnight culture of the <i>Rhodococcus</i> cells followed by a plasmid purification was followed. The plasmid purification eventually yielded plasmid samples with concentrations of 98.6ng/μL to 182.7ng/μL (4 samples obtained overall). Additionally, the catalase biobrick was used to transform some stock competent cells, and samples of some colonies were subsequently PCR'ed. Although, the gel verification showed some potential contamination, and the required banding patterns at around 2200bp was not obtained. </p><br />
<br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<br />
<br />
<p> PCR was attempted to amplify the genes of the <I>dsz</i> operon utilising an adapted PCR protocol with purified <i>Taq</i> polymerase that had been isolated from the host organism. Eventually, some banding pattern was obtained between 1200 and 1500 base pairs when a gradient thermocycler was used with melting temperatures ranging betweeen 55°C to 65°C. This was assumed to be indicative of successful amplification of <i>dszB</i>; however, further purification and gel verification results were inconclusive and no yield was obtained when placed tested using a nanodrop machine. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<br />
<br />
<p>Top 10 E.coli cells were transformed with <a href="http://partsregistry.org/Part:BBa_R0011">BBa_R0011</a><br />
(IPTG inducible promoter in psb1C3 backbone), and resulting colonies were tested using cPCR. <br />
Colony PCR was performed on cells containing the catalase biobrick. Catalase is 2217bp long but since biobrick primers add about 200bp, bands of 2400 bp were expected if the part was present in the biobrick. These bands were observed, indicating that the <a href=" http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a> gene was most likely present.</p><br />
<p></html>[[File:Ucalgary2012 4.7.2012 catalase colony pcr 2.jpg|thumb|500px|center|Figure 4: Colony PCR on potential colonies containing katG-LAA. Biobrick primers were used. Bands at 2200 indicate successful amplification of the catalase part within the biobrick backbone. Smaller bands are indicative of primer degradation and are nonspecific amplification. +C indicates the positive control, and RFP generator, which should amplify at around 1000 bp.]]<html></p> <p><br />
<br />
PCR using Phusion high fidelity polymerase was carried out on <i>dszA</i>, <i>dszB</i>, and <i>dszC</i> in a gradient thermocycler. Amplification of non-specific bands was present for <i>dszA</i> and <i>dszB</i>, however strong banding for the desired size of the gene was observed for both (around 1500 for <i>dszA</i>, 1100 for <i>dszB</i> </p><br />
<br />
<p></html>[[File:Ucalgary2012 6.7.2012.dszABphusionPCR.jpg|thumb|500px|center|Figure 5:DszA and DszB PCR from <i>Rhodococcus</i>. DszA amplicon is around 1300 bp, and is observed to run higher on the gel. DszB amplicon is expected to be 1098 bp, which is observed in addition to multiple nonspecific banding. +C indicates the positive control, and RFP generator, which when PCRed with biobrick primers should be around 1000 bp. As this control is running high as well, it is believed that both amplicons for DszA and DszB have been obtained. No contamination is observed in the NTC (no template control).]]<html><br />
</p> <br />
<p></html>[[File:Ucalgary2012 7.5.2012 dszC.png|thumb|500px|center|Figure 6:DszC PCR from <i>Rhodococcus</i>. The numbers above the figure show the colony number. DszC amplicon is around 1450 bp, and is observed to run at almost the right size on the gel. No contamination is observed in the NTC (no template control).]]<html><br />
<p><br />
Examining the sequences of the <i>dszABC</i> genes led to the discovery that all 4 had multiple illegal enzyme cut-sites in them that we have to eliminate before biobrick composite part construction can occur. <i>dszA</i> has four PstI cut sites, <i>dszB</i> has a PstI and a NotI and <i>dszC</i> has a PstI cut site. The Stratagene QuickChange mutagenesis procedure is going to be used to eliminate illegal cut sites with the only alteration being that Kapa HiFi polymerase would be used during the process. Primers needed for the mutagenesis were designed based on the procedure mentioned above. </p><br />
<br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<br />
<p> Following successful amplification of the <i>dsz</i> operon genes in the previous week, the genes were constructed into the <a href="http://partsregistry.org/Part:pSB1C3"> pSB1C3</a> vector. Colony PCR verifications were observed to be positive. Furthermore, the insertion of part <a href="http://partsregistry.org/Part:BBa_J13002"> BBa_J13002, (P<sub>tetR</sub>-RBS)</a><br />
in front of the previously biobricked <i>hpaC</i> was attempted. Overnight cultures were also prepared using two colonies each for <a href="http://partsregistry.org/Part:BBa_J13002"> <i>P<sub>tetR</sub>-RBS</i></a> and <a href="http://partsregistry.org/Part:BBa_R0011"> <i>P<sub>lacI</sub></i> </a> (an IPTG inducible promoter that we hope to build in front of <ahref="http://partsregistry.org/Part:BBa_B0034">an RBS site, BBa_B0034</a>). These cultures were then miniprepped to yield the respective parts.</p> <br />
<br />
<p>Additionally, <a href="http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a> was built into a <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> backbone. The construction and availability of all these parts will be critical in the construction of our overall circuit for biodesulfurization. Colonies which looked good on cPCR were used to prepare overnight cultures, and were miniprepped and sent in for sequencing verification the following day.<br />
On the side, M9 minimal media was also prepared to carry out growth experimentation and overall desulfurization capability of <i>Rhodococcus</i> when exposed to DBT. The various growth treatments were M9 Media and glucose only, M9+glucose+DBT, M9+glucose+MgSO4+/-DBT, M9+glucose+MgCl2+/-DBT. 0.008g of FeCl2.4H2O was also added to each of the tubes. Samples were then inoculated with colonies of the <i>Rhodococcus</i>.</p><br />
<br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<br />
<p>This week, while awaiting sequencing verification results which were required before we could begin the construction process, the desulfurization team initially aided in some of the tasks related to the other hydrocarbon groups. The success of the construction of <a href="http://partsregistry.org/Part:BBa_J13002"><i>P<sub>tetR</sub></i>-RBS</a><br />
with <a href="http://partsregistry.org/Part:BBa_K902057"><i>hpaC</i></a> was also explored by using forward and reverse primers of <a href="http://partsregistry.org/Part:BBa_R0040">BBa_R0040</a> (the promoter component of the composite part BBa_J13002). However, the eventual gel verification was inconclusive and sequencing results finally indicated an unsuccessful ligation. Additionally, the minimal media M9 preparation had been contaminated in the previous effort so this process was repeated to create tubes of each of the growth condition treatments detailed previously, and two repeats, one with an extra filtration step and one without was used to prepare the cultures.</p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<br />
<p> Mutagenic primers were redesigned after the initial ones were found to have premature stop codons. As part of the redesign process in constructing our overall gene circuits for desulfurization, a backbone switch of <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> into a chloramphenicol (Chlor) resistant vector was necessary. The subsequent transformed products were plated on a Chlor plate and selected colonies were used to prepare O/N cultures, then minipreped before finally being digested with enzymes EcoRI and PstI. The resulting gel verification images were inconclusive as they did not show the required banding pattern around 50bp. Meanwhile, colony PCR was run on colonies transformed with <i>katG-LAA</i> constructed into a <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> backbone, as well as the <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> +<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a> construct. <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></a> was shown to have been successfully amplified, so overnight cultures were prepared and subsequently miniprepped. On the other hand, the construct was not successful so a third attempt was carried out. Colony PCR treatments that used either <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> forward primers or <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
primers were used and the overall constructs were made either on a chlor-resistant, or ampicillin-resistant vectors. Preliminary images of the gel verification appeared to have confirmed the construct, although sequencing verification will be the final indicator of overall success. </p><br />
<br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<br />
<br />
<p>Sequencing results from the previous week's constructs were available confirming that we constructed KatGLAA in a chlor-resistant backbone. However, switching the plasmid backbone of <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> to <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> was not successful. The construction of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>+<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> was finally sent in for sequencing. Site-directed mutagenesis of the <i>dsz</i> operon was also initiated: <i>dszA</i> has four PstI cut sites; <i>dszB</i> has a PstI and a NotI site; <i>dszC</i> has two PstI cut sites. Site directed mutagenesis was started this week to change a single base pair in these genes in a way that eliminates the cut site but preserves the amino acid codons, so as to not mutate the protein coding sequence. Ohshiro 2007 demonstrated that replacing the Tyr residue at position 63 of <i>dszB</i> gene with a Phe increases the activity of the enzyme. Therefore we want to introduce the same mutation into our <i>dszB</i>.</p><p><br />
For the first attempt at mutagenesis we chose to mutate the second PstI site in <i>dszC</i> (PstI2). As a positive control for the procedure, we also performed the mutagenic PCR on a plasmid containing the β-galactosidase gene with a point mutation where the PCR would cause it to regain its function. For both mutagenesis protocols we used the Kappa Hifi kit. After confirming that the PCR worked by running some produce on a gel, the PCR products were DpnI digested, the purpose of which is to degrade the unmodified parental DNA (DpnI degrades methylated DNA only). Control PCR products were plated on an ampicillin plate containing IPTG and X-gal. The colonies that grew on the control plates were blue indicating that the mutagenesis had worked for the β-galactosidase gene. Minipreps of the O/N culture of <i>dszC</i> mutants were digested with PstI enzyme and the results indicated that the mutagenesis was successful.</p><p> <br />
Attempts to simultaneously perform all the mutations in <i>dszC</i> genes in one step using the Knight procedure failed (<a href="http://openwetware.org/wiki/Knight:Site-directed_mutagenesis/Multi_site">Knight Multi-site Mutagenesis Procedure</a>). What enables simultaneous mutations is that Taq ligase closes the gaps in PCR products after each cycle. In the protocol it instructs to use Taq ligase buffer only for the PCR/ligation protocol. We suspected that the reason this procedure did not work might be that the Kappa polymerase is not functional in Taq ligase buffer. Therefore we did some experiments on the controls in Taq ligase kit and kappa polymerase kit to find out which buffer that Kappa polymerase and Taq ligase both work best in. The result was that both enzymes work best in a buffer made of half Taq ligase buffer and half Kappa polymerase buffer. </p><br />
<br />
<br />
<br />
</html>[[File:Ucalgary2012 31.7.2012 dszc mutagenesis 5 20 and 50ng.jpg|thumb|500px|center|Figure 7: DszC PstI2 mutagenesis PCR with varying concentrations of template plasmid was performed. The gel shows the PCR products that were run on a gel. +C lanes show the PCR products of the control. The control was pWhitescript™ 4.5-kb control plasmid from Stratagene mutagenesis kit which was mutated with primers also from the kit. Bands indicate successful amplification of the plasmid.]]<br />
[[File:UCalgary 02.08.12 dszC psti digest mutagenesis.jpg|thumb|700px|center|Figure 8: The control and the mutated plasmids were digested with PstI restriction enzyme. The control is dszC biobrick. Other lanes show the plasmids purified from the cells transformed with PstI2 mutagenesis PCR products followed by digestion. The control shows three bands since it has three PstI cut sites. The other lanes show two bands which indicates one of the cut sites has been eliminated through mutagenesis. ]]<html><br />
<br />
</html>[[File:Ucalgary2012 2.8.2012 finding the right buffer for multisite mutagenesis.png|thumb|800px|center|Figure 9: The multisite mutagenesis using the Knight procedure was tried in different buffers to find out the buffer that Kappa works optimally at. All the PCRs were performed on the pWhitescript™ 4.5-kb control plasmid from Stratagene mutagenesis kit which contains the beta-galactosidase gene. Based on this gel, the optimal buffer is composed of 50% kappa buffer and 50% Taq ligase buffer.]]<html><br />
<br />
</html>[[File:Ucalgary 3.8.2012 optimal buffer for Taq ligase.png|thumb|800px|center|Figure 10: Using the control provided in the NEB Taq ligase buffer (BsteII digested lambda DNA), we tried to find if the Taq ligase enzyme can function in combinations of Kappa Hifi buffer and Taq ligase buffer. Lane two is the control which is only the digested lambda DNA. Lanes 1 and 3 show that some of the bands compared to control have been ligated together. Therefore, Taq ligase functions just as good in the buffer composed of 50% kappa hifi buffer and 50% Taq ligase as it would in its own buffer.]]<html><br />
<br />
<h2>Week 15 (August 6 - August 11)</h2><br />
<br />
<p>Sequencing results for <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A> <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> returned negative, so a 3-part ligation method was used to retry this construction. The following parts were ligated with the restriction enzymes indicated in brackets after each: <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>(EcoRI/SpeI) + <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> (XbaI/PstI) + <A HREF=" http://partsregistry.org/Part:PSB1K3">BBa_PSB1K3</A> (EcoRI/PstI). Also, the more conventional construction (only 1 insert) of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>(SpeI/PstI) + <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> (XbaI/PstI) was reattempted. Furthermore, 3-way ligations were also attempted for <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
+<A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A>+<A HREF="http://partsregistry.org/Part:PSB1K3">BBa_PSB1K3</A>, and <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a>+<a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
+<A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A>, as well as the two-way contruction of just <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> after the <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a>. After plating these transformations, colony PCRs were carried out and samples that gave an indication of being successful on the gels were used to prepare O/N cultures followed by miniprep. With regards to the site-directed mutagenesis side of the experimentation, <i>dszA</i>-PstI1 (the first PstI cut site in <i>dszA</i>) ,<i> dszB</i>-PstI and <i>dszC</i>(PstI2 mutated)-PstI1 mutagenesis were performed following the procedure explained in the previous week. The gel below shows the successful result of digest confirmation (Fig. 8). <br />
Multisite mutagenesis (Knight method) was repeated using the modified buffer (half Taq ligase buffer and half Kappa buffer). However it was not successful again. We also tried doing multisite mutagenesis using Pfu Turbo polymerase and following the Knight procedure without any buffer modifications. No successful results were observed. </p><br />
<br />
<br />
<br />
</html>[[File:Ucalgary2012 Digestion confirmation of mutagenesis in dszAPstI1, dszBPstI and dszCPstI1 (PstI2 site mutated)..jpg|thumb|800px|center|Figure 11: Confirmation of site-directed mutagensis to eliminate PstI cut sites in: <i>dszA</i>PstI1 cut site, <i>dszB</i>PstI cut site and <i>dszC</i>PstI1 (PstI2 site mutated). Note that in each case, the unmutated controls for each gene are to the left of each set of plasmids derived from mutagenic PCRs. Plasmids from cloned <i>dsz</i> genes are smaller than the bands to the right of them (which have undergone mutagenesis), indicating that in the plasmids on the right, the PstI cut site was lost. ]]<html><br />
<br />
<br />
<h2>Week 16 (August 12 - August 18)</h2><br />
<br />
<br />
<p>The progress in mutagenesis of <i>dsz</i> genes was continued from the previous week: <i>dszB</i>(PstI mutated)-Y63F and <i>dszA</i>(PstI1 mutated)-PstI3 mutagenesis. The gel below shows the digest confirmation.</p><br />
<br />
<br />
</html><br />
[[File:Ucalgary2012 15.08.2012 dszAPstI1&amp;3 dszB Psti y63f muta diges-1.jpg|thumb|800px|center|Figure 12: Digestion confirmation of biobricks of a) dszA (PstI1 mutated) PstI3 mutagenesis and b) dszB (PstI mutated) Y63F mutagenesis. Cutting dszA (PstI1 mutated) with PstI is expected to produce fragments of 2790, 333, 255, and 114bp. Cutting dszA (PstI1 and PstI3 mutated) is expected to produce fragments of 2730, 588, and 114bp. The primers for dszB-Y63F mutagenesis introduce an HpyAV cut site. pSB1C3 inteslf has two more HpyAV cut sites. Before the mutation bands of 2858 abd 310bp are expected, and after successful mutation bands of 1502, 1356, 310 are expected. Lane legend: 1- dszA (PstI1 mutated). 2-dszA (PstI1 mutated) digested with PstI. 3-dszA (PstI1 mutated) PstI3 mutagenesis c1 Digested with PstI. 4- dszA (PstI1 mutated) PstI3 mutagenesis c2 Digested with PstI. 5-dszA (PstI1 mutated) PstI3 mutagenesis c3 Digested with PstI. 6- dszA (PstI1 mutated) PstI3 mutagenesis c4 Digested with PstI. 7- Fermentas 1kb Plus Ladder. 8- Empty. 9- dszB (PstI mutated). 10-dszB (PstI mutated) digested with HpyAV. 11- dszB (PstI mutated) Y63F mutagenesis c1 digested with HpyAV. 12- dszB (PstI mutated) Y63F mutagenesis c2 digested with HpyAV. 13- dszB (PstI mutated) Y63F mutagenesis c3 digested with HpyAV. 14-dszB (PstI mutated) Y63F mutagenesis c4 digested with HpyAV.]]<html></p><br />
<br />
<p>We attempted a different approach to speed up the turnover time of the mutagenesis PCR. Briefly, after the PCR mutagenesis the PCR products were purified and then incubated with T4 polynucleotide kinase (PNK) and ligase. After heat inactivating the ligase and T4 PNK, the products were DpnI digested. Subsequently another round of DNA purification was performed. However, the results were unsatisfactory after the digest confirmation.</p><p><br />
Sequencing results came back. <i>dszA</i> (PstI1 and PstI3 mutated) and <i>dszB</i>(PstI and Y63F mutated) were good. However <i>dszC</i> (PstI1 and PstI2 mutated) had an insertion next to the PstI1 cut site. Mutagenesis was repeated on the <i>dszC</i>(PstI2 mutated).<br />
<i>dszB</i>(PstI and Y63F mutated)-NotI and <i>dszA</i>(PstI1 and PstI3 mutated)-PstI4 mutagenesis were also performed.</p><br />
<p> To investigate the desulfurisation capability of the <i>Rhodococcus</i> sp. from which we cloned the <i>dsz</i> operon, a desulfurization assay was prepared by inoculating different treatments of M9 media. We also prepared some solutions that will be needed for analysis in the following week: a conditioning agent composed of 100ml of 95% ethanol, 50ml glycerol, 30ml of 12M HCl (aq) and 70g of NaCl(s) was prepared. The assay relies on the turbidity of a sample containing sulphate ions which are precipitated (hence the turbidometric nature of the assay) upon adding BaCl2(s), therefore if the <i>dsz</i> pathway is active, we expect a more turbid solution to form than in control samples. </p><br />
<br />
<br />
<br />
<h2>Week 17 (August 19 - August 25)</h2><br />
<br />
<br />
<p>This week, progress was made in determining the desulfurization activity of our <i>Rhodococcus</i> strain as measured by the sulfate release using a turbidometric assay. We encountered several challenges in our prescribed protocol as the concentrations that we used to prepare the standard curve may have been too dilute, or the composition of out conditioning agent may have been flawed. Additionally, steps were taken to determine the decomposition of DBT to 2-HBP through Gas Chromatograph-Mass Spectroscopy (GC-MS) analysis, but due to a preparation error, the DBT was added to a growth solution of M9 media prematurely and the autoclaving process decomposed the DBT releasing a yellow colouration into the solution. These two approaches in determining the desulfurization capability of the <i>dsz</i> operon will be further investigated. </p><br />
<br />
<p>Since the <i>dszC</i> second mutagenesis had proven to be unsuccessful last week, the <i>dszC</i>(PstI2 mutated)PstI1 mutagenesis was repeated. Also <i>dszA</i>(PstI1,3,4 mutated) PstI2 mutagenesis was performed. <i>dszA</i> and <i>dszC</i> were sent for sequencing on Wednesday. <i>dszB</i> was sent for sequencing on Friday. Sequencing results of <i>dszA</i> and <i>dszC</i> were back by Friday. <i>dszC</i> was successful. However, <i>dszA</i> contained an insertion next to the binding site of PstI4 cut sit, so the last two mutations must be redone. <i>dszB</i>(PstI and Y63F mutated)-NotI-mutagenesis was also repeated in case the result of the sequencing was not successful. These constructions were repeated. <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>-<A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
-<i>dszC</i> constructions were attempted, however they were not successful as indicated by colony PCR. Constructions of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>/<i>hpaC</i> were carried out and also came back negative in sequencing, however <a href="http://partsregistry.org/Part:BBa_K902059">BBa_B0034<br />
/<i>katG-LAA</i> (BBa_K902059)</a> was sequence confirmed.</p><br />
</html>[[File:Ucalgary2012 20.8.2012 dszC(psti2)psti1 mutagenesis.png|thumb|700px|center|Figure 13: Another attempt to mutagenize dszC(PstI2 mutated)at PstI1 site. The gel shows the confirmation digestion. C is the control which is dszC(PstI2 mutated). Since the control has two PstI cut sites, two bands are observed on the gel. The mutagenesis has been successful since only one band is observed on the other lanes indicating that one of the cut sites has been eliminated during mutagenesis.]]<html><br />
<br />
</html>[[File:Ucalgary2012 21.8.2012 dszA final mutagenesis digest.png|thumb|700px|center|Figure 14: DszA (PstI1, PstI2, PstI3 mutated)PstI4 mutagenesis digestion confirmation gel. All the lanes show the results of the plasmids being cut with PstI enzyme. C indicates the lane containing the control which is dszA(PstI1, PstI3 and PstI4 mutated). The other lanes are the digestions of the plasmids that are mutagenized, from different colonies. Control plasmid has two PstI cut sites and therefore the two bands observed on the gel were expected. The other lanes show only one band of about 3500bp which is about the right size for dszA in a psb1c3 vector. Therefore one of the cutsites has been eliminated during mutagenesis.]]<html><br />
<br />
<br />
<br />
<br />
<h2>Week 18 (August 26 - September 1)</h2><br />
<br />
<p> <A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i> (BBa_K902052)</i></A> sequencing results came back as successful. <i>dszA</i>(PstI1,3 mutated)-PstI2-mutagenesis was performed and sent for sequencing. Also <i>dszA</i>(PstI1,2,3 mutated)-PstI4-mutagenesis was performed, and this was also sent for sequencing. </p><br />
<p>Constructions of <A HREF="http://partsregistry.org/Part:BBa_K902053">P<sub>tetR</sub>-RBS/<i>dszB</i> (BBa_K902053)</a> and <a href="http://partsregistry.org/Part:BBa_K902056">BBa_B0034<br />
/<i>dszC</i> (BBa_K902056)</a> were attempted, verification digested, and sent for sequencing. Sequencing results for these constructs came back as positive, along with successful mutagenesis of <A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i> (BBa_K902050)</A>.</p><br />
<p>At this point, all of the <i>dsz</i> genes have been successfully made biobrick compatible, and <i>hpaC</i> has been biobricked. We have also successfully constructed <a href="http://partsregistry.org/Part:BBa_K902059">BBa_B0034 with <i>katG-LAA</i></a> to be used in the optimization circuit, as well as <A HREF="http://partsregistry.org/Part:BBa_K902053"><i>P<sub>tetR</sub>-RBS</i>/<i>dszB</i> (BBa_K902053) </a>and <a href="http://partsregistry.org/Part:BBa_K902056">BBa_B0034/<i>dszC</i> (BBa_K902056)</a>.<br />
<p> Constructions of <A HREF="http://partsregistry.org/Part:BBa_J04500">P<sub>lacI</sub>-RBS</A>with <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> were performed. As well, attempts to construct <A HREF="http://partsregistry.org/Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></a>/<a href="http://partsregistry.org/Part:BBa_K902056">RBS-<i>dszC</i></a> as well as <A HREF="http://partsregistry.org/Part:BBa_J13002">P<sub>tetR</sub>-RBS</A>/<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
/<A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i></A>, and <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>/<A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> were also carried out. These parts are intended as construction intermediates towards building the final systems, as well as providing a way of testing the genes functionality (namely, to test HpaC for oxidoreductase activity and to test if over-expression of KatG in the cell will increase its ability to survive H<sub>2</sub>O<sub>2</sub> stress). Transformations of all these constructions were carried out at the end of the week.</p><br />
<br />
<h2>Week 19 (September 2- September 8)</h2><br />
<p>Confirmation digests on colonies of the previous constructions that gave bands of the expected size with cPCR were performed. Positive results were found for colonies of <A HREF="http://partsregistry.org/Part:BBa_J04500"><i>P<sub>lacI</sub>-RBS</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <A HREF="http://partsregistry.org/Part:BBa_J04500"><i>P<sub>lacI</sub>-RBS</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
/<A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i></A>. Sequencing was sent, and results indicated that the constructions of <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS/hpaC</i> (BBa_K902058)</a> were successful, meaning that after many months of trying we FINALLY have a promoter in front of the <i>hpaC</i> gene and can proceed to test the parts functionality. Attempts to construct <i>hpaC</i> with the (<A HREF="http://partsregistry.org/Part:BBa_J13002">evil TetR promotor, BBa_J13002</A>) were abandoned, as it was believed that this construction was failing due to toxicity of over-expressing the protein, and it was determined that this part was not necessary after all. <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub>-RBS/dszB</i>, (BBa_K902054)</a> also worked, though this was less exciting. <a href="http://partsregistry.org/Part:BBa_K902051">BBa_B0034<br />
/<i>dszA</i></a> came back as a bad read despite looking very good on the confirmation digest gel, so this part will be resent for sequencing. Constructions of <A HREF="http://partsregistry.org/Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> were reattempted, and confirmation digests for this part looked good, and so samples were sent for sequencing.</p><br />
<br />
<br />
<h2>Week 20 (September 9- September 15)</h2><br />
<p>Construction attempts on <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> were performed. Colonies grew for the constructions, however further confirmation results were dissapointing (only 2 clones of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a> appeared to have been successful). These clones were sent for sequencing, and constructions were reattempted.</p><p> However, when sequencing came back, somehow reads indicated that these clones were in fact a gene from the Denitrogenation project (which is 990bp and a completely different band then what we saw on the gel). We believe, somewhere, something has gone very wrong- further investigation into this will be carried out. In the meantime, the above constructions were reattempted, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902051">BBa_B0034/<i>dszA</i></A> was re-prepped in case a contaminant in the plasmid stock was to blame for the bad reads found in this batch of sequencing as well as the last. In addition, plasmid switches of multiple sequence confirmed parts into a <a href=" http://partsregistry.org/Part:pSB1C3"> pSB1C3</a> backbone were carried out. </p><br />
<br />
<h2>Week 21 (September 16- September 22)</h2><br />
<p>Colonies for the transforms of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> have been few and far between, and cPCR results are always discouraging. Sequencing results for other sections of the project have once again come back very confusing, and further research continues into the source of this madness. </p><br />
<p> The sulfur compound degradation assay was set up to test the desulfurization rate of the original <i>Rhodococcus baikonurensis </i> (refer to the protocol page).</p><br />
<br />
<br />
<h2>Week 22 (September 23- September 29)</h2><br />
<p>Attempts to construct <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, , and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> continue. In the meantime, <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> was tested for functionality. In order to do this, cultures of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> were grown up overnight in LB media. A strain carrying <A HREF="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</A> only was used as a negative control. The following morning, 20 &micro;L of each culture was inoculated into 3 mL of LB with various concentrations of hydrogen peroxide; 0 mM, 1 mM, 5 mM, and 10 mM. These cultures were then allowed to grow overnight, and culture turbidity was observed. It was found that the negative control exhibited no growth after 12h at 1 mM peroxide, however cultures with induced expression of catalase were turbid after 12 h of growth at this concentration (Fig. 10). This demonstrated the ability of the catalase to protect the cells from excessive peroxide concentrations.</p><p><br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 15: Catalase Assay. Overnight cultures of J04500 and J04500-<i>KatGLAA</i> were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbitity was observed.]]<html></p><br />
</p><br />
<p>In addition to this, activity of <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-hpaC</i></a> was tested. In order to do this, cultures of <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-hpaC</i></a> and <a href="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub>-dszB</i></a> were grown up overnight in LB with appropriate antibiotics. Following this, protein expression was induced with IPTG, after which the assay was carried out as described in the following figure and on the protocols page.</p><br />
<p> </html> <br />
[[File:Ucalgary2012 DesulfurizationGroup HpaC assayTake1.png|center|600px|thumb|Figure 16: HpaC Assay. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 2 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, 1 mL of cell lysate was transferred to a cuvette, and a spectrophotometer was blanked at 340 nm (maximal absorbance of NADH) with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p>When the assay was run, it was found that NADH does not convert readily to NAD+ on its own. When cell lysate containing the naturally expressed amounts of oxidoreductase was added, a decrease in absorbance could quickly be observed as the NADH was converted to NAD+. When cultures over-expressing HpaC were tested, the absorbance levels were found to start much lower than the control. We believe that this is because with the amount of cell lysate tested, when the HpaC protein is overexpressed the NADH is consumed almost immediately and therefore the data reflecting the drop in absorbance is missed. Further tests will use differing amounts of cell lysate in order to try to capture data that shows the drop in absorbance for HpaC cultures.</p><br />
<br />
<h2>Week 23 (September 30-October 3): Wiki-Freeze PANIC!!</h2><br />
<br />
<p>The GCMS results of the sulfur assay were received. DBT desulfurization was successful (figures below). Some compounds were not detected by GCMS since they were too polar. However, 2 additional compounds showed degradation in addition to DBT, indicating that the pathway has a wider substrate specificity!</p><br />
<p></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 17: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol in the protocols section for details). Samples were taken out at different time points and were run through GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria, run through the GCMS after 6 days of being in the incubator to account for abiotic degradation. ]]<html></p<br />
<br />
<p></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 18: The peak in this mass spectra demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound ('''A:''' dibenzothiophene, '''B:''' tetrahydro-4h-thiopyran-4-one, and '''C''': benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdownn. Degradation is seen for DBT (the model studied compound) as well as other sulfur containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<p>The HpaC assay was repeated using 100 &micro;L of cell lysate instead of 1 mL of 2x concentrated cell lysate, following the previously used protocol. With this smaller amount, a clear sharp decrease in the absorbance of NADH can be observed, indicating a very fast conversion by HpaC, and further confirming that the part was functional.</p><br />
<br />
<p></html>[[File:Ucalgary2012Desulfurization-Hpacasay2.PNG|center|550px|thumb|Figure 20: HpaC Assay. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, 100 &micro;L of cell lysate was transferred to a cuvette, and a spectrophotometer was blanked at 340 nm (maximal absorbance of NADH) with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<h2>Week 24 (October 4- October 11)</h2><br />
<p>This week, before the Americas West Regional Competition, attempts to construct the previously mentioned parts continued. Unfortunately, they were unsuccessful.<br />
<br />
<h2>Week 25 (October 16- October 20)</h2><br />
<p>Traditional attempts to build the sulfur operon and its sub-parts were repeated, however attempts failed once more. During this week, it was decided that due to time consstraints, alternative approaches to construct the operon would be undertaken. Due to this, splice-overlap-extention (SOE) PCR primers (containing scar-sites where appropriate) were designed according to <b>protocol</b> and ordered.</p><br />
<p><br><br />
<br />
<br />
Primer: 1a (BBK)-J13/DszB F - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGTCCCTATCAGTGATAGAGATTGACATCCC-3'<br />
<br><br><br />
Primer: 1b (BBK)-J04/DszB F - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 2 Jxx/B-(RBS/C) R - 5'-GGTGACAGTGTCATCTAGTATTTCTCCTCTTTCTAGTACTATCGGTGGCGATTGAGGCTGTTG-3'<br />
<br><br><br />
Primer: 3 (Jxx/B)-RBS/C F - 5'-CAACAGCCTCAATCGCCACCGATAGTACTAGAAAGAGGAGAAATACTAGATGACACTGTCACC-3'<br />
<br><br><br />
Primer: 4 RBC/C-(RBS/A) R - 5'-GCCAGATGCATTTGTCGTTGTTGAGTCATCTAGTATTTCTCCTCTTTCTAGTATCAGGAGGTGAAGCCGGGAATCG-3'<br />
<br><br><br />
Primer: 5 (RBS/C)-RBS/A F - 5'-CGATTCCCGGCTTCACCTCCTGATACTAGAAAGAGGAGAAATACTAGATGACTCAACAACGACAAATGCATCTGGC-3'<br />
<br><br><br />
Primer: 6 RBS/A-(J04/HpaC) R - 5'-GGGGAGAGGCGGTTTGCGTATTGCTAGTATCATGAAGGTTGTCCTTGCAGTTGTGG-3'<br />
<br><br><br />
Primer: 7 RBS/A-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATCATGAAGGTTGTCCTTGCAGTTGTGG-3'<br />
<br><br><br />
Primer: 8 (BBK)-J04/HpaC F - 5'-CGAATTCGCGGCCGCTTCTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 9 (RBS/A)-J04/HpaC F - 5'-CCACAACTGCAAGGACAACCTTCATGATACTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 10 J04/HpaC-(RBS/Kat) R - 5'-CTGACGTGCTCATCTAGTATTTCTCCTCTTTCTAGTATTAAATCGCAGCTTCCATTTCCAGCATCAC-3'<br />
<br><br><br />
Primer: 11 J04/HpaC-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTAAATCGCAGCTTCCATTTCCAGCATCAC-3'<br />
<br><br><br />
Primer: 12 (J04/HpaC)-RBS/Kat F - 5'-GTGATGCTGGAAATGGAAGCTGCGATTTAATACTAGAAAGAGGAGAAATACTAGATGAGCACGTCAG-3'<br />
<br><br><br />
Primer: 13 RBS/Kat-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTATTAAGCAGCCAGAGCGTAGTTTTCGTC-3'<br />
</p><br><br />
<p><br />
Colony PCR was done on potential colonies of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, , and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A>. Faint bands around the expected size were seen, and these were stabbed with a pipette tip and stirred into water in order to be used as template in a further round of PCR to get amplification of the part. The plan was that these amplicons could then be used as an insert in further constructions, however no amplification was seen. Further optimization of this procedure could possibly result in a short-cut to construction, however the time needed to do this is a luxury that we do not have.</p><p>Additional attempts to create inserts by doing PCR on ligation reactions in addition to gel extraction of colony PCR bands were attempted as well, however these attempts were unsuccessful as well. SOE PCR and possible Gibson Assembly appear to be the Sulfur Teams last hope at this point, and we are eagerly awaiting our primers.<br />
</p><br />
<br />
<br />
<h2>Week 26 (October 21- October 26)</h2><br />
<p><br />
Much to our delight, SOE PCR primers were recieved this week. Therefore, alongside traditional construction of the aforementioned parts, SOE PCR was carried out according to <b>protocol</b> and these parameters:</p><br />
<br><br />
<h4>Round 1:</h4><p><br />
<ul><br />
<li>Primers 1a and 2 with <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A> as a template = Product L1 (J13002/DszB with BBk prefix and B0034/DszC overlap)<br />
<br></li><li><br />
Primers 1b and 2 with <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A> as a template = Product L2 (J04500/<i>dszB</i> with BBk prefix and B0034/<i>dszC</i> overlap)<br />
<br></li><li><br />
Primers 3 and 4 with <A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a> as a template = Product L3 (B0034/<i>dszC</i> with <i>dszB</i> and B0034/<i>dszA</i> overlap)<br />
<br></li><li><br />
Primers 5 and 6 with <A HREF="http://partsregistry.org/Part:BBa_K902050"><i>dszA</i></a> as a template = Product L4 (B0034/<i>dszA</i> with <i>dszC</i> and J04500/<i>hpaC</i> overlap)<br />
<br></li><li><br />
Primers 5 and 7 with <A HREF="http://partsregistry.org/Part:BBa_K902050"><i>dszA</i></a> as a template = Product L5 (B0034/<i>dszA</i> with <i>dszC</i> overlap and BBk suffix)<br />
<br></li><li><br />
Primers 8 and 10 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L6 (J04500/<i>hpaC</i> with BBk prefix and B0034/<i>katG-LAA</i> overlap)<br />
<br></li><li><br />
Primers 9 and 10 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L7 (J04500/<i>hpaC</i> with <i>dszA</i> and B0034/<i>katG-LAA</i> overlap)<br />
<br></li><li><br />
Primers 9 and 11 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L8 (J04500/<i>hpaC</i> with <i>dszA</i> overlap and BBk suffix)<br />
<br></li><li><br />
Primers 12 and 13 with <A HREF="http://partsregistry.org/Part:BBa_K902059"><i>RBS-katG-LAA</i></a> as a template = Product L9 (B0034/<i>katG-LAA</i> with J04500/<i>hpaC</i> overlap and BBk suffix)<br />
</li></ul><br />
</p><br />
<p><br />
</html>[[File:Ucalgary2012 Spliceoverlapextentionpcr1.png|center|thumb|800px|Figure 21: Round 1 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with varying amounts of DNA template as indicated above the wells. Cycling conditions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 55%deg;C for 1 min, 72%deg;C for 2 min. 30 s.)x 31, Stage 3 (72%deg;C for 10 min.). Bands seen at ~1500 bp for L1 through L5, ~800 bp for L6-L8, and ~2200 bp for L9 indicate successful amplification of the desired product.]]<html><br />
</p><br />
<h4>Round 2</h4><br />
<p><br />
<ul><br />
<li>Primers 1a and 4 with templates L1 and L3 = Product X1 (J13002/<i>dszB</i>/B0034/<i>dszC</i> with BBk prefix and B0034/<i>dszA</i> overlap)</li><br />
<li>Primers 1b and 4 with templates L2 and L3 = Product X2 (J04500/<i>dszB</i>/B0034/<i>dszC</i> with BBk prefix and B0034/<i>dszA</i> overlap)</li><br />
<li>Primers 3 and 6 with templates L3 and L4 = Product X3 (B0034/<i>dszC</i>/B0034/<i>dszA</i> with <i>dszB</i> and J04500/<i>hpaC</i> overlap)</li><br />
<li>Primers 3 and 7 with templates L3 and L5 = Product X4 (B0034/<i>dszC</i>/B0034/<i>dszA</i> with <i>dszB</i> overlap and BBk suffix)</li><br />
<li>Primers 5 and 10 with templates L4 and L7 = Product X5 (B0034/<i>dszA</i>/J04500/<i>hpaC</i> with <i>dszC</i> and B0034/<i>katG-LAA</i> overlap)</li><br />
<li>Primers 5 and 11 with templates L4 and L8 = Product X6 (B0034/<i>dszA</i>/J04500/<i>hpaC</i> with <i>dszC</i> overlap and BBk suffix)</li><br />
<li>Primers 8 and 13 with templates L6 and L9 = Product X7 (J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with BBk prefix and suffix)</li><br />
<li>Primers 9 and 13 with templates L7 and L9 = Product X8 (J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with <i>dszA</i> overlap and BBk suffix)</li><br />
</ul></p><p><br />
</html>[[File:Ucalgary2012 Spliceoverlapextentionpcr2.png|center|thumb|800px|Figure 22: Round 2 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with varying amounts of gel-extracted DNA template from the previous PCR round as indicated above the wells. Bands were extracted using Qiagen QIAquick Gel Extraction Kit. Cycling conditions for the PCR reactions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 65%deg;C for 1 min, 72%deg;C for 3 min. 30 s.)x 31, Stage 3 (72%deg;C for 10 min.). Faint bands seen at ~3000bp indicate amplification of the desired product.]]<html> <br />
</p><br />
<h4>Round 3</h4><br />
<p><br />
<ul><br />
<li>Primers 1a and 10 with templates X1 and X5 = Product Z1 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with B0034/<i>katG-LAA</i> overlap and BBk prefix)</li><br />
<li>Primers 1a and 11 with templates X1 and X6 = Product Z2 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with BBk prefix and suffix)</li><br />
<li>Primers 1a and 7 with templates X1 and L5 = Product Z3 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 10 with templates X2 and X5 = Product Z4 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with B0034/<i>katG-LAA</i> overlap and BBk prefix)</li><br />
<li>Primers 1b and 7 with templates X2 and L5 = Product Z5 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 11 with templates X2 and X6 = Product Z6 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 6 with templates L2 and X3 = Product Z7(J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with J04500/<i>hpaC</i> overlap and BBk prefix)</li><br />
<li>Primers 1b and 7 with templates L2 and X4 = Product Z8 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1a and 6 with templates L1 and X3 = Product Z9 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with J04500/<i>hpaC</i> overlap and BBk prefix)</li><br />
<li>Primers 1a and 7 with templates L1 and X4 = Product Z10 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 5 and 13 with templates X5 and X8 = Product Z11 (B0034/<i>dszA</i>/J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with <i>dszC</i> overlap and BBk suffix)</li><br />
</ul></p><p><br />
</html>[[File:Ucalgary2012 sulfurSpliceoverlapextentionpcr3.png|center|thumb|800px|Figure 23: Round 3 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with 50 ng of gel-extracted DNA template from the previous PCR round. Bands were extracted using Qiagen QIAquick Gel Extraction Kit. Cycling conditions for the PCR reactions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 65%deg;C for 1 min, 72%deg;C for 6 min.)x 31, Stage 3 (72%deg;C for 10 min.). A faint band (~6000bp) can be seen in lane 4, indicating possible amplification of the desired product. Other reactions appear to have failed, as this bands are not present elsewhere.]]<html> <br />
</p><br />
<p><br />
Though it appears that S.O.E. PCR consists of crushed dreams and lies (or that extensive optimization would have to be performed in order to amplify the correct product), extraction was carried out on the single band seen, in the hopes that a subsequent round of PCR with the terminal primers for this product would possibly amplify it to a concentration that can be used in construction in the next week to assemble and test the sulfur operon constructs before competition. Conveniently, the first round of PCR creates <b>Gibson Assembly</b> compatible products. Because a kit is available in the lab, this will be attempted in the following days as well. Meanwhile, a round of traditional construction appears to have finally created the parts <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902079"><i>P<sub>tetR</sub></i>-<i>dszB</i>-B0034-<i>dszC</i></A>, <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902080"><i>P<sub>lacI</sub></i>-<i>dszB</i>-B0034-<i>dszC</i></A>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902081"><i>P<sub>lacI</sub></i>-<i>hpaC</i>-B0034-<i>katG</i></A>. Though sequence confirmation could not be obtained before Wiki-Freeze, we are confident that these parts are correct due to the clear size difference seen between these parts and their sub-parts. A plasmid switch was attempted the night before DNA submission had to be sent, however due to technical issues the incubator stayed at a low temperature overnight, and cultures did not grow enough to obtain the first two parts in a pSB1C3 backbone. Because of the difficulty assembling these parts, the deadline for DNA submission, and the fact that having them in an alternate backbone in the registry would allow for easier and quicker assembly of the sulfur operon by others (no plasmid switches of large parts would have to be performed, as we have found that plasmid switching the larger constructs is quite difficult.) we decided to submit these parts in an ampicillin backbone. Further attempts to switch the backbone for these parts will continue, and hopefully we will be able to submit them in the standard backbone at a later date before the competition. In addition, a reconstruction of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902051">B0034-<i>dszA</i></A> was obtained and finally, at long last, sequence confirmed. Because of the verification of this part and the excellent sequence read when compared to those obtained for the previously submitted version, we decided to also resubmit this part to the registry.</p><p>Construction attempts of the operon will continue into the following week, as only 1 additional construction is needed to assemble a testable version of the operon. Hopefully, this data will be obtained before the competition, and documented on the respective parts pages.</p><br />
<p><br><br>This is the Desulfurization Team, signing off.</p><br />
</h><br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/PromoterScreenTeam:Calgary/Notebook/PromoterScreen2012-10-26T21:45:10Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookGreen|<br />
TITLE=Transposon Library Notebook|<br />
CONTENT =<br />
<html><br />
<!--<br />
NOTE: This is a template for entering things for the time being. All dates should be enclosed in <h2> tags and all paragraphs should be enclosed in <p> tags. For bulleted lists, <ul> tags will create the list and <li> tags will surround each list item. If there are any questions, please let me know.<br />
<br />
Patrick.<br />
--><br />
<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>&nbsp;&nbsp;This was the first week where we met with other team members and summarized the primary subprojects the team will be tackling this coming summer.</p><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>&nbsp;&nbsp;During this week literature searches were performed.</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>&nbsp;&nbsp;During this week literature searches were performed.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>&nbsp;&nbsp;During this week, strains of <i> Pseudomonas fluorescens</i> PF-5 were obtained. Two cultures were started by adding 500 &micro;L stock to 10 mL LB media containing 50 mg/L ACROS Naphthenic Acids. These cultures were grown at 30&deg;C overnight, shaking at 110 rpm.</p><br />
<p>&nbsp;&nbsp;Overnight cultures were then streaked on LB agar the following day with various types and concentrations of antibiotics in order to determine the susceptibility profile of the organism. This was necessary in order to determine what marker could be used on a transposon to allow for selection of organisms with sucessful transposon insertions. These plates were grown overnight to look for death or growth, and the following results were obtained; </p><br />
<br />
<center><table><br />
<table border="2" CELLPADDING=3 CELLSPACING=1 <br />
RULES=COLS FRAME=VSIDES><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Gentamycin</b></td><br />
<td><b>Kanamycin</b></td><br />
<td><b>Chloramphenicol</b></td><br />
<td><b>Tetracycline</b></td><br />
</tr><br />
<tr><br />
<td>25 &micro;g/ml = no growth</td><br />
<td>5 &micro;g/ml = slight growth</td><br />
<td>5 &micro;g/ml = growth</td><br />
<td>50 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td>50 &micro;g/ml = no growth</td><br />
<td>10 &micro;g/ml = slight growth</td><br />
<td>10 &micro;g/ml = growth</td><br />
<td>100 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td>100 &micro;g/ml = no growth</td><br />
<td>25 &micro;g/ml = no growth</td><br />
<td>25 &micro;g/ml = growth</td><br />
<td>200 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td>50 &micro;g/ml = no growth</td><br />
<td>50 &micro;g/ml = growth</td><br />
<td> </td><br />
</tr><br />
</table></center><br />
<br />
<br />
<p><br />
<br></br> &nbsp;&nbsp;Based on these results, it was determined that Kanamycin, Gentamycin, and Tetracycline could be used as the selectable marker on the transposon, while Chloramphenicol could not as the strain is naturally resistant. Glycerol stocks of the strains were also made at this time from a fresh overnight culture.</p><br />
<br><br><br />
<h2>Week 5 (May 28-June 1)</h2><br />
<p>&nbsp;&nbsp;At the beginning of this week we spoke to Dr. Michael Hynes, who was able to give us <i> E. coli </i> SM10 and SM17-1 cells containing the plasmid pOT182. This plasmid contains an <i> E. coli </i> origin of replication, allowing it to act as a suicide vector when transferred to a different bacterial species. pOT182 contains a Tn5 transposon element containing a promotorless <i>lacZ</i> gene, genes for tetracycline resistance as well as a beta-lactamase, transposase and an <i>E. coli</i> origin of replication. These elements are bordered by insertion element sequences which are recognised by the transposase. When transferred to a different host through conjugation, the plasmid itself can no longer replicate. The transposase however can recognise and transfer the sequence between the insertion elements in a cut-and-paste fashion randomly into the genome. In this fashion, the tetracycline and beta-lactam resistant traits would only persist in cells in which the transposon has jumped into the genome, allowing these antibiotics to select for transposon positive cells. The lacZ protein will only be produced if the transposon jumps in frame downstream of a promotor, and in this case would allow for a lacZ based assay of the promotors response. The <i>E. coli</i> origin of replication present in the transposon allows for the self-cloning of the transposon in a plasmid format after genomic digestion and circularization and transformation into <i>E. coli</i>, allowing for the sequence of bordering gene fragments to be determined easily and therefore mapping the transposon in the genome.</P><br />
<br />
<br />
<br />
<p>&nbsp;&nbsp;Cultures of <i>E. coli</i> SM10 and <i>P. fluorescens</i> PF-5 were grown up overnight in shakers at 37&deg;C and 30&deg;C respectively. SM10 was grown in LB + 10 &micro;g/ml Tet, and PF-5 was grown in LB + 50 mg/L ACROS naphthenic acids. In the morning, the SM10 culture was subcultured (1/4) into LB without antibiotics and allowed to grow for an additional 4h. After this, 3 replicates of mating mixtures were made with 500 &micro;l of each culture were mixed, and the cells were spun down and resuspended in 50 &micro;l of LB. These samples were then plated in separate spots on LB agar. Additional spots were made in the same fashion with just PF-5 culture and just SM10 culture as controls, and the plate was incubated at 37&deg;C overnight. In the morning, each spot was resuspended in 500&micro;l sterile water + 25 &micro;g/ml Tet, and dilutions of 1, 1/10, 1/100, and 1/1000 of each mating mixture was plated on Pseudomonas Isolation Agar (PIA) + 10&micro;g/l Tet + 50 mg/L ACROS naphthenic acids. These cultures were allowed to grow at 30&deg;C over the weekend. </p><br />
<p>&nbsp;&nbsp;The purpose of the PIA is to selectively allow the growth of the PF-5 strain, while killing off the donor SM10 strain. The tetracycline is designed to select for the positive transposon mutants in the PF-5 strain, as the only way that tetracycline resistance would be acquired (barring spontaneous mutation events) would be if the transposable element had jumped into the genome of the cell. We decided to use 10 &micro;g/ml as the concentration of the tetracycline in the plates because we believed that 50 &micro;g/ml would be too high for even strains carrying resistance to survive. Seeing as 10 &micro;g/ml was effective for <i>E. coli</i>, we chose to try this. After streaking the PF-5 culture on the plate to test for its resistance however, it was found that the strain without the transposon was able to grow slightly on the plates, meaning that the concentration of antibiotic is not high enough to properly select for transposon mutants v.s. untransformed cells. In order to try to remedy this, the mating spots were resuspended in a mixture containing a higher dose of antibiotics. The results of this experiment are pending. </p><br />
<br />
<h2>Week 6 (June 4-8)</h2><br />
<br />
<p>&nbsp;&nbsp;When the plates from last week were examined, it was found that though the PIA was sucessful in inhibiting the growth of the <i>E. coli</i> donor strain, the original PF-5 strain was capable of growth on the media. Because of this, the plates were not selective towards cells containing the transposon insertion, and thus lawns of bacteria were seen on each of the plates.</p><br />
<p>&nbsp;&nbsp;Because of this, new selective media plates were prepared. These contained LB agar + 50 mg/L ACROS NA's + 50 &micro;g each chloramphenicol and tetracycline. The chloramphenicol was used in order to kill off the SM10 donor strain, while the tetracycline was used at a concentration previously shown to kill off the PF-5 cells that did not contain a transposon insertion. The conjugation procedure previously described was repeated, with 1 replicate mating spot being plated in 4 different dilutions on the new selective media. These plates were grown overnight at 30&deg;C. It was found that both SM10 and unmodified PF-5 were not capable of growth on the new selective plates, and colonies were found growing on all 4 of the dilutions for the mating spot.<br />
<br />
</html>[[File:Ucalgary2012_TMscreenpractice.png|center|thumb|400px|Figure 1: Transposon selective plates. WT Pf-5 and the donor strain are not capable of growth, however colonies of Pf-5 containing the transposon are capable of growth.]]<html><br />
<br />
<p>&nbsp;&nbsp;Because the host and the untransformed cells were not capable of growth on the selective plates, it is believed that these colonies must represent sucessful transposition events, as this would be the only way that the tetracycline resistance would be transfered to the PF-5 cells (chloramphenicol would have no effect, as PF-5 cells are naturally resistant at this concentration, as previously shown). Because the cell density was too high, the 1 and 1/10 dilution plates were discarded, while the 1/100 and 1/1000 plates were stored at 4&deg;C until the next step of the procedure, in which screening for naphthenic acid response will be performed. </p><br />
<br />
<h2>Week 7 (June 11-15) </h2> <br />
<br />
<p>&nbsp;&nbsp;In order to test for a naphthenic acid response, a lacZ reporter system in the transposon will be utilized. Because the lacZ enzyme is capable of degrading X-gal, a dissacharide sugar containing lactose, into lactose, and an insoluble sugar (5,5'-dibromo-4,4'-dichloro-indigo), which appears blue. Therefore, in response to activation of a native gene promotor, in frame transposon insertions will produce lacZ at levels corresponding to the activation level of the gene, and these colonies will be able to utilize lactose as a carbon source as well as utilize Xgal as a substrate. Cells responding to naphthenic acids will therefore show blue pigmentation and be capable of growth on lactose. Those that do not respond should remain white, and perish when lactose is given as the only sugar source.</p><br />
<p>&nbsp;&nbsp;The concentration of naphthenic acids used in the test plates will be 4x less than the minimal inhibitory concentration (MIC). Therefore, a MIC assay for naphthenic acids with PF-5 cells was carried out overnight at 30&deg;C at 2%, 1%, 0.5%, 0.25%, and 0.125% concentrations in LB media. It was found that PF-5 grew well at all these concentrations; therefore 1% was arbitrarily picked for the test conditions for naphthenic acid response plates.</p><br />
<p><br />
Plates were made as follows, with Xgal spread on top:</p><br />
<br />
<center><table><br />
<table border="1" CELLPADDING=3 CELLSPACING=1 <br />
RULES=NONE FRAME=BOX table width=600px height=150px><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Wild-Type PF-5 Controls</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media Alone, M9 Minimal Media + 0.4% Glucose, M9 Minimal Media + 0.4% Lactose, M9 Minimal Media + 1% NA's</td><br />
</tr><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Transposon Replica Plates</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media + 0.4% Glucose + Chlor, M9 Minimal Media + 0.4% Glucose + Chlor + 1% NA's, M9 Minimal Media + 0.4% Lactose + Tet, M9 Minimal Media + 0.4% Lactose + Tet + 1% NA's </td><br />
</tr><br />
</table></center><br />
<br />
<br />
<p><br />
<br></br><br />
&nbsp;&nbsp;Colonies from the 1/1000 transposon plate were replica stamped using velvet cloth onto the transposon test plates, and WT PF-5 was streaked onto the controls. The plates were allowed to grow over the weekend at 30 &deg;C. </p><br />
<br />
<h2>Week 8 (June 18-22)</h2><br />
<br />
<p>&nbsp;&nbsp;The plates from the previous week were observed on Monday. WT PF-5 grew on M9 + Glucose, but not on any of the other controls, indicating that the wild-type strain could not utilize lactose, indicating that this could possibly be used as a selection measure for responses from the transposon mutants. </p><br />
<p>&nbsp;&nbsp;No growth was observed on any of the transposon test plates. This was believed to be likely from poor transfer during stamping, resulting from a poor contact with the plates in addition to a grease layer formed by the undissolved naphthenic acids. Because of this, a new method of creating plates had to be determined.</P><br />
<p>&nbsp;&nbsp;We tried to dissolve NAs in DMF and spread this mixture on top of agar plates as well as mix it into the liquid agar before pouring plates. Though this helped to solubilize the naphthenic acids, it was not as effective as increasing the pH of the media (pH 8 was found to be effective). We made M9 media at pH 8 before making the same set of test plates as before. It was found that the NAs stayed in solution when mixed, however came out of solution when the plates dried, leading to a grease layer on the plates as previously seen. </P><br />
<p>&nbsp;&nbsp;In another attempt to raise the pH, stock solutions of NAs dissolved in NaOH at pH 12 were made, and these were autoclaved alongside the media. When NAs were added to the NaOH, the solutions became cloudy and uniform, indicating they were in solution. When the NA stocks were added to the media before plate pouring, the NAs remained in solution even after the plates dried, indicating that this method would be sucessful in making test plates. Using this method, the following plates were created:<br />
<br />
<center><table><br />
<table border="1" CELLPADDING=3 CELLSPACING=1 <br />
RULES=NONE FRAME=BOX table width=600px height=150px><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Wild-Type PF-5 Controls</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media Alone, M9 Minimal Media + 0.2% Glucose, M9 Minimal Media + 0.2% Lactose</td><br />
</tr><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Transposon Replica Plates</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media + 0.2% Glucose + Chlor, M9 Minimal Media + 0.2% Glucose + Chlor + 0.05% NA's, M9 Minimal Media + 0.2% Glucose + Chlor + 1% NA's, M9 Minimal Media + 0.2% Lactose + Tet, 0.2% Lactose + Tet + 0.05% NA's, M9 Minimal Media + 0.2% Lactose + Tet + 1% NA's </td><br />
</tr><br />
</table></center><br />
<br />
<p><br />
<br></br><br />
&nbsp;&nbsp;Xgal was spread on all the plates with NA's, however it was left out of the controls by mistake. Stamping was carried out as previously described from both the previously created 1/100 and 1/1000 dilution plates of transposon mutants (A new round of transposon mutagenesis was initiated, but the mating mixture was incubated at 30&deg;C instead of 37&deg;C, and no mutants were obtained on the selective plates). These plates were allowed to grow over the weekend at 30&deg;C.</P><br />
<br />
<h2>Week 9 (June 25-June 29)</h2><br />
<p>Growth was observed from the selection plates incubated over the weekend at 30&deg;C. Screening replica plates are made as before. The 1/1000 diluted transposon plate was used for the replica plating on all the screening conditions. The screening plates are incubated overnight at 30&deg;C. The next day, no growth was observed on the negative control plates (M9 alone, M9 with 0.05% NAs), and no growth was observed on the positive control plates containing glucose. Similarly, no growth was observed on the M9+lactose (+Tet, with/without X-GAL, with/without 0.05% NAs). This suggests that either a large number of matings is needed before a desirable transconjugant is seen, or that the media condition requires modification. The plate surface is not as greasy as before, which suggests that the replica plating transfer process works. However, the pH of the media may affect the growth of transconjugants. Plans are made to modify the pH of the media before replica plating. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>Replica plating screening plates are made so that NA stock solutions (1% at pH 12 diluted to 0.05% final concentration) were added to the M9 agar solution. The pH is adjusted to 7.4 before autoclaving. The pH prior to adjusting is approximately 9.0. This may explain the poor growth observed in the previous trial. A new series of mating were started (2 mating spots), and incubated overnight at 37&deg;C. The mating spots are scraped and serial diluted up to 1/1000, plated on LB+Chlor50+Tet50, and incubated overnight at 30&deg;C. This time, the results are consistent with the previous attempt, where no growth was observed on the negative control plates (M9 alone, M9 with 0.05% NAs), and ample growth was observed on the positive control plates containing glucose. However, still no growth was observed on the M9+lactose (+Tet, with/without X-GAL, with/without 0.05% NAs)plates. This seems to confirm with previous hypothesis that a large number of matings are needed to screen the genome for NA sensitive elements. Alternatively, perhaps the NA concentration used here is too high for proper selection of the transconjugants that have a promoter element upstream of the transposon insertion. If the NA concentration used for the screen is too high, the promoter may be suppressed, and the lacZ is not expressed for cell survival. In fact, the lowest NA concentration used up to this point is 0.05% or 500 mg/L. The culturing conditions required to maintain Pseudomonas Pf-5's NA degrading abilities is LB+50mg/L. Therefore, to allow both cell survival and screen for the most robust and sensitive system, maybe the NA concentration should be lowered.</p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The exact same experiment from the previous week has been repeated with two more mating mixtures (and their dilutions). However, we obtained the same results, with the positive and negative controls yielding predicted growth and no growth, respectively. But no growth was observed on the lactose plates. Furthermore, upon close examination of the selection plates after the conjugating/bipartite mating reaction (LB+Chlor+Tet), two colony morphologies can be seen: drier and larger colonies (resembling Pseudomonas), and smaller moist colonies (resembling E. coli). This suggests perhaps the LB+Chlor+Tet plates may be useful for selecting Pseudomonas transconjugants, but it does not prevent the donor E. coli to be efficiently killed. A final replica plating experiment is designed for next week, and Pseudomonas Isolation agar (PIA, just received)will be used for selection instead. </p> <br />
<br />
<h2>Week 12 (July 16-July 20)</h2><br />
<p>Another mating reaction was set up, and plated on PIA+Tet50 to select for positive transconjugants. The colonies from the resulting mutants show only a single colony morphology consistent with Pseudomonas. From this, we know the selection is appropriate. The 1/1000 dilution of the mating reaction dilution is replica-plated on two sets of screening plates. The first set is prepared the same way as the previous week (NAs added and the media pH adjusted prior to autoclaving); the second set is the same except the NAs are added by spreading 100uL of a 50mg/L sterile stock NA solution in NaOH (pH12) prior to replica plating. The 1/1000 dilution plate of the mating reaction is replica plated on both plates (with the same controls as before). However, the same results are obtained as before, where no growth is observed on the M9+0.2% lactose+X-GAL or the M9+0.2% lactose+X-GAL+50mg/L NAs plates. Lastly, we had an interesting observation where overnight growth of the lactose screening plates showed no growth, if they are left for up to 48hr, some colonies can be observed; however, none of the colonies are blue. The problem may be that the M9 media conditions are not suitable for selecting the Tn mutants, or that the replica plating approach we used cannot effectively transfer all colonies. At this point, since no NA-sensitive strains are isolated, a new approach needs to be taken in order to find a tranposon insertion mutant that has an NA-sensitive promoter upstream. Since all possible approaches have been taken using the replica plating strategy, another mass screening method is needed. </p><br />
<br />
<h2>Week 13 (July 23-July 27)</h2><br />
<p> Even though, the previous screening method is quick and convenient, the waiting period is too long, and no real time data in terms of growth or lactose utilization can be observed/discerned. Therefore, a 96-well mass screening method is devised, where the conjugation mating reaction, and the selection on PIA+Tet50 are the same, but the screening step is different. The screening media is a M9 minimal media with 50 µg/mL tetracycline to maintain the genomic Tn insertions, 0.2% lactose to select for lactose utilization due to Tn insertions, and 50 mg/L NAs to maintain the strain's ability to use/degrade NAs (in short the media is M9+0.2%lactose+50µg/mL Tet+50mg/L NAs). On each 96-well plate, 94 colonies are inoculated and screened; the other two wells are used for negative and positive controls. The negative control well has the same media (M9+0.2%lactose+50µg/mL Tet+50mg/L NAs), with no colonies inoculated. The positive control has M9+0.2%glucose+50µg/mL Tet+50mg/L NAs, and is inoculated with a random colony.<br />
<br />
</p><p><br />
<br />
Using this new method, any inoculated colony that grows in the liquid media can utilize lactose, and this should be because of a transposon insertion downstream of a promoter. To select for a NA sensitive promoter, each colony is inoculated into two plates with the same media components (as described above), except one has NAs, and the other does not. If a promoter is NA-sensitive, then it should not grow if NAs are absent from the media, as the NA mixture would activate the promoter. When NAs are present however, the NA-sensitive promoter would turn on, allowing it to utilize lactose, survive, and grow. <br />
<br />
As a first run of this method, a plate is set up using mutant colonies from a previous mating reaction. This plate contained M9+0.2%lactose+50µg/mL Tet+50mg/L NAs. The appropriate control plate with no NAs was not included as this is only to test feasibility of this approach. The plate is incubated over the weekend for 60hrs in a plate reader at 30&deg;C. </p><br />
<br />
<h2>Week 14 (July 30-August 3)</h2><br />
<p>The data from the plate incubated from last week was collected. First, since the lid of the 96-well plate was kept on the plate to prevent dehydration of the media, there was significant condensation on the middle of the lid (covering about 30 wells), which prevented proper absorbance measurements of those wells (data from these wells were omitted). However, six wells/colonies demonstrated growth from a baseline. The following table is a display of the 96-well plate at the end point, with the colonies/wells that grow were highlighted.</p><br />
<br />
</html>[[File:Ucalgary2012 0801data1.png|center|thumb|400px|Figure 2: A 60hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media]]<html><br />
<br />
<p>The cultures from these six wells are washed with M9+0.2%lactose+50µg/mL Tet (no NAs) three times, and resuspended in 1 mL of M9+0.2%lactose+50µg/mL Tet. 500µl of each colony was inoculated into 5mL cultures with M9+0.2%lactose+50µg/mL Tet or M9+0.2%lactose+50µg/mL Tet+50mg/L NAs. These cultures are grown over 48 hours to observe growth (any strain that grows only when NAs are added would be noted). Also, the colonies are restreaked on PIA+Tet50 plates to ensure that the absorbance increase actually indicated growth (since the increase was so small at around +0.200). The results were not promising, on the restreaked plates, only 2 colonies (from H12 and B1) showed growth, this suggests some of the measurements were not very accurate. Also, in the culturing experiment, only 1 set of cultures (from 1 colony, H12) showed growth, but in both media conditions, which is not desirable.</p><br />
<br />
<p>Even though, no colonies were shown as NA sensitive, this result is still very promising, as this experiment shows that some colonies from the transposon mutagenesis can actually utilize lacZ, which demonstrates that the transposable element approach is an appropriate and feasible approach for screen promoters sensitive to environmental stimuli. (Please note, that mating reactions and mutant selection are conducted regularly every week, usually twice per week, with two mating spots each time, to provide mutants for these screens.)</p><br />
<br />
<h2>Week 15 (August 6-August 10)</h2><br />
<p><br />
The same experiment from the previous week was repeated. However, the plate was not incubated in the plate reader, but incubated in a 30&deg;C shaker, at 140rpm. At the baseline (beginning of incubation), 24hr, and 48hr, timepoint measurements were made. The results are as follows.<br />
</p><p><br />
</html>[[File:Ucalgary2012_080312.png|center|thumb|400px|Figure 3: A 48hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media at time points (baseline, 24hr, and 48hr)]]<html><br />
</p> <br />
<br />
<p><br />
However, these measurements were made with the lid of the plate on. Even though there was no clear trend in terms of the growth in the absorbance data, it is possible to observe some wells become cloudy by eye. For instance, well F9 was cloudy at the end of the 48hr incubation, but the data showed no change in absorbance. The lid should be taken off in the future when making timepoint measurements. Also, the absorbance data can support the visual data.<br />
</p><br />
<br />
<p><br />
Also, this week, two runs of the screening experiment was conducted with two plates run in parallel, one with NAs and the other without (the proper screening setup). However, it was found that the lactose solution (which was filter sterilized) used to make the M9 media for the screen was contaminated with yeast. Therefore, new media solutions and lactose solutions needed to be made. These data are not used because of this.<br />
</p><br />
<br />
<h2>Week 16 (August 13-August 17)</h2><br />
<p><br />
New lactose and glucose stock solutions as well as new M9 screening media were prepared. The experiments from last week were repeated using proper solutions. Also, to prevent fogging of the plate lid and cross-contamination, all media solutions and plates were pre-warmed in a 30&deg;C incubator before dispensing the media into the plates and colony inoculation. The new trial, again, has two plates, with the same wells (e.g. A1) inoculated with the same colony, where one plate has M9 screening media (with Tet and lactose) alone and the other plate containing 50mg/L NAs. Two runs were completed this week. The data from one of those trials are shown below.<br />
</p><p><br />
</html>[[File:Ucalgary2012 081612.png|center|thumb|800px| Figure 4: A 48hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media at time points (baseline, 24hr, and 48hr), comparing the same colonies in M9 screening media with/without 50mg/L NAs]]<html><br />
<br />
<p><br />
In this particular run, only two colonies showed growth (D8 and H11), these were subcultured in 5mL of M9 screening media with NAs. An interesting observation is that there is more growth in the lactose+NAs plate than the lactose alone plate. This suggests that the NAs may play a role in activating the lacZ gene, and improving the survival rates. These results are consistently observed in the other trials conducted. <br />
<br />
<p><br />
At this point, since time is limited, the entire mutant selection process needs to be more efficient. Therefore, the mating spots are diluted, and plated on a more selective media, containing PIA+50ug/mL tetracycline+50mg/L NAs+20 μg/mL X-Gal. This way, the NA-responsive and/or lactose-utilizing Pseudomonas transconjugants can be selected as they would appear blue on the media. This reduces the number of colonies to be inoculated, and improves the efficiency of the whole process. (Note: Wildtype Pseudomonas fluorescens pf-5 are always plated as positive control to observe the natural forward mutation rates, and to ensure that the amount of spontaneous tetracycline resistant Pf-5 mutants are at a reasonable level.<br />
</p><br />
<br />
<p><br />
At this point, a large number of matings should be conducted and mutants selected, in order to provide enough of lactose-utilizing and NA-sensitive Tn-insertion mutants, and to cover as much of the Pseudomonas genome as possible<br />
</p><br />
<br />
<h2>Week 17 (August 20-August 24)</h2><br />
<p>No work on the transposon library was done this week, other than planning for the upcoming screen.</p><br />
<br />
<h2>Week 18 (August 27-August 31)</h2><br />
<p>This week, a large screen was accomplished. First, cultures of <i>P. fluorescens</i> Pf-5 and <i>E. coli</i> SM10 were grown up overnight at 30&deg;C and 37&deg;C respectively to a high optical density. The following morning, the <i>E. coli</i> was subcultured in a 1/4 dilution to dilute out the tetracycline, and grown for approximately 2 more hours. After this, 100 &micro;L of each culture were mixed, spun down, and resuspended in 20 &micro;L of media before being spotted onto LB plates.</p><br />
<p>In total, 500 separate mating spots were plated. These spots were allowed to grow at 37&deg;C overnight. The following day, 2 mating spots were scraped up, combined, and resuspended in 500 &micro;L PBS. 1/400 dilutions of these resuspensions were made, and 100 &micro;L of these was plated onto selective plates consisting of PIA, 100mg/L NAs, and tetracycline. These plates had 40 &micro;L of 20 mg/mL X-gal spread on their surface in order to allow for blue-white screening. 250 plates were made in total.</p><br />
<br />
<p></html>[[File:2012-08-29 21-32-07 236 Calgary.jpg|center|500px|thumb|Figure 5: Plates in incubator]]<html> <p><br />
<br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<br />
<p>Plates were left to grow for 2 days, after which blue colonies (24 in total) were selected and pinned in duplicate into 96-well plates for response testing. Initially, minimal media + lactose with and without NAs was used, however no growth in any of the wells was observed. Because of this, the screening protocol was altered such that LB with or without NAs was used instead of minimal media, and X-gal was used instead of lactose for screening- the idea being that if naphthenic acids were sensed, a blue color change would be observed relative to the negative LB control.</p><br />
<br />
<p></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 6: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16h. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><br />
<br />
<p>When results were observed it was found that 4 colonies showed clear differential regulation in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. Therefore, these colonies will be used in further screening to test the specificity of the response.</p><br />
<br />
<br />
<h2>Week 19 (September 10- September 14)</h2><br />
<p>This week, further screens on the previously identified four hits were performed. These involved the use of different toxins at environmentally relevant concentrations to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used in one of the media samples in order to attempt to rule out a general stress response by the cell.<br />
<br />
<p></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 7: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:170-1data.png|thumb|600px|center|Figure 8: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 9: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 10: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p>Due to these results, further screens on these two colonies will be performed, using lower hydrogen peroxide concentrations to rule out a general stress response, and decanoic acid to rule out a response to fatty acids.</p><br />
<br />
<h2>Week 20 (September 17- September 21)</h2><p><br />
Further screens were conducted, however due to cells drying out in the plate the results were invalidated. Because of this, the screen will be repeated, and explained in further detail when this is done.</p><br />
<br />
<h2>Week 21 (September 24- September 28)</h2><p><br />
Genomes of 66-1 and 170-1 were isolated, digested with BglII and with XbaI, and religated before being transformed into <i>E.coli</i>. Cells were plated onto tetracycline plates to isolate cells containing a ligation product with the transposon present. These colonies have been miniprepped, and are awaiting being sent for sequencing to determine which genes the transposon has been inserted into. </p><br />
<br />
<h2>Week 22 (October 1-October 5)</h2><p><br />
Minipreps from previous weeks of the transposon self-cloned plasmid were nanodropped, and it was determined that no DNA had been isolated. Therefore, colonies were re-prepped, and sent for sequencing (as sequencing primers have arrived from Dr. Hynes).</p><p><br />
In addition, the assay to test the specificity of the response of the transposon clones was repeated. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The assay was carried out according to the <b>protocol</b> on our protocols section. This data was recieved after Wiki-Freeze had happened, and therefore will be analyzed at a later date. </p><br />
<br />
<h2>Week 23 (October 8-October 11)</h2><p><br />
Unfortunately, no transposon work was accomplished this week.<br />
<br />
<h2>Week 24 (October 15-October 19)</h2><p><br />
Sequencing reactions came back as having no reads, so sequencing samples were resent. This happened multiple times this week, and it is unclear why reactions continue to fail.<br />
<br />
<h2>Week 25 (October 22-October 26)</h2><p><br />
This week, data from the previously run assay was analyzed. The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure 8:Stress response screen on <i>P. fluorescens</i> Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions (shown are '''A:''' 66-1 undiluted, '''B:''' 66-1 at 1/10 dilution, '''C:''' 170-1 undiluted, '''D:''' 170-1 at 1/10 dilution) into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 uL of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.]]<html><p><br />
These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.</p><br />
<br />
<p>In addition, electrochemical tests were conducted on the transposon clones this week. The results can be seen on the <b>page</b>.<br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/FRED/DetectingTeam:Calgary/Project/FRED/Detecting2012-10-26T21:44:12Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Transposon-Mediated Mutant Library for Toxin Detection|<br />
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CONTENT=<br />
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<img src="https://static.igem.org/mediawiki/2012/5/52/UCalgary2012_FRED_Detecting.png" style="float: right; padding: 10px; height: 280px;"></img><br />
<p align="justify"><br />
This year, our team wanted to identify a novel responsive element capable of detecting and quantifying different tailings ponds toxins (e.g. naphthenic acids, NAs) in solution. While numerous studies have begun to identify species of bacteria capable of surviving and sensing a variety of toxic compounds (e.g. NAs), the degradation pathways have not yet been fully characterized. Therefore, we needed to design and implement novel approaches to efficiently isolate the genetic elements that detect and potentially lead to the breakdown of these toxins.<br />
</p><br />
<h2>Transposons: What, How, Why?</h2><br />
<p align="justify"><br />
The transposable element (TE), Tn5, is a conservative transposon that can insert a segment of genes bordered by specific 19bp insertion sequences from one part of the genome (e.g. plasmid vector) randomly to another location like a chromosome (Reznikoff, 2008). The transposition event is catalyzed by a transposase enzyme encoded by <i>tnp</i> gene included in the TE. Using the appropriate selective pressure, the insertion can be maintained permanently in the genome.</p><br />
<br />
</html>[[File:Transposon.jpg|thumb|700px|center|Figure 1: "Transposition reaction from plasmid entry into the recipient cell to integration of the transposon into the genome. Modified from Transposons: Shifting Segments of the Genome" by McGraw Hill]]<html><br />
<br />
<br />
<p align="justify">By inserting a vector construct containing the TE with selectable markers (such as tetracyclin resistance and lacZ) into an organism with a desirable phenotype, we can find out what genetic elements (e.g. genes and promoters) are responsible for that particular function. This can happen via a random insertion of a TE containing a promoterless reporter gene downstream of promoter elements that creates a transcriptional fusion, providing activity in response to specific environmental stimuli. Using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">bipartite-mating (conjugation) method</a> to transfer the TE vector into the organism of choice is an efficient method for creating the massive number of mutants required.</p><br />
<p align="justify"><br />
Due to the complexity of biological systems, our team focused our efforts on utilizing a system for identification of promoter elements that respond specifically in the presence of environmental stimuli. Our hypothesis requires that the organisms we use respond specifically to particular toxins and result in upregulation of metabolic genes with little background effect in the cell. We recognize that any number of biological molecules may play a role in toxin sensing, such as enzymes, transcription factors, and even RNA elements (e.g. riboswitches). However, the identification of a promoter sequence takes us further in that we can better understand the degradation mechanism by elucidating the genes involved.<br />
</p><br />
<br />
<br />
<br><br />
<h2>Toxin-Degrading Organism Used</h2><br />
<p align="justify"><br />
<i>Pseudomonas spp. </i>have been isolated from oil sands tailings ponds and shown to biodegrade model and tailings-associated NAs and nitrogen- and sulfur-containing heterocyclic aromatic compounds (Ramos-Padrón <i>et al</i>. 2010; Herman <i>et al</i>., 1994; Del Rio <i>et al</i>., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins.</p><p> We wanted to use a commercially available strain of <i>Pseudomonas fluorescens</i> characterized for a response to toxins found in tailings pond water (TPW). The <i>P. fluorescens </i>PF-5 strain (Paulsen <i>et al</i>., 2005) is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, the genome sequence is available for this strain with annotations (Pseudomonas Genome Database V2, http://pseudomonas.com/). This allows us to use sequencing data from the mutants and identify where in the genome the TE insertion occurred, and what genes (if present) are located downstream of it.<br />
</p><br />
<br />
<br><br />
<br />
<br />
<a name="library"></a><h2>Method Design</h2><br />
<h3>Mutant Library Generation</h3><br />
<p align="justify"><br />
To construct the promoter library, a pOT182 vector construct (containing a IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR transposable element) is introduced into commercially purchased <i>E. coli SM10</i> donor strain.</p><br />
<br />
<p align="justify"><br />
</html>[[File:Transposonproject Tn5OT182constructucalgary.png|thumb|750px|center|Figure 2: The transposable Tn5 element used in the pOT182 plasmid, containing a lacZ reporter gene, ampicillin and tetracycline resistance, an<br />
<i> E. coli</i> origin of replication for use during downstream sequencing protocols, and transposase. The genes are flanked by the transposon insertion elements]]<html><br />
</p><br />
<br />
<p align="justify">The plasmid contains a RP4 mob conjugation region and a p15A origin of replication (ori), and is engineered to only replicate in <i>E. coli</i>. The TE construct is transferred from the <i>E. coli</i> donor strain to the recipient <i>P. fluorescens </i> PF-5 using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in <i>P. fluorescens</i>, and selected by isolating the recipients that have a genomic TE insertion on Pseudomonas Isolation Agar/PIA with tetracycline. If a promoter element is fused upstream of the TE construct, then promoter activation will turn on the expression of lacZ, which can be detected by the degradation of a colorless compound, X-Gal, to an insoluble blue pigment product (an indoxyl compound) (Juers <i>et al</i>., 2012). If the fused promoter is activated in response to a stimulus, then the lacZ enzyme will be produced in response. Mutant strains sensitive to the particular toxic stimulus will appear as blue colonies on the selective plate.</p><br />
<br />
<h3>Mutant Strain Characterization</h3><br />
<p align="justify">Mutants generated are characterized for their roles in the response to toxins with dose response experiments, and compared to general stress-inducing agents (e.g. H<font style="text-transform: lowercase;">2</font>O<font style="text-transform: lowercase;">2</font>) and compounds such as fatty acids to ensure the specificity of the response. These measurements help to determine thresholds of detection, robustness of the signal, and specificity of response. The dose response curves will also assess the usefulness of correlating the concentration of NA to the level of response, and the possibility of measuring NA concentrations in a sample, rather than simply by presence/absence.</p><br />
</p><br />
<h3>Self-Cloning and Sequencing</h3><br />
<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman and Lamont, 1993). The TE used is a self-cloning construct because it contains all the elements required for plasmid replication (i.e. origin of replication) and selection (Tet resistance). Genomic DNA from a desirable mutant is isolated, and restriction digested with BglII (a restriction enzyme that does not cut within the TE but numerous times within the genome). The resulting fragments may contain the TE construct with flanking sequences. The genomic fragments are circularized by self-ligation and transformed into <i>E. coli</i>. Plasmids from the transformed cells contain the TE construct with the upstream and downstream flanking sequencing connected by the BglII restriction site. Sequencing primers designed against the 19 bp recognition sequence in the TE to sequence the isolated plasmids.</p><br />
<br />
<p align="justify">For a detailed protocol, please consult our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">methods section</a>.</p><br />
<br />
<h2>Results</h2><br />
<h3>Detection by Mutant <i>Pseudomonas fluorescens</i> PF-5</h3><br />
<br />
<br />
<p align="justify">After mating experiments and plating on selective media (Pseudomonas isolation agar, with tetracycline and naphthenic acids), 24 responsive (blue) colonies were found. Screens were conducted on these blue colonies found on selective plates comparing a response in LB and LB with 100mg/L naphthenic acids (both with X-Gal). When results were observed it was found that 4 mutant strains are differentially regulated in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. These colonies were further screened to test the specificity of their responses.</p><br />
<br />
<p align="justify"></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 3: Transposons: Shifting Segments of the Genome: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16hr. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><p align="justify"><br />
<br />
<br />
Screens involving the use of different toxins at environmentally relevant concentrations were performed to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used as one testing condition to determine if the response is simply stress-induced.<br />
</p><br />
<p align="justify"></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 4: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:170-1data.png|thumb|650px|center|Figure 5: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 6: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 7: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<p align="justify"><br />
From these screens, it was seen that both colony 66-1 and colony 170-1 appear to respond to toxins when compared to a response in LB media. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure 8:Stress response screen on <i>P. fluorescens</i>Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 %micro&L of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.]]<html></p><br />
<p><br />
These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.</p><br />
<br />
<h2>Promoter Constructs Isolated</h2><br />
<p align="justify">To determine the location of the transposon insertion, we utilized the self-cloning properties of the transposon. By digesting the genome, religating, and transforming the ligated genomic fragments into <i>E. coli</i>, plasmids containing the transposon and flanking gene sequences were isolated. These plasmids have been isolated and sent for sequencing. However, we are having difficulty with getting sequencing reactions to produce a read. The results so far are a promising step towards finding a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds. </p><p> <br />
Our next steps were to test these strains in conjunction with our electrochemical detector as well as see if they could detect tailings toxins. <b>The results of this can be seen on the x page</b>.</p><br />
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}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/PromoterScreenTeam:Calgary/Notebook/PromoterScreen2012-10-26T21:42:24Z<p>Lisa.O: </p>
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<div>{{Team:Calgary/TemplateNotebookGreen|<br />
TITLE=Transposon Library Notebook|<br />
CONTENT =<br />
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Patrick.<br />
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<h2>Week 1 (May 1-4)</h2><br />
<p>&nbsp;&nbsp;This was the first week where we met with other team members and summarized the primary subprojects the team will be tackling this coming summer.</p><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>&nbsp;&nbsp;During this week literature searches were performed.</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>&nbsp;&nbsp;During this week literature searches were performed.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>&nbsp;&nbsp;During this week, strains of <i> Pseudomonas fluorescens</i> PF-5 were obtained. Two cultures were started by adding 500 &micro;L stock to 10 mL LB media containing 50 mg/L ACROS Naphthenic Acids. These cultures were grown at 30&deg;C overnight, shaking at 110 rpm.</p><br />
<p>&nbsp;&nbsp;Overnight cultures were then streaked on LB agar the following day with various types and concentrations of antibiotics in order to determine the susceptibility profile of the organism. This was necessary in order to determine what marker could be used on a transposon to allow for selection of organisms with sucessful transposon insertions. These plates were grown overnight to look for death or growth, and the following results were obtained; </p><br />
<br />
<center><table><br />
<table border="2" CELLPADDING=3 CELLSPACING=1 <br />
RULES=COLS FRAME=VSIDES><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Gentamycin</b></td><br />
<td><b>Kanamycin</b></td><br />
<td><b>Chloramphenicol</b></td><br />
<td><b>Tetracycline</b></td><br />
</tr><br />
<tr><br />
<td>25 &micro;g/ml = no growth</td><br />
<td>5 &micro;g/ml = slight growth</td><br />
<td>5 &micro;g/ml = growth</td><br />
<td>50 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td>50 &micro;g/ml = no growth</td><br />
<td>10 &micro;g/ml = slight growth</td><br />
<td>10 &micro;g/ml = growth</td><br />
<td>100 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td>100 &micro;g/ml = no growth</td><br />
<td>25 &micro;g/ml = no growth</td><br />
<td>25 &micro;g/ml = growth</td><br />
<td>200 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td>50 &micro;g/ml = no growth</td><br />
<td>50 &micro;g/ml = growth</td><br />
<td> </td><br />
</tr><br />
</table></center><br />
<br />
<br />
<p><br />
<br></br> &nbsp;&nbsp;Based on these results, it was determined that Kanamycin, Gentamycin, and Tetracycline could be used as the selectable marker on the transposon, while Chloramphenicol could not as the strain is naturally resistant. Glycerol stocks of the strains were also made at this time from a fresh overnight culture.</p><br />
<br><br><br />
<h2>Week 5 (May 28-June 1)</h2><br />
<p>&nbsp;&nbsp;At the beginning of this week we spoke to Dr. Michael Hynes, who was able to give us <i> E. coli </i> SM10 and SM17-1 cells containing the plasmid pOT182. This plasmid contains an <i> E. coli </i> origin of replication, allowing it to act as a suicide vector when transferred to a different bacterial species. pOT182 contains a Tn5 transposon element containing a promotorless <i>lacZ</i> gene, genes for tetracycline resistance as well as a beta-lactamase, transposase and an <i>E. coli</i> origin of replication. These elements are bordered by insertion element sequences which are recognised by the transposase. When transferred to a different host through conjugation, the plasmid itself can no longer replicate. The transposase however can recognise and transfer the sequence between the insertion elements in a cut-and-paste fashion randomly into the genome. In this fashion, the tetracycline and beta-lactam resistant traits would only persist in cells in which the transposon has jumped into the genome, allowing these antibiotics to select for transposon positive cells. The lacZ protein will only be produced if the transposon jumps in frame downstream of a promotor, and in this case would allow for a lacZ based assay of the promotors response. The <i>E. coli</i> origin of replication present in the transposon allows for the self-cloning of the transposon in a plasmid format after genomic digestion and circularization and transformation into <i>E. coli</i>, allowing for the sequence of bordering gene fragments to be determined easily and therefore mapping the transposon in the genome.</P><br />
<br />
<br />
<br />
<p>&nbsp;&nbsp;Cultures of <i>E. coli</i> SM10 and <i>P. fluorescens</i> PF-5 were grown up overnight in shakers at 37&deg;C and 30&deg;C respectively. SM10 was grown in LB + 10 &micro;g/ml Tet, and PF-5 was grown in LB + 50 mg/L ACROS naphthenic acids. In the morning, the SM10 culture was subcultured (1/4) into LB without antibiotics and allowed to grow for an additional 4h. After this, 3 replicates of mating mixtures were made with 500 &micro;l of each culture were mixed, and the cells were spun down and resuspended in 50 &micro;l of LB. These samples were then plated in separate spots on LB agar. Additional spots were made in the same fashion with just PF-5 culture and just SM10 culture as controls, and the plate was incubated at 37&deg;C overnight. In the morning, each spot was resuspended in 500&micro;l sterile water + 25 &micro;g/ml Tet, and dilutions of 1, 1/10, 1/100, and 1/1000 of each mating mixture was plated on Pseudomonas Isolation Agar (PIA) + 10&micro;g/l Tet + 50 mg/L ACROS naphthenic acids. These cultures were allowed to grow at 30&deg;C over the weekend. </p><br />
<p>&nbsp;&nbsp;The purpose of the PIA is to selectively allow the growth of the PF-5 strain, while killing off the donor SM10 strain. The tetracycline is designed to select for the positive transposon mutants in the PF-5 strain, as the only way that tetracycline resistance would be acquired (barring spontaneous mutation events) would be if the transposable element had jumped into the genome of the cell. We decided to use 10 &micro;g/ml as the concentration of the tetracycline in the plates because we believed that 50 &micro;g/ml would be too high for even strains carrying resistance to survive. Seeing as 10 &micro;g/ml was effective for <i>E. coli</i>, we chose to try this. After streaking the PF-5 culture on the plate to test for its resistance however, it was found that the strain without the transposon was able to grow slightly on the plates, meaning that the concentration of antibiotic is not high enough to properly select for transposon mutants v.s. untransformed cells. In order to try to remedy this, the mating spots were resuspended in a mixture containing a higher dose of antibiotics. The results of this experiment are pending. </p><br />
<br />
<h2>Week 6 (June 4-8)</h2><br />
<br />
<p>&nbsp;&nbsp;When the plates from last week were examined, it was found that though the PIA was sucessful in inhibiting the growth of the <i>E. coli</i> donor strain, the original PF-5 strain was capable of growth on the media. Because of this, the plates were not selective towards cells containing the transposon insertion, and thus lawns of bacteria were seen on each of the plates.</p><br />
<p>&nbsp;&nbsp;Because of this, new selective media plates were prepared. These contained LB agar + 50 mg/L ACROS NA's + 50 &micro;g each chloramphenicol and tetracycline. The chloramphenicol was used in order to kill off the SM10 donor strain, while the tetracycline was used at a concentration previously shown to kill off the PF-5 cells that did not contain a transposon insertion. The conjugation procedure previously described was repeated, with 1 replicate mating spot being plated in 4 different dilutions on the new selective media. These plates were grown overnight at 30&deg;C. It was found that both SM10 and unmodified PF-5 were not capable of growth on the new selective plates, and colonies were found growing on all 4 of the dilutions for the mating spot.<br />
<br />
</html>[[File:Ucalgary2012_TMscreenpractice.png|center|thumb|400px|Figure 1: Transposon selective plates. WT Pf-5 and the donor strain are not capable of growth, however colonies of Pf-5 containing the transposon are capable of growth.]]<html><br />
<br />
<p>&nbsp;&nbsp;Because the host and the untransformed cells were not capable of growth on the selective plates, it is believed that these colonies must represent sucessful transposition events, as this would be the only way that the tetracycline resistance would be transfered to the PF-5 cells (chloramphenicol would have no effect, as PF-5 cells are naturally resistant at this concentration, as previously shown). Because the cell density was too high, the 1 and 1/10 dilution plates were discarded, while the 1/100 and 1/1000 plates were stored at 4&deg;C until the next step of the procedure, in which screening for naphthenic acid response will be performed. </p><br />
<br />
<h2>Week 7 (June 11-15) </h2> <br />
<br />
<p>&nbsp;&nbsp;In order to test for a naphthenic acid response, a lacZ reporter system in the transposon will be utilized. Because the lacZ enzyme is capable of degrading X-gal, a dissacharide sugar containing lactose, into lactose, and an insoluble sugar (5,5'-dibromo-4,4'-dichloro-indigo), which appears blue. Therefore, in response to activation of a native gene promotor, in frame transposon insertions will produce lacZ at levels corresponding to the activation level of the gene, and these colonies will be able to utilize lactose as a carbon source as well as utilize Xgal as a substrate. Cells responding to naphthenic acids will therefore show blue pigmentation and be capable of growth on lactose. Those that do not respond should remain white, and perish when lactose is given as the only sugar source.</p><br />
<p>&nbsp;&nbsp;The concentration of naphthenic acids used in the test plates will be 4x less than the minimal inhibitory concentration (MIC). Therefore, a MIC assay for naphthenic acids with PF-5 cells was carried out overnight at 30&deg;C at 2%, 1%, 0.5%, 0.25%, and 0.125% concentrations in LB media. It was found that PF-5 grew well at all these concentrations; therefore 1% was arbitrarily picked for the test conditions for naphthenic acid response plates.</p><br />
<p><br />
Plates were made as follows, with Xgal spread on top:</p><br />
<br />
<center><table><br />
<table border="1" CELLPADDING=3 CELLSPACING=1 <br />
RULES=NONE FRAME=BOX table width=600px height=150px><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Wild-Type PF-5 Controls</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media Alone, M9 Minimal Media + 0.4% Glucose, M9 Minimal Media + 0.4% Lactose, M9 Minimal Media + 1% NA's</td><br />
</tr><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Transposon Replica Plates</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media + 0.4% Glucose + Chlor, M9 Minimal Media + 0.4% Glucose + Chlor + 1% NA's, M9 Minimal Media + 0.4% Lactose + Tet, M9 Minimal Media + 0.4% Lactose + Tet + 1% NA's </td><br />
</tr><br />
</table></center><br />
<br />
<br />
<p><br />
<br></br><br />
&nbsp;&nbsp;Colonies from the 1/1000 transposon plate were replica stamped using velvet cloth onto the transposon test plates, and WT PF-5 was streaked onto the controls. The plates were allowed to grow over the weekend at 30 &deg;C. </p><br />
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<h2>Week 8 (June 18-22)</h2><br />
<br />
<p>&nbsp;&nbsp;The plates from the previous week were observed on Monday. WT PF-5 grew on M9 + Glucose, but not on any of the other controls, indicating that the wild-type strain could not utilize lactose, indicating that this could possibly be used as a selection measure for responses from the transposon mutants. </p><br />
<p>&nbsp;&nbsp;No growth was observed on any of the transposon test plates. This was believed to be likely from poor transfer during stamping, resulting from a poor contact with the plates in addition to a grease layer formed by the undissolved naphthenic acids. Because of this, a new method of creating plates had to be determined.</P><br />
<p>&nbsp;&nbsp;We tried to dissolve NAs in DMF and spread this mixture on top of agar plates as well as mix it into the liquid agar before pouring plates. Though this helped to solubilize the naphthenic acids, it was not as effective as increasing the pH of the media (pH 8 was found to be effective). We made M9 media at pH 8 before making the same set of test plates as before. It was found that the NAs stayed in solution when mixed, however came out of solution when the plates dried, leading to a grease layer on the plates as previously seen. </P><br />
<p>&nbsp;&nbsp;In another attempt to raise the pH, stock solutions of NAs dissolved in NaOH at pH 12 were made, and these were autoclaved alongside the media. When NAs were added to the NaOH, the solutions became cloudy and uniform, indicating they were in solution. When the NA stocks were added to the media before plate pouring, the NAs remained in solution even after the plates dried, indicating that this method would be sucessful in making test plates. Using this method, the following plates were created:<br />
<br />
<center><table><br />
<table border="1" CELLPADDING=3 CELLSPACING=1 <br />
RULES=NONE FRAME=BOX table width=600px height=150px><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Wild-Type PF-5 Controls</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media Alone, M9 Minimal Media + 0.2% Glucose, M9 Minimal Media + 0.2% Lactose</td><br />
</tr><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Transposon Replica Plates</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media + 0.2% Glucose + Chlor, M9 Minimal Media + 0.2% Glucose + Chlor + 0.05% NA's, M9 Minimal Media + 0.2% Glucose + Chlor + 1% NA's, M9 Minimal Media + 0.2% Lactose + Tet, 0.2% Lactose + Tet + 0.05% NA's, M9 Minimal Media + 0.2% Lactose + Tet + 1% NA's </td><br />
</tr><br />
</table></center><br />
<br />
<p><br />
<br></br><br />
&nbsp;&nbsp;Xgal was spread on all the plates with NA's, however it was left out of the controls by mistake. Stamping was carried out as previously described from both the previously created 1/100 and 1/1000 dilution plates of transposon mutants (A new round of transposon mutagenesis was initiated, but the mating mixture was incubated at 30&deg;C instead of 37&deg;C, and no mutants were obtained on the selective plates). These plates were allowed to grow over the weekend at 30&deg;C.</P><br />
<br />
<h2>Week 9 (June 25-June 29)</h2><br />
<p>Growth was observed from the selection plates incubated over the weekend at 30&deg;C. Screening replica plates are made as before. The 1/1000 diluted transposon plate was used for the replica plating on all the screening conditions. The screening plates are incubated overnight at 30&deg;C. The next day, no growth was observed on the negative control plates (M9 alone, M9 with 0.05% NAs), and no growth was observed on the positive control plates containing glucose. Similarly, no growth was observed on the M9+lactose (+Tet, with/without X-GAL, with/without 0.05% NAs). This suggests that either a large number of matings is needed before a desirable transconjugant is seen, or that the media condition requires modification. The plate surface is not as greasy as before, which suggests that the replica plating transfer process works. However, the pH of the media may affect the growth of transconjugants. Plans are made to modify the pH of the media before replica plating. </p><br />
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<h2>Week 10 (July 2-July 6)</h2><br />
<p>Replica plating screening plates are made so that NA stock solutions (1% at pH 12 diluted to 0.05% final concentration) were added to the M9 agar solution. The pH is adjusted to 7.4 before autoclaving. The pH prior to adjusting is approximately 9.0. This may explain the poor growth observed in the previous trial. A new series of mating were started (2 mating spots), and incubated overnight at 37&deg;C. The mating spots are scraped and serial diluted up to 1/1000, plated on LB+Chlor50+Tet50, and incubated overnight at 30&deg;C. This time, the results are consistent with the previous attempt, where no growth was observed on the negative control plates (M9 alone, M9 with 0.05% NAs), and ample growth was observed on the positive control plates containing glucose. However, still no growth was observed on the M9+lactose (+Tet, with/without X-GAL, with/without 0.05% NAs)plates. This seems to confirm with previous hypothesis that a large number of matings are needed to screen the genome for NA sensitive elements. Alternatively, perhaps the NA concentration used here is too high for proper selection of the transconjugants that have a promoter element upstream of the transposon insertion. If the NA concentration used for the screen is too high, the promoter may be suppressed, and the lacZ is not expressed for cell survival. In fact, the lowest NA concentration used up to this point is 0.05% or 500 mg/L. The culturing conditions required to maintain Pseudomonas Pf-5's NA degrading abilities is LB+50mg/L. Therefore, to allow both cell survival and screen for the most robust and sensitive system, maybe the NA concentration should be lowered.</p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The exact same experiment from the previous week has been repeated with two more mating mixtures (and their dilutions). However, we obtained the same results, with the positive and negative controls yielding predicted growth and no growth, respectively. But no growth was observed on the lactose plates. Furthermore, upon close examination of the selection plates after the conjugating/bipartite mating reaction (LB+Chlor+Tet), two colony morphologies can be seen: drier and larger colonies (resembling Pseudomonas), and smaller moist colonies (resembling E. coli). This suggests perhaps the LB+Chlor+Tet plates may be useful for selecting Pseudomonas transconjugants, but it does not prevent the donor E. coli to be efficiently killed. A final replica plating experiment is designed for next week, and Pseudomonas Isolation agar (PIA, just received)will be used for selection instead. </p> <br />
<br />
<h2>Week 12 (July 16-July 20)</h2><br />
<p>Another mating reaction was set up, and plated on PIA+Tet50 to select for positive transconjugants. The colonies from the resulting mutants show only a single colony morphology consistent with Pseudomonas. From this, we know the selection is appropriate. The 1/1000 dilution of the mating reaction dilution is replica-plated on two sets of screening plates. The first set is prepared the same way as the previous week (NAs added and the media pH adjusted prior to autoclaving); the second set is the same except the NAs are added by spreading 100uL of a 50mg/L sterile stock NA solution in NaOH (pH12) prior to replica plating. The 1/1000 dilution plate of the mating reaction is replica plated on both plates (with the same controls as before). However, the same results are obtained as before, where no growth is observed on the M9+0.2% lactose+X-GAL or the M9+0.2% lactose+X-GAL+50mg/L NAs plates. Lastly, we had an interesting observation where overnight growth of the lactose screening plates showed no growth, if they are left for up to 48hr, some colonies can be observed; however, none of the colonies are blue. The problem may be that the M9 media conditions are not suitable for selecting the Tn mutants, or that the replica plating approach we used cannot effectively transfer all colonies. At this point, since no NA-sensitive strains are isolated, a new approach needs to be taken in order to find a tranposon insertion mutant that has an NA-sensitive promoter upstream. Since all possible approaches have been taken using the replica plating strategy, another mass screening method is needed. </p><br />
<br />
<h2>Week 13 (July 23-July 27)</h2><br />
<p> Even though, the previous screening method is quick and convenient, the waiting period is too long, and no real time data in terms of growth or lactose utilization can be observed/discerned. Therefore, a 96-well mass screening method is devised, where the conjugation mating reaction, and the selection on PIA+Tet50 are the same, but the screening step is different. The screening media is a M9 minimal media with 50 µg/mL tetracycline to maintain the genomic Tn insertions, 0.2% lactose to select for lactose utilization due to Tn insertions, and 50 mg/L NAs to maintain the strain's ability to use/degrade NAs (in short the media is M9+0.2%lactose+50µg/mL Tet+50mg/L NAs). On each 96-well plate, 94 colonies are inoculated and screened; the other two wells are used for negative and positive controls. The negative control well has the same media (M9+0.2%lactose+50µg/mL Tet+50mg/L NAs), with no colonies inoculated. The positive control has M9+0.2%glucose+50µg/mL Tet+50mg/L NAs, and is inoculated with a random colony.<br />
<br />
</p><p><br />
<br />
Using this new method, any inoculated colony that grows in the liquid media can utilize lactose, and this should be because of a transposon insertion downstream of a promoter. To select for a NA sensitive promoter, each colony is inoculated into two plates with the same media components (as described above), except one has NAs, and the other does not. If a promoter is NA-sensitive, then it should not grow if NAs are absent from the media, as the NA mixture would activate the promoter. When NAs are present however, the NA-sensitive promoter would turn on, allowing it to utilize lactose, survive, and grow. <br />
<br />
As a first run of this method, a plate is set up using mutant colonies from a previous mating reaction. This plate contained M9+0.2%lactose+50µg/mL Tet+50mg/L NAs. The appropriate control plate with no NAs was not included as this is only to test feasibility of this approach. The plate is incubated over the weekend for 60hrs in a plate reader at 30&deg;C. </p><br />
<br />
<h2>Week 14 (July 30-August 3)</h2><br />
<p>The data from the plate incubated from last week was collected. First, since the lid of the 96-well plate was kept on the plate to prevent dehydration of the media, there was significant condensation on the middle of the lid (covering about 30 wells), which prevented proper absorbance measurements of those wells (data from these wells were omitted). However, six wells/colonies demonstrated growth from a baseline. The following table is a display of the 96-well plate at the end point, with the colonies/wells that grow were highlighted.</p><br />
<br />
</html>[[File:Ucalgary2012 0801data1.png|center|thumb|400px|Figure 2: A 60hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media]]<html><br />
<br />
<p>The cultures from these six wells are washed with M9+0.2%lactose+50µg/mL Tet (no NAs) three times, and resuspended in 1 mL of M9+0.2%lactose+50µg/mL Tet. 500µl of each colony was inoculated into 5mL cultures with M9+0.2%lactose+50µg/mL Tet or M9+0.2%lactose+50µg/mL Tet+50mg/L NAs. These cultures are grown over 48 hours to observe growth (any strain that grows only when NAs are added would be noted). Also, the colonies are restreaked on PIA+Tet50 plates to ensure that the absorbance increase actually indicated growth (since the increase was so small at around +0.200). The results were not promising, on the restreaked plates, only 2 colonies (from H12 and B1) showed growth, this suggests some of the measurements were not very accurate. Also, in the culturing experiment, only 1 set of cultures (from 1 colony, H12) showed growth, but in both media conditions, which is not desirable.</p><br />
<br />
<p>Even though, no colonies were shown as NA sensitive, this result is still very promising, as this experiment shows that some colonies from the transposon mutagenesis can actually utilize lacZ, which demonstrates that the transposable element approach is an appropriate and feasible approach for screen promoters sensitive to environmental stimuli. (Please note, that mating reactions and mutant selection are conducted regularly every week, usually twice per week, with two mating spots each time, to provide mutants for these screens.)</p><br />
<br />
<h2>Week 15 (August 6-August 10)</h2><br />
<p><br />
The same experiment from the previous week was repeated. However, the plate was not incubated in the plate reader, but incubated in a 30&deg;C shaker, at 140rpm. At the baseline (beginning of incubation), 24hr, and 48hr, timepoint measurements were made. The results are as follows.<br />
</p><p><br />
</html>[[File:Ucalgary2012_080312.png|center|thumb|400px|Figure 3: A 48hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media at time points (baseline, 24hr, and 48hr)]]<html><br />
</p> <br />
<br />
<p><br />
However, these measurements were made with the lid of the plate on. Even though there was no clear trend in terms of the growth in the absorbance data, it is possible to observe some wells become cloudy by eye. For instance, well F9 was cloudy at the end of the 48hr incubation, but the data showed no change in absorbance. The lid should be taken off in the future when making timepoint measurements. Also, the absorbance data can support the visual data.<br />
</p><br />
<br />
<p><br />
Also, this week, two runs of the screening experiment was conducted with two plates run in parallel, one with NAs and the other without (the proper screening setup). However, it was found that the lactose solution (which was filter sterilized) used to make the M9 media for the screen was contaminated with yeast. Therefore, new media solutions and lactose solutions needed to be made. These data are not used because of this.<br />
</p><br />
<br />
<h2>Week 16 (August 13-August 17)</h2><br />
<p><br />
New lactose and glucose stock solutions as well as new M9 screening media were prepared. The experiments from last week were repeated using proper solutions. Also, to prevent fogging of the plate lid and cross-contamination, all media solutions and plates were pre-warmed in a 30&deg;C incubator before dispensing the media into the plates and colony inoculation. The new trial, again, has two plates, with the same wells (e.g. A1) inoculated with the same colony, where one plate has M9 screening media (with Tet and lactose) alone and the other plate containing 50mg/L NAs. Two runs were completed this week. The data from one of those trials are shown below.<br />
</p><p><br />
</html>[[File:Ucalgary2012 081612.png|center|thumb|800px| Figure 4: A 48hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media at time points (baseline, 24hr, and 48hr), comparing the same colonies in M9 screening media with/without 50mg/L NAs]]<html><br />
<br />
<p><br />
In this particular run, only two colonies showed growth (D8 and H11), these were subcultured in 5mL of M9 screening media with NAs. An interesting observation is that there is more growth in the lactose+NAs plate than the lactose alone plate. This suggests that the NAs may play a role in activating the lacZ gene, and improving the survival rates. These results are consistently observed in the other trials conducted. <br />
<br />
<p><br />
At this point, since time is limited, the entire mutant selection process needs to be more efficient. Therefore, the mating spots are diluted, and plated on a more selective media, containing PIA+50ug/mL tetracycline+50mg/L NAs+20 μg/mL X-Gal. This way, the NA-responsive and/or lactose-utilizing Pseudomonas transconjugants can be selected as they would appear blue on the media. This reduces the number of colonies to be inoculated, and improves the efficiency of the whole process. (Note: Wildtype Pseudomonas fluorescens pf-5 are always plated as positive control to observe the natural forward mutation rates, and to ensure that the amount of spontaneous tetracycline resistant Pf-5 mutants are at a reasonable level.<br />
</p><br />
<br />
<p><br />
At this point, a large number of matings should be conducted and mutants selected, in order to provide enough of lactose-utilizing and NA-sensitive Tn-insertion mutants, and to cover as much of the Pseudomonas genome as possible<br />
</p><br />
<br />
<h2>Week 17 (August 20-August 24)</h2><br />
<p>No work on the transposon library was done this week, other than planning for the upcoming screen.</p><br />
<br />
<h2>Week 18 (August 27-August 31)</h2><br />
<p>This week, a large screen was accomplished. First, cultures of <i>P. fluorescens</i> Pf-5 and <i>E. coli</i> SM10 were grown up overnight at 30&deg;C and 37&deg;C respectively to a high optical density. The following morning, the <i>E. coli</i> was subcultured in a 1/4 dilution to dilute out the tetracycline, and grown for approximately 2 more hours. After this, 100 &micro;L of each culture were mixed, spun down, and resuspended in 20 &micro;L of media before being spotted onto LB plates.</p><br />
<p>In total, 500 separate mating spots were plated. These spots were allowed to grow at 37&deg;C overnight. The following day, 2 mating spots were scraped up, combined, and resuspended in 500 &micro;L PBS. 1/400 dilutions of these resuspensions were made, and 100 &micro;L of these was plated onto selective plates consisting of PIA, 100mg/L NAs, and tetracycline. These plates had 40 &micro;L of 20 mg/mL X-gal spread on their surface in order to allow for blue-white screening. 250 plates were made in total.</p><br />
<br />
<p></html>[[File:2012-08-29 21-32-07 236 Calgary.jpg|center|500px|thumb|Figure 5: Plates in incubator]]<html> <p><br />
<br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<br />
<p>Plates were left to grow for 2 days, after which blue colonies (24 in total) were selected and pinned in duplicate into 96-well plates for response testing. Initially, minimal media + lactose with and without NAs was used, however no growth in any of the wells was observed. Because of this, the screening protocol was altered such that LB with or without NAs was used instead of minimal media, and X-gal was used instead of lactose for screening- the idea being that if naphthenic acids were sensed, a blue color change would be observed relative to the negative LB control.</p><br />
<br />
<p></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 6: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16h. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><br />
<br />
<p>When results were observed it was found that 4 colonies showed clear differential regulation in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. Therefore, these colonies will be used in further screening to test the specificity of the response.</p><br />
<br />
<br />
<h2>Week 19 (September 10- September 14)</h2><br />
<p>This week, further screens on the previously identified four hits were performed. These involved the use of different toxins at environmentally relevant concentrations to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used in one of the media samples in order to attempt to rule out a general stress response by the cell.<br />
<br />
<p></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 7: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:170-1data.png|thumb|600px|center|Figure 8: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 9: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 10: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p>Due to these results, further screens on these two colonies will be performed, using lower hydrogen peroxide concentrations to rule out a general stress response, and decanoic acid to rule out a response to fatty acids.</p><br />
<br />
<h2>Week 20 (September 17- September 21)</h2><p><br />
Further screens were conducted, however due to cells drying out in the plate the results were invalidated. Because of this, the screen will be repeated, and explained in further detail when this is done.</p><br />
<br />
<h2>Week 21 (September 24- September 28)</h2><p><br />
Genomes of 66-1 and 170-1 were isolated, digested with BglII and with XbaI, and religated before being transformed into <i>E.coli</i>. Cells were plated onto tetracycline plates to isolate cells containing a ligation product with the transposon present. These colonies have been miniprepped, and are awaiting being sent for sequencing to determine which genes the transposon has been inserted into. </p><br />
<br />
<h2>Week 22 (October 1-October 5)</h2><p><br />
Minipreps from previous weeks of the transposon self-cloned plasmid were nanodropped, and it was determined that no DNA had been isolated. Therefore, colonies were re-prepped, and sent for sequencing (as sequencing primers have arrived from Dr. Hynes).</p><p><br />
In addition, the assay to test the specificity of the response of the transposon clones was repeated. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The assay was carried out according to the <b>protocol</b> on our protocols section. This data was recieved after Wiki-Freeze had happened, and therefore will be analyzed at a later date. </p><br />
<br />
<h2>Week 23 (October 8-October 11)</h2><p><br />
Unfortunately, no transposon work was accomplished this week.<br />
<br />
<h2>Week 24 (October 15-October 19)</h2><p><br />
Sequencing reactions came back as having no reads, so sequencing samples were resent. This happened multiple times this week, and it is unclear why reactions continue to fail.<br />
<br />
<h2>Week 25 (October 22-October 26)</h2><p><br />
This week, data from the previously run assay was analyzed. The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure 8:Stress response screen on <i>P. fluorescens</i>Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions (shown are '''A:''' 66-1 undiluted, '''B:''' 66-1 at 1/10 dilution, '''C:''' 170-1 undiluted, '''D:''' 170-1 at 1/10 dilution) into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 %micro&L of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.]]<html><p><br />
These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.</p><br />
<br />
<p>In addition, electrochemical tests were conducted on the transposon clones this week. The results can be seen on the <b>page</b>.<br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/PromoterScreenTeam:Calgary/Notebook/PromoterScreen2012-10-26T21:40:50Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookGreen|<br />
TITLE=Transposon Library Notebook|<br />
CONTENT =<br />
<html><br />
<!--<br />
NOTE: This is a template for entering things for the time being. All dates should be enclosed in <h2> tags and all paragraphs should be enclosed in <p> tags. For bulleted lists, <ul> tags will create the list and <li> tags will surround each list item. If there are any questions, please let me know.<br />
<br />
Patrick.<br />
--><br />
<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>&nbsp;&nbsp;This was the first week where we met with other team members and summarized the primary subprojects the team will be tackling this coming summer.</p><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>&nbsp;&nbsp;During this week literature searches were performed.</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>&nbsp;&nbsp;During this week literature searches were performed.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>&nbsp;&nbsp;During this week, strains of <i> Pseudomonas fluorescens</i> PF-5 were obtained. Two cultures were started by adding 500 &micro;L stock to 10 mL LB media containing 50 mg/L ACROS Naphthenic Acids. These cultures were grown at 30&deg;C overnight, shaking at 110 rpm.</p><br />
<p>&nbsp;&nbsp;Overnight cultures were then streaked on LB agar the following day with various types and concentrations of antibiotics in order to determine the susceptibility profile of the organism. This was necessary in order to determine what marker could be used on a transposon to allow for selection of organisms with sucessful transposon insertions. These plates were grown overnight to look for death or growth, and the following results were obtained; </p><br />
<br />
<center><table><br />
<table border="2" CELLPADDING=3 CELLSPACING=1 <br />
RULES=COLS FRAME=VSIDES><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Gentamycin</b></td><br />
<td><b>Kanamycin</b></td><br />
<td><b>Chloramphenicol</b></td><br />
<td><b>Tetracycline</b></td><br />
</tr><br />
<tr><br />
<td>25 &micro;g/ml = no growth</td><br />
<td>5 &micro;g/ml = slight growth</td><br />
<td>5 &micro;g/ml = growth</td><br />
<td>50 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td>50 &micro;g/ml = no growth</td><br />
<td>10 &micro;g/ml = slight growth</td><br />
<td>10 &micro;g/ml = growth</td><br />
<td>100 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td>100 &micro;g/ml = no growth</td><br />
<td>25 &micro;g/ml = no growth</td><br />
<td>25 &micro;g/ml = growth</td><br />
<td>200 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td>50 &micro;g/ml = no growth</td><br />
<td>50 &micro;g/ml = growth</td><br />
<td> </td><br />
</tr><br />
</table></center><br />
<br />
<br />
<p><br />
<br></br> &nbsp;&nbsp;Based on these results, it was determined that Kanamycin, Gentamycin, and Tetracycline could be used as the selectable marker on the transposon, while Chloramphenicol could not as the strain is naturally resistant. Glycerol stocks of the strains were also made at this time from a fresh overnight culture.</p><br />
<br><br><br />
<h2>Week 5 (May 28-June 1)</h2><br />
<p>&nbsp;&nbsp;At the beginning of this week we spoke to Dr. Michael Hynes, who was able to give us <i> E. coli </i> SM10 and SM17-1 cells containing the plasmid pOT182. This plasmid contains an <i> E. coli </i> origin of replication, allowing it to act as a suicide vector when transferred to a different bacterial species. pOT182 contains a Tn5 transposon element containing a promotorless <i>lacZ</i> gene, genes for tetracycline resistance as well as a beta-lactamase, transposase and an <i>E. coli</i> origin of replication. These elements are bordered by insertion element sequences which are recognised by the transposase. When transferred to a different host through conjugation, the plasmid itself can no longer replicate. The transposase however can recognise and transfer the sequence between the insertion elements in a cut-and-paste fashion randomly into the genome. In this fashion, the tetracycline and beta-lactam resistant traits would only persist in cells in which the transposon has jumped into the genome, allowing these antibiotics to select for transposon positive cells. The lacZ protein will only be produced if the transposon jumps in frame downstream of a promotor, and in this case would allow for a lacZ based assay of the promotors response. The <i>E. coli</i> origin of replication present in the transposon allows for the self-cloning of the transposon in a plasmid format after genomic digestion and circularization and transformation into <i>E. coli</i>, allowing for the sequence of bordering gene fragments to be determined easily and therefore mapping the transposon in the genome.</P><br />
<br />
<br />
<br />
<p>&nbsp;&nbsp;Cultures of <i>E. coli</i> SM10 and <i>P. fluorescens</i> PF-5 were grown up overnight in shakers at 37&deg;C and 30&deg;C respectively. SM10 was grown in LB + 10 &micro;g/ml Tet, and PF-5 was grown in LB + 50 mg/L ACROS naphthenic acids. In the morning, the SM10 culture was subcultured (1/4) into LB without antibiotics and allowed to grow for an additional 4h. After this, 3 replicates of mating mixtures were made with 500 &micro;l of each culture were mixed, and the cells were spun down and resuspended in 50 &micro;l of LB. These samples were then plated in separate spots on LB agar. Additional spots were made in the same fashion with just PF-5 culture and just SM10 culture as controls, and the plate was incubated at 37&deg;C overnight. In the morning, each spot was resuspended in 500&micro;l sterile water + 25 &micro;g/ml Tet, and dilutions of 1, 1/10, 1/100, and 1/1000 of each mating mixture was plated on Pseudomonas Isolation Agar (PIA) + 10&micro;g/l Tet + 50 mg/L ACROS naphthenic acids. These cultures were allowed to grow at 30&deg;C over the weekend. </p><br />
<p>&nbsp;&nbsp;The purpose of the PIA is to selectively allow the growth of the PF-5 strain, while killing off the donor SM10 strain. The tetracycline is designed to select for the positive transposon mutants in the PF-5 strain, as the only way that tetracycline resistance would be acquired (barring spontaneous mutation events) would be if the transposable element had jumped into the genome of the cell. We decided to use 10 &micro;g/ml as the concentration of the tetracycline in the plates because we believed that 50 &micro;g/ml would be too high for even strains carrying resistance to survive. Seeing as 10 &micro;g/ml was effective for <i>E. coli</i>, we chose to try this. After streaking the PF-5 culture on the plate to test for its resistance however, it was found that the strain without the transposon was able to grow slightly on the plates, meaning that the concentration of antibiotic is not high enough to properly select for transposon mutants v.s. untransformed cells. In order to try to remedy this, the mating spots were resuspended in a mixture containing a higher dose of antibiotics. The results of this experiment are pending. </p><br />
<br />
<h2>Week 6 (June 4-8)</h2><br />
<br />
<p>&nbsp;&nbsp;When the plates from last week were examined, it was found that though the PIA was sucessful in inhibiting the growth of the <i>E. coli</i> donor strain, the original PF-5 strain was capable of growth on the media. Because of this, the plates were not selective towards cells containing the transposon insertion, and thus lawns of bacteria were seen on each of the plates.</p><br />
<p>&nbsp;&nbsp;Because of this, new selective media plates were prepared. These contained LB agar + 50 mg/L ACROS NA's + 50 &micro;g each chloramphenicol and tetracycline. The chloramphenicol was used in order to kill off the SM10 donor strain, while the tetracycline was used at a concentration previously shown to kill off the PF-5 cells that did not contain a transposon insertion. The conjugation procedure previously described was repeated, with 1 replicate mating spot being plated in 4 different dilutions on the new selective media. These plates were grown overnight at 30&deg;C. It was found that both SM10 and unmodified PF-5 were not capable of growth on the new selective plates, and colonies were found growing on all 4 of the dilutions for the mating spot.<br />
<br />
</html>[[File:Ucalgary2012_TMscreenpractice.png|center|thumb|400px|Figure 1: Transposon selective plates. WT Pf-5 and the donor strain are not capable of growth, however colonies of Pf-5 containing the transposon are capable of growth.]]<html><br />
<br />
<p>&nbsp;&nbsp;Because the host and the untransformed cells were not capable of growth on the selective plates, it is believed that these colonies must represent sucessful transposition events, as this would be the only way that the tetracycline resistance would be transfered to the PF-5 cells (chloramphenicol would have no effect, as PF-5 cells are naturally resistant at this concentration, as previously shown). Because the cell density was too high, the 1 and 1/10 dilution plates were discarded, while the 1/100 and 1/1000 plates were stored at 4&deg;C until the next step of the procedure, in which screening for naphthenic acid response will be performed. </p><br />
<br />
<h2>Week 7 (June 11-15) </h2> <br />
<br />
<p>&nbsp;&nbsp;In order to test for a naphthenic acid response, a lacZ reporter system in the transposon will be utilized. Because the lacZ enzyme is capable of degrading X-gal, a dissacharide sugar containing lactose, into lactose, and an insoluble sugar (5,5'-dibromo-4,4'-dichloro-indigo), which appears blue. Therefore, in response to activation of a native gene promotor, in frame transposon insertions will produce lacZ at levels corresponding to the activation level of the gene, and these colonies will be able to utilize lactose as a carbon source as well as utilize Xgal as a substrate. Cells responding to naphthenic acids will therefore show blue pigmentation and be capable of growth on lactose. Those that do not respond should remain white, and perish when lactose is given as the only sugar source.</p><br />
<p>&nbsp;&nbsp;The concentration of naphthenic acids used in the test plates will be 4x less than the minimal inhibitory concentration (MIC). Therefore, a MIC assay for naphthenic acids with PF-5 cells was carried out overnight at 30&deg;C at 2%, 1%, 0.5%, 0.25%, and 0.125% concentrations in LB media. It was found that PF-5 grew well at all these concentrations; therefore 1% was arbitrarily picked for the test conditions for naphthenic acid response plates.</p><br />
<p><br />
Plates were made as follows, with Xgal spread on top:</p><br />
<br />
<center><table><br />
<table border="1" CELLPADDING=3 CELLSPACING=1 <br />
RULES=NONE FRAME=BOX table width=600px height=150px><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Wild-Type PF-5 Controls</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media Alone, M9 Minimal Media + 0.4% Glucose, M9 Minimal Media + 0.4% Lactose, M9 Minimal Media + 1% NA's</td><br />
</tr><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Transposon Replica Plates</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media + 0.4% Glucose + Chlor, M9 Minimal Media + 0.4% Glucose + Chlor + 1% NA's, M9 Minimal Media + 0.4% Lactose + Tet, M9 Minimal Media + 0.4% Lactose + Tet + 1% NA's </td><br />
</tr><br />
</table></center><br />
<br />
<br />
<p><br />
<br></br><br />
&nbsp;&nbsp;Colonies from the 1/1000 transposon plate were replica stamped using velvet cloth onto the transposon test plates, and WT PF-5 was streaked onto the controls. The plates were allowed to grow over the weekend at 30 &deg;C. </p><br />
<br />
<h2>Week 8 (June 18-22)</h2><br />
<br />
<p>&nbsp;&nbsp;The plates from the previous week were observed on Monday. WT PF-5 grew on M9 + Glucose, but not on any of the other controls, indicating that the wild-type strain could not utilize lactose, indicating that this could possibly be used as a selection measure for responses from the transposon mutants. </p><br />
<p>&nbsp;&nbsp;No growth was observed on any of the transposon test plates. This was believed to be likely from poor transfer during stamping, resulting from a poor contact with the plates in addition to a grease layer formed by the undissolved naphthenic acids. Because of this, a new method of creating plates had to be determined.</P><br />
<p>&nbsp;&nbsp;We tried to dissolve NAs in DMF and spread this mixture on top of agar plates as well as mix it into the liquid agar before pouring plates. Though this helped to solubilize the naphthenic acids, it was not as effective as increasing the pH of the media (pH 8 was found to be effective). We made M9 media at pH 8 before making the same set of test plates as before. It was found that the NAs stayed in solution when mixed, however came out of solution when the plates dried, leading to a grease layer on the plates as previously seen. </P><br />
<p>&nbsp;&nbsp;In another attempt to raise the pH, stock solutions of NAs dissolved in NaOH at pH 12 were made, and these were autoclaved alongside the media. When NAs were added to the NaOH, the solutions became cloudy and uniform, indicating they were in solution. When the NA stocks were added to the media before plate pouring, the NAs remained in solution even after the plates dried, indicating that this method would be sucessful in making test plates. Using this method, the following plates were created:<br />
<br />
<center><table><br />
<table border="1" CELLPADDING=3 CELLSPACING=1 <br />
RULES=NONE FRAME=BOX table width=600px height=150px><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Wild-Type PF-5 Controls</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media Alone, M9 Minimal Media + 0.2% Glucose, M9 Minimal Media + 0.2% Lactose</td><br />
</tr><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Transposon Replica Plates</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media + 0.2% Glucose + Chlor, M9 Minimal Media + 0.2% Glucose + Chlor + 0.05% NA's, M9 Minimal Media + 0.2% Glucose + Chlor + 1% NA's, M9 Minimal Media + 0.2% Lactose + Tet, 0.2% Lactose + Tet + 0.05% NA's, M9 Minimal Media + 0.2% Lactose + Tet + 1% NA's </td><br />
</tr><br />
</table></center><br />
<br />
<p><br />
<br></br><br />
&nbsp;&nbsp;Xgal was spread on all the plates with NA's, however it was left out of the controls by mistake. Stamping was carried out as previously described from both the previously created 1/100 and 1/1000 dilution plates of transposon mutants (A new round of transposon mutagenesis was initiated, but the mating mixture was incubated at 30&deg;C instead of 37&deg;C, and no mutants were obtained on the selective plates). These plates were allowed to grow over the weekend at 30&deg;C.</P><br />
<br />
<h2>Week 9 (June 25-June 29)</h2><br />
<p>Growth was observed from the selection plates incubated over the weekend at 30&deg;C. Screening replica plates are made as before. The 1/1000 diluted transposon plate was used for the replica plating on all the screening conditions. The screening plates are incubated overnight at 30&deg;C. The next day, no growth was observed on the negative control plates (M9 alone, M9 with 0.05% NAs), and no growth was observed on the positive control plates containing glucose. Similarly, no growth was observed on the M9+lactose (+Tet, with/without X-GAL, with/without 0.05% NAs). This suggests that either a large number of matings is needed before a desirable transconjugant is seen, or that the media condition requires modification. The plate surface is not as greasy as before, which suggests that the replica plating transfer process works. However, the pH of the media may affect the growth of transconjugants. Plans are made to modify the pH of the media before replica plating. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>Replica plating screening plates are made so that NA stock solutions (1% at pH 12 diluted to 0.05% final concentration) were added to the M9 agar solution. The pH is adjusted to 7.4 before autoclaving. The pH prior to adjusting is approximately 9.0. This may explain the poor growth observed in the previous trial. A new series of mating were started (2 mating spots), and incubated overnight at 37&deg;C. The mating spots are scraped and serial diluted up to 1/1000, plated on LB+Chlor50+Tet50, and incubated overnight at 30&deg;C. This time, the results are consistent with the previous attempt, where no growth was observed on the negative control plates (M9 alone, M9 with 0.05% NAs), and ample growth was observed on the positive control plates containing glucose. However, still no growth was observed on the M9+lactose (+Tet, with/without X-GAL, with/without 0.05% NAs)plates. This seems to confirm with previous hypothesis that a large number of matings are needed to screen the genome for NA sensitive elements. Alternatively, perhaps the NA concentration used here is too high for proper selection of the transconjugants that have a promoter element upstream of the transposon insertion. If the NA concentration used for the screen is too high, the promoter may be suppressed, and the lacZ is not expressed for cell survival. In fact, the lowest NA concentration used up to this point is 0.05% or 500 mg/L. The culturing conditions required to maintain Pseudomonas Pf-5's NA degrading abilities is LB+50mg/L. Therefore, to allow both cell survival and screen for the most robust and sensitive system, maybe the NA concentration should be lowered.</p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The exact same experiment from the previous week has been repeated with two more mating mixtures (and their dilutions). However, we obtained the same results, with the positive and negative controls yielding predicted growth and no growth, respectively. But no growth was observed on the lactose plates. Furthermore, upon close examination of the selection plates after the conjugating/bipartite mating reaction (LB+Chlor+Tet), two colony morphologies can be seen: drier and larger colonies (resembling Pseudomonas), and smaller moist colonies (resembling E. coli). This suggests perhaps the LB+Chlor+Tet plates may be useful for selecting Pseudomonas transconjugants, but it does not prevent the donor E. coli to be efficiently killed. A final replica plating experiment is designed for next week, and Pseudomonas Isolation agar (PIA, just received)will be used for selection instead. </p> <br />
<br />
<h2>Week 12 (July 16-July 20)</h2><br />
<p>Another mating reaction was set up, and plated on PIA+Tet50 to select for positive transconjugants. The colonies from the resulting mutants show only a single colony morphology consistent with Pseudomonas. From this, we know the selection is appropriate. The 1/1000 dilution of the mating reaction dilution is replica-plated on two sets of screening plates. The first set is prepared the same way as the previous week (NAs added and the media pH adjusted prior to autoclaving); the second set is the same except the NAs are added by spreading 100uL of a 50mg/L sterile stock NA solution in NaOH (pH12) prior to replica plating. The 1/1000 dilution plate of the mating reaction is replica plated on both plates (with the same controls as before). However, the same results are obtained as before, where no growth is observed on the M9+0.2% lactose+X-GAL or the M9+0.2% lactose+X-GAL+50mg/L NAs plates. Lastly, we had an interesting observation where overnight growth of the lactose screening plates showed no growth, if they are left for up to 48hr, some colonies can be observed; however, none of the colonies are blue. The problem may be that the M9 media conditions are not suitable for selecting the Tn mutants, or that the replica plating approach we used cannot effectively transfer all colonies. At this point, since no NA-sensitive strains are isolated, a new approach needs to be taken in order to find a tranposon insertion mutant that has an NA-sensitive promoter upstream. Since all possible approaches have been taken using the replica plating strategy, another mass screening method is needed. </p><br />
<br />
<h2>Week 13 (July 23-July 27)</h2><br />
<p> Even though, the previous screening method is quick and convenient, the waiting period is too long, and no real time data in terms of growth or lactose utilization can be observed/discerned. Therefore, a 96-well mass screening method is devised, where the conjugation mating reaction, and the selection on PIA+Tet50 are the same, but the screening step is different. The screening media is a M9 minimal media with 50 µg/mL tetracycline to maintain the genomic Tn insertions, 0.2% lactose to select for lactose utilization due to Tn insertions, and 50 mg/L NAs to maintain the strain's ability to use/degrade NAs (in short the media is M9+0.2%lactose+50µg/mL Tet+50mg/L NAs). On each 96-well plate, 94 colonies are inoculated and screened; the other two wells are used for negative and positive controls. The negative control well has the same media (M9+0.2%lactose+50µg/mL Tet+50mg/L NAs), with no colonies inoculated. The positive control has M9+0.2%glucose+50µg/mL Tet+50mg/L NAs, and is inoculated with a random colony.<br />
<br />
</p><p><br />
<br />
Using this new method, any inoculated colony that grows in the liquid media can utilize lactose, and this should be because of a transposon insertion downstream of a promoter. To select for a NA sensitive promoter, each colony is inoculated into two plates with the same media components (as described above), except one has NAs, and the other does not. If a promoter is NA-sensitive, then it should not grow if NAs are absent from the media, as the NA mixture would activate the promoter. When NAs are present however, the NA-sensitive promoter would turn on, allowing it to utilize lactose, survive, and grow. <br />
<br />
As a first run of this method, a plate is set up using mutant colonies from a previous mating reaction. This plate contained M9+0.2%lactose+50µg/mL Tet+50mg/L NAs. The appropriate control plate with no NAs was not included as this is only to test feasibility of this approach. The plate is incubated over the weekend for 60hrs in a plate reader at 30&deg;C. </p><br />
<br />
<h2>Week 14 (July 30-August 3)</h2><br />
<p>The data from the plate incubated from last week was collected. First, since the lid of the 96-well plate was kept on the plate to prevent dehydration of the media, there was significant condensation on the middle of the lid (covering about 30 wells), which prevented proper absorbance measurements of those wells (data from these wells were omitted). However, six wells/colonies demonstrated growth from a baseline. The following table is a display of the 96-well plate at the end point, with the colonies/wells that grow were highlighted.</p><br />
<br />
</html>[[File:Ucalgary2012 0801data1.png|center|thumb|400px|Figure 2: A 60hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media]]<html><br />
<br />
<p>The cultures from these six wells are washed with M9+0.2%lactose+50µg/mL Tet (no NAs) three times, and resuspended in 1 mL of M9+0.2%lactose+50µg/mL Tet. 500µl of each colony was inoculated into 5mL cultures with M9+0.2%lactose+50µg/mL Tet or M9+0.2%lactose+50µg/mL Tet+50mg/L NAs. These cultures are grown over 48 hours to observe growth (any strain that grows only when NAs are added would be noted). Also, the colonies are restreaked on PIA+Tet50 plates to ensure that the absorbance increase actually indicated growth (since the increase was so small at around +0.200). The results were not promising, on the restreaked plates, only 2 colonies (from H12 and B1) showed growth, this suggests some of the measurements were not very accurate. Also, in the culturing experiment, only 1 set of cultures (from 1 colony, H12) showed growth, but in both media conditions, which is not desirable.</p><br />
<br />
<p>Even though, no colonies were shown as NA sensitive, this result is still very promising, as this experiment shows that some colonies from the transposon mutagenesis can actually utilize lacZ, which demonstrates that the transposable element approach is an appropriate and feasible approach for screen promoters sensitive to environmental stimuli. (Please note, that mating reactions and mutant selection are conducted regularly every week, usually twice per week, with two mating spots each time, to provide mutants for these screens.)</p><br />
<br />
<h2>Week 15 (August 6-August 10)</h2><br />
<p><br />
The same experiment from the previous week was repeated. However, the plate was not incubated in the plate reader, but incubated in a 30&deg;C shaker, at 140rpm. At the baseline (beginning of incubation), 24hr, and 48hr, timepoint measurements were made. The results are as follows.<br />
</p><p><br />
</html>[[File:Ucalgary2012_080312.png|center|thumb|400px|Figure 3: A 48hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media at time points (baseline, 24hr, and 48hr)]]<html><br />
</p> <br />
<br />
<p><br />
However, these measurements were made with the lid of the plate on. Even though there was no clear trend in terms of the growth in the absorbance data, it is possible to observe some wells become cloudy by eye. For instance, well F9 was cloudy at the end of the 48hr incubation, but the data showed no change in absorbance. The lid should be taken off in the future when making timepoint measurements. Also, the absorbance data can support the visual data.<br />
</p><br />
<br />
<p><br />
Also, this week, two runs of the screening experiment was conducted with two plates run in parallel, one with NAs and the other without (the proper screening setup). However, it was found that the lactose solution (which was filter sterilized) used to make the M9 media for the screen was contaminated with yeast. Therefore, new media solutions and lactose solutions needed to be made. These data are not used because of this.<br />
</p><br />
<br />
<h2>Week 16 (August 13-August 17)</h2><br />
<p><br />
New lactose and glucose stock solutions as well as new M9 screening media were prepared. The experiments from last week were repeated using proper solutions. Also, to prevent fogging of the plate lid and cross-contamination, all media solutions and plates were pre-warmed in a 30&deg;C incubator before dispensing the media into the plates and colony inoculation. The new trial, again, has two plates, with the same wells (e.g. A1) inoculated with the same colony, where one plate has M9 screening media (with Tet and lactose) alone and the other plate containing 50mg/L NAs. Two runs were completed this week. The data from one of those trials are shown below.<br />
</p><p><br />
</html>[[File:Ucalgary2012 081612.png|center|thumb|800px| Figure 4: A 48hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media at time points (baseline, 24hr, and 48hr), comparing the same colonies in M9 screening media with/without 50mg/L NAs]]<html><br />
<br />
<p><br />
In this particular run, only two colonies showed growth (D8 and H11), these were subcultured in 5mL of M9 screening media with NAs. An interesting observation is that there is more growth in the lactose+NAs plate than the lactose alone plate. This suggests that the NAs may play a role in activating the lacZ gene, and improving the survival rates. These results are consistently observed in the other trials conducted. <br />
<br />
<p><br />
At this point, since time is limited, the entire mutant selection process needs to be more efficient. Therefore, the mating spots are diluted, and plated on a more selective media, containing PIA+50ug/mL tetracycline+50mg/L NAs+20 μg/mL X-Gal. This way, the NA-responsive and/or lactose-utilizing Pseudomonas transconjugants can be selected as they would appear blue on the media. This reduces the number of colonies to be inoculated, and improves the efficiency of the whole process. (Note: Wildtype Pseudomonas fluorescens pf-5 are always plated as positive control to observe the natural forward mutation rates, and to ensure that the amount of spontaneous tetracycline resistant Pf-5 mutants are at a reasonable level.<br />
</p><br />
<br />
<p><br />
At this point, a large number of matings should be conducted and mutants selected, in order to provide enough of lactose-utilizing and NA-sensitive Tn-insertion mutants, and to cover as much of the Pseudomonas genome as possible<br />
</p><br />
<br />
<h2>Week 17 (August 20-August 24)</h2><br />
<p>No work on the transposon library was done this week, other than planning for the upcoming screen.</p><br />
<br />
<h2>Week 18 (August 27-August 31)</h2><br />
<p>This week, a large screen was accomplished. First, cultures of <i>P. fluorescens</i> Pf-5 and <i>E. coli</i> SM10 were grown up overnight at 30&deg;C and 37&deg;C respectively to a high optical density. The following morning, the <i>E. coli</i> was subcultured in a 1/4 dilution to dilute out the tetracycline, and grown for approximately 2 more hours. After this, 100 &micro;L of each culture were mixed, spun down, and resuspended in 20 &micro;L of media before being spotted onto LB plates.</p><br />
<p>In total, 500 separate mating spots were plated. These spots were allowed to grow at 37&deg;C overnight. The following day, 2 mating spots were scraped up, combined, and resuspended in 500 &micro;L PBS. 1/400 dilutions of these resuspensions were made, and 100 &micro;L of these was plated onto selective plates consisting of PIA, 100mg/L NAs, and tetracycline. These plates had 40 &micro;L of 20 mg/mL X-gal spread on their surface in order to allow for blue-white screening. 250 plates were made in total.</p><br />
<br />
<p></html>[[File:2012-08-29 21-32-07 236 Calgary.jpg|center|500px|thumb|Figure 5: Plates in incubator]]<html> <p><br />
<br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<br />
<p>Plates were left to grow for 2 days, after which blue colonies (24 in total) were selected and pinned in duplicate into 96-well plates for response testing. Initially, minimal media + lactose with and without NAs was used, however no growth in any of the wells was observed. Because of this, the screening protocol was altered such that LB with or without NAs was used instead of minimal media, and X-gal was used instead of lactose for screening- the idea being that if naphthenic acids were sensed, a blue color change would be observed relative to the negative LB control.</p><br />
<br />
<p></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 6: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16h. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><br />
<br />
<p>When results were observed it was found that 4 colonies showed clear differential regulation in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. Therefore, these colonies will be used in further screening to test the specificity of the response.</p><br />
<br />
<br />
<h2>Week 19 (September 10- September 14)</h2><br />
<p>This week, further screens on the previously identified four hits were performed. These involved the use of different toxins at environmentally relevant concentrations to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used in one of the media samples in order to attempt to rule out a general stress response by the cell.<br />
<br />
<p></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 7: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:170-1data.png|thumb|600px|center|Figure 8: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 9: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 10: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p>Due to these results, further screens on these two colonies will be performed, using lower hydrogen peroxide concentrations to rule out a general stress response, and decanoic acid to rule out a response to fatty acids.</p><br />
<br />
<h2>Week 20 (September 17- September 21)</h2><p><br />
Further screens were conducted, however due to cells drying out in the plate the results were invalidated. Because of this, the screen will be repeated, and explained in further detail when this is done.</p><br />
<br />
<h2>Week 21 (September 24- September 28)</h2><p><br />
Genomes of 66-1 and 170-1 were isolated, digested with BglII and with XbaI, and religated before being transformed into <i>E.coli</i>. Cells were plated onto tetracycline plates to isolate cells containing a ligation product with the transposon present. These colonies have been miniprepped, and are awaiting being sent for sequencing to determine which genes the transposon has been inserted into. </p><br />
<br />
<h2>Week 22 (October 1-October 5)</h2><p><br />
Minipreps from previous weeks of the transposon self-cloned plasmid were nanodropped, and it was determined that no DNA had been isolated. Therefore, colonies were re-prepped, and sent for sequencing (as sequencing primers have arrived from Dr. Hynes).</p><p><br />
In addition, the assay to test the specificity of the response of the transposon clones was repeated. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The assay was carried out according to the <b>protocol</b> on our protocols section. This data was recieved after Wiki-Freeze had happened, and therefore will be analyzed at a later date. </p><br />
<br />
<h2>Week 23 (October 8-October 11)</h2><p><br />
Unfortunately, no transposon work was accomplished this week.<br />
<br />
<h2>Week 24 (October 15-October 19)</h2><p><br />
Sequencing reactions came back as having no reads, so sequencing samples were resent. This happened multiple times this week, and it is unclear why reactions continue to fail.<br />
<br />
<h2>Week 25 (October 22-October 26)</h2><p><br />
This week, data from the previously run assay was analyzed. The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure 8:Stress response screen on <i>P. fluorescens</i>Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions (shown are '''A:''' 66-1 undiluted, '''B:''' 66-1 at 1/10 dilution, '''C:''' 170-1 undiluted, '''D:''' 170-1 at 1/10 dilution) into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 %micro&L of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.]]<html><p><br />
These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.</p><br />
<br />
<br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/PromoterScreenTeam:Calgary/Notebook/PromoterScreen2012-10-26T21:38:40Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookGreen|<br />
TITLE=Transposon Library Notebook|<br />
CONTENT =<br />
<html><br />
<!--<br />
NOTE: This is a template for entering things for the time being. All dates should be enclosed in <h2> tags and all paragraphs should be enclosed in <p> tags. For bulleted lists, <ul> tags will create the list and <li> tags will surround each list item. If there are any questions, please let me know.<br />
<br />
Patrick.<br />
--><br />
<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>&nbsp;&nbsp;This was the first week where we met with other team members and summarized the primary subprojects the team will be tackling this coming summer.</p><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>&nbsp;&nbsp;During this week literature searches were performed.</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>&nbsp;&nbsp;During this week literature searches were performed.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>&nbsp;&nbsp;During this week, strains of <i> Pseudomonas fluorescens</i> PF-5 were obtained. Two cultures were started by adding 500 &micro;L stock to 10 mL LB media containing 50 mg/L ACROS Naphthenic Acids. These cultures were grown at 30&deg;C overnight, shaking at 110 rpm.</p><br />
<p>&nbsp;&nbsp;Overnight cultures were then streaked on LB agar the following day with various types and concentrations of antibiotics in order to determine the susceptibility profile of the organism. This was necessary in order to determine what marker could be used on a transposon to allow for selection of organisms with sucessful transposon insertions. These plates were grown overnight to look for death or growth, and the following results were obtained; </p><br />
<br />
<center><table><br />
<table border="2" CELLPADDING=3 CELLSPACING=1 <br />
RULES=COLS FRAME=VSIDES><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Gentamycin</b></td><br />
<td><b>Kanamycin</b></td><br />
<td><b>Chloramphenicol</b></td><br />
<td><b>Tetracycline</b></td><br />
</tr><br />
<tr><br />
<td>25 &micro;g/ml = no growth</td><br />
<td>5 &micro;g/ml = slight growth</td><br />
<td>5 &micro;g/ml = growth</td><br />
<td>50 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td>50 &micro;g/ml = no growth</td><br />
<td>10 &micro;g/ml = slight growth</td><br />
<td>10 &micro;g/ml = growth</td><br />
<td>100 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td>100 &micro;g/ml = no growth</td><br />
<td>25 &micro;g/ml = no growth</td><br />
<td>25 &micro;g/ml = growth</td><br />
<td>200 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td>50 &micro;g/ml = no growth</td><br />
<td>50 &micro;g/ml = growth</td><br />
<td> </td><br />
</tr><br />
</table></center><br />
<br />
<br />
<p><br />
<br></br> &nbsp;&nbsp;Based on these results, it was determined that Kanamycin, Gentamycin, and Tetracycline could be used as the selectable marker on the transposon, while Chloramphenicol could not as the strain is naturally resistant. Glycerol stocks of the strains were also made at this time from a fresh overnight culture.</p><br />
<br><br><br />
<h2>Week 5 (May 28-June 1)</h2><br />
<p>&nbsp;&nbsp;At the beginning of this week we spoke to Dr. Michael Hynes, who was able to give us <i> E. coli </i> SM10 and SM17-1 cells containing the plasmid pOT182. This plasmid contains an <i> E. coli </i> origin of replication, allowing it to act as a suicide vector when transferred to a different bacterial species. pOT182 contains a Tn5 transposon element containing a promotorless <i>lacZ</i> gene, genes for tetracycline resistance as well as a beta-lactamase, transposase and an <i>E. coli</i> origin of replication. These elements are bordered by insertion element sequences which are recognised by the transposase. When transferred to a different host through conjugation, the plasmid itself can no longer replicate. The transposase however can recognise and transfer the sequence between the insertion elements in a cut-and-paste fashion randomly into the genome. In this fashion, the tetracycline and beta-lactam resistant traits would only persist in cells in which the transposon has jumped into the genome, allowing these antibiotics to select for transposon positive cells. The lacZ protein will only be produced if the transposon jumps in frame downstream of a promotor, and in this case would allow for a lacZ based assay of the promotors response. The <i>E. coli</i> origin of replication present in the transposon allows for the self-cloning of the transposon in a plasmid format after genomic digestion and circularization and transformation into <i>E. coli</i>, allowing for the sequence of bordering gene fragments to be determined easily and therefore mapping the transposon in the genome.</P><br />
<br />
<br />
<br />
<p>&nbsp;&nbsp;Cultures of <i>E. coli</i> SM10 and <i>P. fluorescens</i> PF-5 were grown up overnight in shakers at 37&deg;C and 30&deg;C respectively. SM10 was grown in LB + 10 &micro;g/ml Tet, and PF-5 was grown in LB + 50 mg/L ACROS naphthenic acids. In the morning, the SM10 culture was subcultured (1/4) into LB without antibiotics and allowed to grow for an additional 4h. After this, 3 replicates of mating mixtures were made with 500 &micro;l of each culture were mixed, and the cells were spun down and resuspended in 50 &micro;l of LB. These samples were then plated in separate spots on LB agar. Additional spots were made in the same fashion with just PF-5 culture and just SM10 culture as controls, and the plate was incubated at 37&deg;C overnight. In the morning, each spot was resuspended in 500&micro;l sterile water + 25 &micro;g/ml Tet, and dilutions of 1, 1/10, 1/100, and 1/1000 of each mating mixture was plated on Pseudomonas Isolation Agar (PIA) + 10&micro;g/l Tet + 50 mg/L ACROS naphthenic acids. These cultures were allowed to grow at 30&deg;C over the weekend. </p><br />
<p>&nbsp;&nbsp;The purpose of the PIA is to selectively allow the growth of the PF-5 strain, while killing off the donor SM10 strain. The tetracycline is designed to select for the positive transposon mutants in the PF-5 strain, as the only way that tetracycline resistance would be acquired (barring spontaneous mutation events) would be if the transposable element had jumped into the genome of the cell. We decided to use 10 &micro;g/ml as the concentration of the tetracycline in the plates because we believed that 50 &micro;g/ml would be too high for even strains carrying resistance to survive. Seeing as 10 &micro;g/ml was effective for <i>E. coli</i>, we chose to try this. After streaking the PF-5 culture on the plate to test for its resistance however, it was found that the strain without the transposon was able to grow slightly on the plates, meaning that the concentration of antibiotic is not high enough to properly select for transposon mutants v.s. untransformed cells. In order to try to remedy this, the mating spots were resuspended in a mixture containing a higher dose of antibiotics. The results of this experiment are pending. </p><br />
<br />
<h2>Week 6 (June 4-8)</h2><br />
<br />
<p>&nbsp;&nbsp;When the plates from last week were examined, it was found that though the PIA was sucessful in inhibiting the growth of the <i>E. coli</i> donor strain, the original PF-5 strain was capable of growth on the media. Because of this, the plates were not selective towards cells containing the transposon insertion, and thus lawns of bacteria were seen on each of the plates.</p><br />
<p>&nbsp;&nbsp;Because of this, new selective media plates were prepared. These contained LB agar + 50 mg/L ACROS NA's + 50 &micro;g each chloramphenicol and tetracycline. The chloramphenicol was used in order to kill off the SM10 donor strain, while the tetracycline was used at a concentration previously shown to kill off the PF-5 cells that did not contain a transposon insertion. The conjugation procedure previously described was repeated, with 1 replicate mating spot being plated in 4 different dilutions on the new selective media. These plates were grown overnight at 30&deg;C. It was found that both SM10 and unmodified PF-5 were not capable of growth on the new selective plates, and colonies were found growing on all 4 of the dilutions for the mating spot.<br />
<br />
</html>[[File:Ucalgary2012_TMscreenpractice.png|center|thumb|400px|Figure 1: Transposon selective plates. WT Pf-5 and the donor strain are not capable of growth, however colonies of Pf-5 containing the transposon are capable of growth.]]<html><br />
<br />
<p>&nbsp;&nbsp;Because the host and the untransformed cells were not capable of growth on the selective plates, it is believed that these colonies must represent sucessful transposition events, as this would be the only way that the tetracycline resistance would be transfered to the PF-5 cells (chloramphenicol would have no effect, as PF-5 cells are naturally resistant at this concentration, as previously shown). Because the cell density was too high, the 1 and 1/10 dilution plates were discarded, while the 1/100 and 1/1000 plates were stored at 4&deg;C until the next step of the procedure, in which screening for naphthenic acid response will be performed. </p><br />
<br />
<h2>Week 7 (June 11-15) </h2> <br />
<br />
<p>&nbsp;&nbsp;In order to test for a naphthenic acid response, a lacZ reporter system in the transposon will be utilized. Because the lacZ enzyme is capable of degrading X-gal, a dissacharide sugar containing lactose, into lactose, and an insoluble sugar (5,5'-dibromo-4,4'-dichloro-indigo), which appears blue. Therefore, in response to activation of a native gene promotor, in frame transposon insertions will produce lacZ at levels corresponding to the activation level of the gene, and these colonies will be able to utilize lactose as a carbon source as well as utilize Xgal as a substrate. Cells responding to naphthenic acids will therefore show blue pigmentation and be capable of growth on lactose. Those that do not respond should remain white, and perish when lactose is given as the only sugar source.</p><br />
<p>&nbsp;&nbsp;The concentration of naphthenic acids used in the test plates will be 4x less than the minimal inhibitory concentration (MIC). Therefore, a MIC assay for naphthenic acids with PF-5 cells was carried out overnight at 30&deg;C at 2%, 1%, 0.5%, 0.25%, and 0.125% concentrations in LB media. It was found that PF-5 grew well at all these concentrations; therefore 1% was arbitrarily picked for the test conditions for naphthenic acid response plates.</p><br />
<p><br />
Plates were made as follows, with Xgal spread on top:</p><br />
<br />
<center><table><br />
<table border="1" CELLPADDING=3 CELLSPACING=1 <br />
RULES=NONE FRAME=BOX table width=600px height=150px><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Wild-Type PF-5 Controls</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media Alone, M9 Minimal Media + 0.4% Glucose, M9 Minimal Media + 0.4% Lactose, M9 Minimal Media + 1% NA's</td><br />
</tr><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Transposon Replica Plates</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media + 0.4% Glucose + Chlor, M9 Minimal Media + 0.4% Glucose + Chlor + 1% NA's, M9 Minimal Media + 0.4% Lactose + Tet, M9 Minimal Media + 0.4% Lactose + Tet + 1% NA's </td><br />
</tr><br />
</table></center><br />
<br />
<br />
<p><br />
<br></br><br />
&nbsp;&nbsp;Colonies from the 1/1000 transposon plate were replica stamped using velvet cloth onto the transposon test plates, and WT PF-5 was streaked onto the controls. The plates were allowed to grow over the weekend at 30 &deg;C. </p><br />
<br />
<h2>Week 8 (June 18-22)</h2><br />
<br />
<p>&nbsp;&nbsp;The plates from the previous week were observed on Monday. WT PF-5 grew on M9 + Glucose, but not on any of the other controls, indicating that the wild-type strain could not utilize lactose, indicating that this could possibly be used as a selection measure for responses from the transposon mutants. </p><br />
<p>&nbsp;&nbsp;No growth was observed on any of the transposon test plates. This was believed to be likely from poor transfer during stamping, resulting from a poor contact with the plates in addition to a grease layer formed by the undissolved naphthenic acids. Because of this, a new method of creating plates had to be determined.</P><br />
<p>&nbsp;&nbsp;We tried to dissolve NAs in DMF and spread this mixture on top of agar plates as well as mix it into the liquid agar before pouring plates. Though this helped to solubilize the naphthenic acids, it was not as effective as increasing the pH of the media (pH 8 was found to be effective). We made M9 media at pH 8 before making the same set of test plates as before. It was found that the NAs stayed in solution when mixed, however came out of solution when the plates dried, leading to a grease layer on the plates as previously seen. </P><br />
<p>&nbsp;&nbsp;In another attempt to raise the pH, stock solutions of NAs dissolved in NaOH at pH 12 were made, and these were autoclaved alongside the media. When NAs were added to the NaOH, the solutions became cloudy and uniform, indicating they were in solution. When the NA stocks were added to the media before plate pouring, the NAs remained in solution even after the plates dried, indicating that this method would be sucessful in making test plates. Using this method, the following plates were created:<br />
<br />
<center><table><br />
<table border="1" CELLPADDING=3 CELLSPACING=1 <br />
RULES=NONE FRAME=BOX table width=600px height=150px><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Wild-Type PF-5 Controls</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media Alone, M9 Minimal Media + 0.2% Glucose, M9 Minimal Media + 0.2% Lactose</td><br />
</tr><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Transposon Replica Plates</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media + 0.2% Glucose + Chlor, M9 Minimal Media + 0.2% Glucose + Chlor + 0.05% NA's, M9 Minimal Media + 0.2% Glucose + Chlor + 1% NA's, M9 Minimal Media + 0.2% Lactose + Tet, 0.2% Lactose + Tet + 0.05% NA's, M9 Minimal Media + 0.2% Lactose + Tet + 1% NA's </td><br />
</tr><br />
</table></center><br />
<br />
<p><br />
<br></br><br />
&nbsp;&nbsp;Xgal was spread on all the plates with NA's, however it was left out of the controls by mistake. Stamping was carried out as previously described from both the previously created 1/100 and 1/1000 dilution plates of transposon mutants (A new round of transposon mutagenesis was initiated, but the mating mixture was incubated at 30&deg;C instead of 37&deg;C, and no mutants were obtained on the selective plates). These plates were allowed to grow over the weekend at 30&deg;C.</P><br />
<br />
<h2>Week 9 (June 25-June 29)</h2><br />
<p>Growth was observed from the selection plates incubated over the weekend at 30&deg;C. Screening replica plates are made as before. The 1/1000 diluted transposon plate was used for the replica plating on all the screening conditions. The screening plates are incubated overnight at 30&deg;C. The next day, no growth was observed on the negative control plates (M9 alone, M9 with 0.05% NAs), and no growth was observed on the positive control plates containing glucose. Similarly, no growth was observed on the M9+lactose (+Tet, with/without X-GAL, with/without 0.05% NAs). This suggests that either a large number of matings is needed before a desirable transconjugant is seen, or that the media condition requires modification. The plate surface is not as greasy as before, which suggests that the replica plating transfer process works. However, the pH of the media may affect the growth of transconjugants. Plans are made to modify the pH of the media before replica plating. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>Replica plating screening plates are made so that NA stock solutions (1% at pH 12 diluted to 0.05% final concentration) were added to the M9 agar solution. The pH is adjusted to 7.4 before autoclaving. The pH prior to adjusting is approximately 9.0. This may explain the poor growth observed in the previous trial. A new series of mating were started (2 mating spots), and incubated overnight at 37&deg;C. The mating spots are scraped and serial diluted up to 1/1000, plated on LB+Chlor50+Tet50, and incubated overnight at 30&deg;C. This time, the results are consistent with the previous attempt, where no growth was observed on the negative control plates (M9 alone, M9 with 0.05% NAs), and ample growth was observed on the positive control plates containing glucose. However, still no growth was observed on the M9+lactose (+Tet, with/without X-GAL, with/without 0.05% NAs)plates. This seems to confirm with previous hypothesis that a large number of matings are needed to screen the genome for NA sensitive elements. Alternatively, perhaps the NA concentration used here is too high for proper selection of the transconjugants that have a promoter element upstream of the transposon insertion. If the NA concentration used for the screen is too high, the promoter may be suppressed, and the lacZ is not expressed for cell survival. In fact, the lowest NA concentration used up to this point is 0.05% or 500 mg/L. The culturing conditions required to maintain Pseudomonas Pf-5's NA degrading abilities is LB+50mg/L. Therefore, to allow both cell survival and screen for the most robust and sensitive system, maybe the NA concentration should be lowered.</p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The exact same experiment from the previous week has been repeated with two more mating mixtures (and their dilutions). However, we obtained the same results, with the positive and negative controls yielding predicted growth and no growth, respectively. But no growth was observed on the lactose plates. Furthermore, upon close examination of the selection plates after the conjugating/bipartite mating reaction (LB+Chlor+Tet), two colony morphologies can be seen: drier and larger colonies (resembling Pseudomonas), and smaller moist colonies (resembling E. coli). This suggests perhaps the LB+Chlor+Tet plates may be useful for selecting Pseudomonas transconjugants, but it does not prevent the donor E. coli to be efficiently killed. A final replica plating experiment is designed for next week, and Pseudomonas Isolation agar (PIA, just received)will be used for selection instead. </p> <br />
<br />
<h2>Week 12 (July 16-July 20)</h2><br />
<p>Another mating reaction was set up, and plated on PIA+Tet50 to select for positive transconjugants. The colonies from the resulting mutants show only a single colony morphology consistent with Pseudomonas. From this, we know the selection is appropriate. The 1/1000 dilution of the mating reaction dilution is replica-plated on two sets of screening plates. The first set is prepared the same way as the previous week (NAs added and the media pH adjusted prior to autoclaving); the second set is the same except the NAs are added by spreading 100uL of a 50mg/L sterile stock NA solution in NaOH (pH12) prior to replica plating. The 1/1000 dilution plate of the mating reaction is replica plated on both plates (with the same controls as before). However, the same results are obtained as before, where no growth is observed on the M9+0.2% lactose+X-GAL or the M9+0.2% lactose+X-GAL+50mg/L NAs plates. Lastly, we had an interesting observation where overnight growth of the lactose screening plates showed no growth, if they are left for up to 48hr, some colonies can be observed; however, none of the colonies are blue. The problem may be that the M9 media conditions are not suitable for selecting the Tn mutants, or that the replica plating approach we used cannot effectively transfer all colonies. At this point, since no NA-sensitive strains are isolated, a new approach needs to be taken in order to find a tranposon insertion mutant that has an NA-sensitive promoter upstream. Since all possible approaches have been taken using the replica plating strategy, another mass screening method is needed. </p><br />
<br />
<h2>Week 13 (July 23-July 27)</h2><br />
<p> Even though, the previous screening method is quick and convenient, the waiting period is too long, and no real time data in terms of growth or lactose utilization can be observed/discerned. Therefore, a 96-well mass screening method is devised, where the conjugation mating reaction, and the selection on PIA+Tet50 are the same, but the screening step is different. The screening media is a M9 minimal media with 50 µg/mL tetracycline to maintain the genomic Tn insertions, 0.2% lactose to select for lactose utilization due to Tn insertions, and 50 mg/L NAs to maintain the strain's ability to use/degrade NAs (in short the media is M9+0.2%lactose+50µg/mL Tet+50mg/L NAs). On each 96-well plate, 94 colonies are inoculated and screened; the other two wells are used for negative and positive controls. The negative control well has the same media (M9+0.2%lactose+50µg/mL Tet+50mg/L NAs), with no colonies inoculated. The positive control has M9+0.2%glucose+50µg/mL Tet+50mg/L NAs, and is inoculated with a random colony.<br />
<br />
</p><p><br />
<br />
Using this new method, any inoculated colony that grows in the liquid media can utilize lactose, and this should be because of a transposon insertion downstream of a promoter. To select for a NA sensitive promoter, each colony is inoculated into two plates with the same media components (as described above), except one has NAs, and the other does not. If a promoter is NA-sensitive, then it should not grow if NAs are absent from the media, as the NA mixture would activate the promoter. When NAs are present however, the NA-sensitive promoter would turn on, allowing it to utilize lactose, survive, and grow. <br />
<br />
As a first run of this method, a plate is set up using mutant colonies from a previous mating reaction. This plate contained M9+0.2%lactose+50µg/mL Tet+50mg/L NAs. The appropriate control plate with no NAs was not included as this is only to test feasibility of this approach. The plate is incubated over the weekend for 60hrs in a plate reader at 30&deg;C. </p><br />
<br />
<h2>Week 14 (July 30-August 3)</h2><br />
<p>The data from the plate incubated from last week was collected. First, since the lid of the 96-well plate was kept on the plate to prevent dehydration of the media, there was significant condensation on the middle of the lid (covering about 30 wells), which prevented proper absorbance measurements of those wells (data from these wells were omitted). However, six wells/colonies demonstrated growth from a baseline. The following table is a display of the 96-well plate at the end point, with the colonies/wells that grow were highlighted.</p><br />
<br />
</html>[[File:Ucalgary2012 0801data1.png|center|thumb|400px|Figure 2: A 60hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media]]<html><br />
<br />
<p>The cultures from these six wells are washed with M9+0.2%lactose+50µg/mL Tet (no NAs) three times, and resuspended in 1 mL of M9+0.2%lactose+50µg/mL Tet. 500µl of each colony was inoculated into 5mL cultures with M9+0.2%lactose+50µg/mL Tet or M9+0.2%lactose+50µg/mL Tet+50mg/L NAs. These cultures are grown over 48 hours to observe growth (any strain that grows only when NAs are added would be noted). Also, the colonies are restreaked on PIA+Tet50 plates to ensure that the absorbance increase actually indicated growth (since the increase was so small at around +0.200). The results were not promising, on the restreaked plates, only 2 colonies (from H12 and B1) showed growth, this suggests some of the measurements were not very accurate. Also, in the culturing experiment, only 1 set of cultures (from 1 colony, H12) showed growth, but in both media conditions, which is not desirable.</p><br />
<br />
<p>Even though, no colonies were shown as NA sensitive, this result is still very promising, as this experiment shows that some colonies from the transposon mutagenesis can actually utilize lacZ, which demonstrates that the transposable element approach is an appropriate and feasible approach for screen promoters sensitive to environmental stimuli. (Please note, that mating reactions and mutant selection are conducted regularly every week, usually twice per week, with two mating spots each time, to provide mutants for these screens.)</p><br />
<br />
<h2>Week 15 (August 6-August 10)</h2><br />
<p><br />
The same experiment from the previous week was repeated. However, the plate was not incubated in the plate reader, but incubated in a 30&deg;C shaker, at 140rpm. At the baseline (beginning of incubation), 24hr, and 48hr, timepoint measurements were made. The results are as follows.<br />
</p><p><br />
</html>[[File:Ucalgary2012_080312.png|center|thumb|400px|Figure 3: A 48hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media at time points (baseline, 24hr, and 48hr)]]<html><br />
</p> <br />
<br />
<p><br />
However, these measurements were made with the lid of the plate on. Even though there was no clear trend in terms of the growth in the absorbance data, it is possible to observe some wells become cloudy by eye. For instance, well F9 was cloudy at the end of the 48hr incubation, but the data showed no change in absorbance. The lid should be taken off in the future when making timepoint measurements. Also, the absorbance data can support the visual data.<br />
</p><br />
<br />
<p><br />
Also, this week, two runs of the screening experiment was conducted with two plates run in parallel, one with NAs and the other without (the proper screening setup). However, it was found that the lactose solution (which was filter sterilized) used to make the M9 media for the screen was contaminated with yeast. Therefore, new media solutions and lactose solutions needed to be made. These data are not used because of this.<br />
</p><br />
<br />
<h2>Week 16 (August 13-August 17)</h2><br />
<p><br />
New lactose and glucose stock solutions as well as new M9 screening media were prepared. The experiments from last week were repeated using proper solutions. Also, to prevent fogging of the plate lid and cross-contamination, all media solutions and plates were pre-warmed in a 30&deg;C incubator before dispensing the media into the plates and colony inoculation. The new trial, again, has two plates, with the same wells (e.g. A1) inoculated with the same colony, where one plate has M9 screening media (with Tet and lactose) alone and the other plate containing 50mg/L NAs. Two runs were completed this week. The data from one of those trials are shown below.<br />
</p><p><br />
</html>[[File:Ucalgary2012 081612.png|center|thumb|800px| Figure 4: A 48hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media at time points (baseline, 24hr, and 48hr), comparing the same colonies in M9 screening media with/without 50mg/L NAs]]<html><br />
<br />
<p><br />
In this particular run, only two colonies showed growth (D8 and H11), these were subcultured in 5mL of M9 screening media with NAs. An interesting observation is that there is more growth in the lactose+NAs plate than the lactose alone plate. This suggests that the NAs may play a role in activating the lacZ gene, and improving the survival rates. These results are consistently observed in the other trials conducted. <br />
<br />
<p><br />
At this point, since time is limited, the entire mutant selection process needs to be more efficient. Therefore, the mating spots are diluted, and plated on a more selective media, containing PIA+50ug/mL tetracycline+50mg/L NAs+20 μg/mL X-Gal. This way, the NA-responsive and/or lactose-utilizing Pseudomonas transconjugants can be selected as they would appear blue on the media. This reduces the number of colonies to be inoculated, and improves the efficiency of the whole process. (Note: Wildtype Pseudomonas fluorescens pf-5 are always plated as positive control to observe the natural forward mutation rates, and to ensure that the amount of spontaneous tetracycline resistant Pf-5 mutants are at a reasonable level.<br />
</p><br />
<br />
<p><br />
At this point, a large number of matings should be conducted and mutants selected, in order to provide enough of lactose-utilizing and NA-sensitive Tn-insertion mutants, and to cover as much of the Pseudomonas genome as possible<br />
</p><br />
<br />
<h2>Week 17 (August 20-August 24)</h2><br />
<p>No work on the transposon library was done this week, other than planning for the upcoming screen.</p><br />
<br />
<h2>Week 18 (August 27-August 31)</h2><br />
<p>This week, a large screen was accomplished. First, cultures of <i>P. fluorescens</i> Pf-5 and <i>E. coli</i> SM10 were grown up overnight at 30&deg;C and 37&deg;C respectively to a high optical density. The following morning, the <i>E. coli</i> was subcultured in a 1/4 dilution to dilute out the tetracycline, and grown for approximately 2 more hours. After this, 100 &micro;L of each culture were mixed, spun down, and resuspended in 20 &micro;L of media before being spotted onto LB plates.</p><br />
<p>In total, 500 separate mating spots were plated. These spots were allowed to grow at 37&deg;C overnight. The following day, 2 mating spots were scraped up, combined, and resuspended in 500 &micro;L PBS. 1/400 dilutions of these resuspensions were made, and 100 &micro;L of these was plated onto selective plates consisting of PIA, 100mg/L NAs, and tetracycline. These plates had 40 &micro;L of 20 mg/mL X-gal spread on their surface in order to allow for blue-white screening. 250 plates were made in total.</p><br />
<br />
<p></html>[[File:2012-08-29 21-32-07 236 Calgary.jpg|center|500px|thumb|Figure 5: Plates in incubator]]<html> <p><br />
<br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<br />
<p>Plates were left to grow for 2 days, after which blue colonies (24 in total) were selected and pinned in duplicate into 96-well plates for response testing. Initially, minimal media + lactose with and without NAs was used, however no growth in any of the wells was observed. Because of this, the screening protocol was altered such that LB with or without NAs was used instead of minimal media, and X-gal was used instead of lactose for screening- the idea being that if naphthenic acids were sensed, a blue color change would be observed relative to the negative LB control.</p><br />
<br />
<p></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 6: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16h. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><br />
<br />
<p>When results were observed it was found that 4 colonies showed clear differential regulation in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. Therefore, these colonies will be used in further screening to test the specificity of the response.</p><br />
<br />
<br />
<h2>Week 19 (September 10- September 14)</h2><br />
<p>This week, further screens on the previously identified four hits were performed. These involved the use of different toxins at environmentally relevant concentrations to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used in one of the media samples in order to attempt to rule out a general stress response by the cell.<br />
<br />
<p></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 7: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:170-1data.png|thumb|600px|center|Figure 8: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 9: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 10: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p>Due to these results, further screens on these two colonies will be performed, using lower hydrogen peroxide concentrations to rule out a general stress response, and decanoic acid to rule out a response to fatty acids.</p><br />
<br />
<h2>Week 20 (September 17- September 21)</h2><p><br />
Further screens were conducted, however due to cells drying out in the plate the results were invalidated. Because of this, the screen will be repeated, and explained in further detail when this is done.</p><br />
<br />
<h2>Week 21 (September 24- September 28)</h2><p><br />
Genomes of 66-1 and 170-1 were isolated, digested with BglII and with XbaI, and religated before being transformed into <i>E.coli</i>. Cells were plated onto tetracycline plates to isolate cells containing a ligation product with the transposon present. These colonies have been miniprepped, and are awaiting being sent for sequencing to determine which genes the transposon has been inserted into. </p><br />
<br />
<h2>Week 22 (October 1-October 5)</h2><p><br />
Minipreps from previous weeks of the transposon self-cloned plasmid were nanodropped, and it was determined that no DNA had been isolated. Therefore, colonies were re-prepped, and sent for sequencing (as sequencing primers have arrived from Dr. Hynes).</p><p><br />
In addition, the assay to test the specificity of the response of the transposon clones was repeated. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The assay was carried out according to the <b>protocol</b> on our protocols section. This data was recieved after Wiki-Freeze had happened, and therefore will be analyzed at a later date. </p><br />
<br />
<h2>Week 23 (October 8-October 11)</h2><p><br />
Unfortunately, no transposon work was accomplished this week.<br />
<br />
<h2>Week 24 (October 15-October 19)</h2><p><br />
Sequencing reactions came back as having no reads, so sequencing samples were resent. This happened multiple times this week, and it is unclear why reactions continue to fail.<br />
<br />
<h2>Week 25 (October 22-October 26)</h2><p><br />
This week, data from the previously run assay was analyzed. The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure 8:Stress response screen on <i>P. fluorescens</i>Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 %micro&L of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.]]<html><p><br />
These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.</p><br />
<br />
<br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/PromoterScreenTeam:Calgary/Notebook/PromoterScreen2012-10-26T21:36:13Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookGreen|<br />
TITLE=Transposon Library Notebook|<br />
CONTENT =<br />
<html><br />
<!--<br />
NOTE: This is a template for entering things for the time being. All dates should be enclosed in <h2> tags and all paragraphs should be enclosed in <p> tags. For bulleted lists, <ul> tags will create the list and <li> tags will surround each list item. If there are any questions, please let me know.<br />
<br />
Patrick.<br />
--><br />
<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>&nbsp;&nbsp;This was the first week where we met with other team members and summarized the primary subprojects the team will be tackling this coming summer.</p><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>&nbsp;&nbsp;During this week literature searches were performed.</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>&nbsp;&nbsp;During this week literature searches were performed.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p>&nbsp;&nbsp;During this week, strains of <i> Pseudomonas fluorescens</i> PF-5 were obtained. Two cultures were started by adding 500 &micro;L stock to 10 mL LB media containing 50 mg/L ACROS Naphthenic Acids. These cultures were grown at 30&deg;C overnight, shaking at 110 rpm.</p><br />
<p>&nbsp;&nbsp;Overnight cultures were then streaked on LB agar the following day with various types and concentrations of antibiotics in order to determine the susceptibility profile of the organism. This was necessary in order to determine what marker could be used on a transposon to allow for selection of organisms with sucessful transposon insertions. These plates were grown overnight to look for death or growth, and the following results were obtained; </p><br />
<br />
<center><table><br />
<table border="2" CELLPADDING=3 CELLSPACING=1 <br />
RULES=COLS FRAME=VSIDES><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Gentamycin</b></td><br />
<td><b>Kanamycin</b></td><br />
<td><b>Chloramphenicol</b></td><br />
<td><b>Tetracycline</b></td><br />
</tr><br />
<tr><br />
<td>25 &micro;g/ml = no growth</td><br />
<td>5 &micro;g/ml = slight growth</td><br />
<td>5 &micro;g/ml = growth</td><br />
<td>50 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td>50 &micro;g/ml = no growth</td><br />
<td>10 &micro;g/ml = slight growth</td><br />
<td>10 &micro;g/ml = growth</td><br />
<td>100 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td>100 &micro;g/ml = no growth</td><br />
<td>25 &micro;g/ml = no growth</td><br />
<td>25 &micro;g/ml = growth</td><br />
<td>200 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td>50 &micro;g/ml = no growth</td><br />
<td>50 &micro;g/ml = growth</td><br />
<td> </td><br />
</tr><br />
</table></center><br />
<br />
<br />
<p><br />
<br></br> &nbsp;&nbsp;Based on these results, it was determined that Kanamycin, Gentamycin, and Tetracycline could be used as the selectable marker on the transposon, while Chloramphenicol could not as the strain is naturally resistant. Glycerol stocks of the strains were also made at this time from a fresh overnight culture.</p><br />
<br><br><br />
<h2>Week 5 (May 28-June 1)</h2><br />
<p>&nbsp;&nbsp;At the beginning of this week we spoke to Dr. Michael Hynes, who was able to give us <i> E. coli </i> SM10 and SM17-1 cells containing the plasmid pOT182. This plasmid contains an <i> E. coli </i> origin of replication, allowing it to act as a suicide vector when transferred to a different bacterial species. pOT182 contains a Tn5 transposon element containing a promotorless <i>lacZ</i> gene, genes for tetracycline resistance as well as a beta-lactamase, transposase and an <i>E. coli</i> origin of replication. These elements are bordered by insertion element sequences which are recognised by the transposase. When transferred to a different host through conjugation, the plasmid itself can no longer replicate. The transposase however can recognise and transfer the sequence between the insertion elements in a cut-and-paste fashion randomly into the genome. In this fashion, the tetracycline and beta-lactam resistant traits would only persist in cells in which the transposon has jumped into the genome, allowing these antibiotics to select for transposon positive cells. The lacZ protein will only be produced if the transposon jumps in frame downstream of a promotor, and in this case would allow for a lacZ based assay of the promotors response. The <i>E. coli</i> origin of replication present in the transposon allows for the self-cloning of the transposon in a plasmid format after genomic digestion and circularization and transformation into <i>E. coli</i>, allowing for the sequence of bordering gene fragments to be determined easily and therefore mapping the transposon in the genome.</P><br />
<br />
<br />
<br />
<p>&nbsp;&nbsp;Cultures of <i>E. coli</i> SM10 and <i>P. fluorescens</i> PF-5 were grown up overnight in shakers at 37&deg;C and 30&deg;C respectively. SM10 was grown in LB + 10 &micro;g/ml Tet, and PF-5 was grown in LB + 50 mg/L ACROS naphthenic acids. In the morning, the SM10 culture was subcultured (1/4) into LB without antibiotics and allowed to grow for an additional 4h. After this, 3 replicates of mating mixtures were made with 500 &micro;l of each culture were mixed, and the cells were spun down and resuspended in 50 &micro;l of LB. These samples were then plated in separate spots on LB agar. Additional spots were made in the same fashion with just PF-5 culture and just SM10 culture as controls, and the plate was incubated at 37&deg;C overnight. In the morning, each spot was resuspended in 500&micro;l sterile water + 25 &micro;g/ml Tet, and dilutions of 1, 1/10, 1/100, and 1/1000 of each mating mixture was plated on Pseudomonas Isolation Agar (PIA) + 10&micro;g/l Tet + 50 mg/L ACROS naphthenic acids. These cultures were allowed to grow at 30&deg;C over the weekend. </p><br />
<p>&nbsp;&nbsp;The purpose of the PIA is to selectively allow the growth of the PF-5 strain, while killing off the donor SM10 strain. The tetracycline is designed to select for the positive transposon mutants in the PF-5 strain, as the only way that tetracycline resistance would be acquired (barring spontaneous mutation events) would be if the transposable element had jumped into the genome of the cell. We decided to use 10 &micro;g/ml as the concentration of the tetracycline in the plates because we believed that 50 &micro;g/ml would be too high for even strains carrying resistance to survive. Seeing as 10 &micro;g/ml was effective for <i>E. coli</i>, we chose to try this. After streaking the PF-5 culture on the plate to test for its resistance however, it was found that the strain without the transposon was able to grow slightly on the plates, meaning that the concentration of antibiotic is not high enough to properly select for transposon mutants v.s. untransformed cells. In order to try to remedy this, the mating spots were resuspended in a mixture containing a higher dose of antibiotics. The results of this experiment are pending. </p><br />
<br />
<h2>Week 6 (June 4-8)</h2><br />
<br />
<p>&nbsp;&nbsp;When the plates from last week were examined, it was found that though the PIA was sucessful in inhibiting the growth of the <i>E. coli</i> donor strain, the original PF-5 strain was capable of growth on the media. Because of this, the plates were not selective towards cells containing the transposon insertion, and thus lawns of bacteria were seen on each of the plates.</p><br />
<p>&nbsp;&nbsp;Because of this, new selective media plates were prepared. These contained LB agar + 50 mg/L ACROS NA's + 50 &micro;g each chloramphenicol and tetracycline. The chloramphenicol was used in order to kill off the SM10 donor strain, while the tetracycline was used at a concentration previously shown to kill off the PF-5 cells that did not contain a transposon insertion. The conjugation procedure previously described was repeated, with 1 replicate mating spot being plated in 4 different dilutions on the new selective media. These plates were grown overnight at 30&deg;C. It was found that both SM10 and unmodified PF-5 were not capable of growth on the new selective plates, and colonies were found growing on all 4 of the dilutions for the mating spot.<br />
<br />
</html>[[File:Ucalgary2012_TMscreenpractice.png|center|thumb|400px|Figure 1: Transposon selective plates. WT Pf-5 and the donor strain are not capable of growth, however colonies of Pf-5 containing the transposon are capable of growth.]]<html><br />
<br />
<p>&nbsp;&nbsp;Because the host and the untransformed cells were not capable of growth on the selective plates, it is believed that these colonies must represent sucessful transposition events, as this would be the only way that the tetracycline resistance would be transfered to the PF-5 cells (chloramphenicol would have no effect, as PF-5 cells are naturally resistant at this concentration, as previously shown). Because the cell density was too high, the 1 and 1/10 dilution plates were discarded, while the 1/100 and 1/1000 plates were stored at 4&deg;C until the next step of the procedure, in which screening for naphthenic acid response will be performed. </p><br />
<br />
<h2>Week 7 (June 11-15) </h2> <br />
<br />
<p>&nbsp;&nbsp;In order to test for a naphthenic acid response, a lacZ reporter system in the transposon will be utilized. Because the lacZ enzyme is capable of degrading X-gal, a dissacharide sugar containing lactose, into lactose, and an insoluble sugar (5,5'-dibromo-4,4'-dichloro-indigo), which appears blue. Therefore, in response to activation of a native gene promotor, in frame transposon insertions will produce lacZ at levels corresponding to the activation level of the gene, and these colonies will be able to utilize lactose as a carbon source as well as utilize Xgal as a substrate. Cells responding to naphthenic acids will therefore show blue pigmentation and be capable of growth on lactose. Those that do not respond should remain white, and perish when lactose is given as the only sugar source.</p><br />
<p>&nbsp;&nbsp;The concentration of naphthenic acids used in the test plates will be 4x less than the minimal inhibitory concentration (MIC). Therefore, a MIC assay for naphthenic acids with PF-5 cells was carried out overnight at 30&deg;C at 2%, 1%, 0.5%, 0.25%, and 0.125% concentrations in LB media. It was found that PF-5 grew well at all these concentrations; therefore 1% was arbitrarily picked for the test conditions for naphthenic acid response plates.</p><br />
<p><br />
Plates were made as follows, with Xgal spread on top:</p><br />
<br />
<center><table><br />
<table border="1" CELLPADDING=3 CELLSPACING=1 <br />
RULES=NONE FRAME=BOX table width=600px height=150px><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Wild-Type PF-5 Controls</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media Alone, M9 Minimal Media + 0.4% Glucose, M9 Minimal Media + 0.4% Lactose, M9 Minimal Media + 1% NA's</td><br />
</tr><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Transposon Replica Plates</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media + 0.4% Glucose + Chlor, M9 Minimal Media + 0.4% Glucose + Chlor + 1% NA's, M9 Minimal Media + 0.4% Lactose + Tet, M9 Minimal Media + 0.4% Lactose + Tet + 1% NA's </td><br />
</tr><br />
</table></center><br />
<br />
<br />
<p><br />
<br></br><br />
&nbsp;&nbsp;Colonies from the 1/1000 transposon plate were replica stamped using velvet cloth onto the transposon test plates, and WT PF-5 was streaked onto the controls. The plates were allowed to grow over the weekend at 30 &deg;C. </p><br />
<br />
<h2>Week 8 (June 18-22)</h2><br />
<br />
<p>&nbsp;&nbsp;The plates from the previous week were observed on Monday. WT PF-5 grew on M9 + Glucose, but not on any of the other controls, indicating that the wild-type strain could not utilize lactose, indicating that this could possibly be used as a selection measure for responses from the transposon mutants. </p><br />
<p>&nbsp;&nbsp;No growth was observed on any of the transposon test plates. This was believed to be likely from poor transfer during stamping, resulting from a poor contact with the plates in addition to a grease layer formed by the undissolved naphthenic acids. Because of this, a new method of creating plates had to be determined.</P><br />
<p>&nbsp;&nbsp;We tried to dissolve NAs in DMF and spread this mixture on top of agar plates as well as mix it into the liquid agar before pouring plates. Though this helped to solubilize the naphthenic acids, it was not as effective as increasing the pH of the media (pH 8 was found to be effective). We made M9 media at pH 8 before making the same set of test plates as before. It was found that the NAs stayed in solution when mixed, however came out of solution when the plates dried, leading to a grease layer on the plates as previously seen. </P><br />
<p>&nbsp;&nbsp;In another attempt to raise the pH, stock solutions of NAs dissolved in NaOH at pH 12 were made, and these were autoclaved alongside the media. When NAs were added to the NaOH, the solutions became cloudy and uniform, indicating they were in solution. When the NA stocks were added to the media before plate pouring, the NAs remained in solution even after the plates dried, indicating that this method would be sucessful in making test plates. Using this method, the following plates were created:<br />
<br />
<center><table><br />
<table border="1" CELLPADDING=3 CELLSPACING=1 <br />
RULES=NONE FRAME=BOX table width=600px height=150px><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Wild-Type PF-5 Controls</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media Alone, M9 Minimal Media + 0.2% Glucose, M9 Minimal Media + 0.2% Lactose</td><br />
</tr><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Transposon Replica Plates</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media + 0.2% Glucose + Chlor, M9 Minimal Media + 0.2% Glucose + Chlor + 0.05% NA's, M9 Minimal Media + 0.2% Glucose + Chlor + 1% NA's, M9 Minimal Media + 0.2% Lactose + Tet, 0.2% Lactose + Tet + 0.05% NA's, M9 Minimal Media + 0.2% Lactose + Tet + 1% NA's </td><br />
</tr><br />
</table></center><br />
<br />
<p><br />
<br></br><br />
&nbsp;&nbsp;Xgal was spread on all the plates with NA's, however it was left out of the controls by mistake. Stamping was carried out as previously described from both the previously created 1/100 and 1/1000 dilution plates of transposon mutants (A new round of transposon mutagenesis was initiated, but the mating mixture was incubated at 30&deg;C instead of 37&deg;C, and no mutants were obtained on the selective plates). These plates were allowed to grow over the weekend at 30&deg;C.</P><br />
<br />
<h2>Week 9 (June 25-June 29)</h2><br />
<p>Growth was observed from the selection plates incubated over the weekend at 30&deg;C. Screening replica plates are made as before. The 1/1000 diluted transposon plate was used for the replica plating on all the screening conditions. The screening plates are incubated overnight at 30&deg;C. The next day, no growth was observed on the negative control plates (M9 alone, M9 with 0.05% NAs), and no growth was observed on the positive control plates containing glucose. Similarly, no growth was observed on the M9+lactose (+Tet, with/without X-GAL, with/without 0.05% NAs). This suggests that either a large number of matings is needed before a desirable transconjugant is seen, or that the media condition requires modification. The plate surface is not as greasy as before, which suggests that the replica plating transfer process works. However, the pH of the media may affect the growth of transconjugants. Plans are made to modify the pH of the media before replica plating. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>Replica plating screening plates are made so that NA stock solutions (1% at pH 12 diluted to 0.05% final concentration) were added to the M9 agar solution. The pH is adjusted to 7.4 before autoclaving. The pH prior to adjusting is approximately 9.0. This may explain the poor growth observed in the previous trial. A new series of mating were started (2 mating spots), and incubated overnight at 37&deg;C. The mating spots are scraped and serial diluted up to 1/1000, plated on LB+Chlor50+Tet50, and incubated overnight at 30&deg;C. This time, the results are consistent with the previous attempt, where no growth was observed on the negative control plates (M9 alone, M9 with 0.05% NAs), and ample growth was observed on the positive control plates containing glucose. However, still no growth was observed on the M9+lactose (+Tet, with/without X-GAL, with/without 0.05% NAs)plates. This seems to confirm with previous hypothesis that a large number of matings are needed to screen the genome for NA sensitive elements. Alternatively, perhaps the NA concentration used here is too high for proper selection of the transconjugants that have a promoter element upstream of the transposon insertion. If the NA concentration used for the screen is too high, the promoter may be suppressed, and the lacZ is not expressed for cell survival. In fact, the lowest NA concentration used up to this point is 0.05% or 500 mg/L. The culturing conditions required to maintain Pseudomonas Pf-5's NA degrading abilities is LB+50mg/L. Therefore, to allow both cell survival and screen for the most robust and sensitive system, maybe the NA concentration should be lowered.</p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The exact same experiment from the previous week has been repeated with two more mating mixtures (and their dilutions). However, we obtained the same results, with the positive and negative controls yielding predicted growth and no growth, respectively. But no growth was observed on the lactose plates. Furthermore, upon close examination of the selection plates after the conjugating/bipartite mating reaction (LB+Chlor+Tet), two colony morphologies can be seen: drier and larger colonies (resembling Pseudomonas), and smaller moist colonies (resembling E. coli). This suggests perhaps the LB+Chlor+Tet plates may be useful for selecting Pseudomonas transconjugants, but it does not prevent the donor E. coli to be efficiently killed. A final replica plating experiment is designed for next week, and Pseudomonas Isolation agar (PIA, just received)will be used for selection instead. </p> <br />
<br />
<h2>Week 12 (July 16-July 20)</h2><br />
<p>Another mating reaction was set up, and plated on PIA+Tet50 to select for positive transconjugants. The colonies from the resulting mutants show only a single colony morphology consistent with Pseudomonas. From this, we know the selection is appropriate. The 1/1000 dilution of the mating reaction dilution is replica-plated on two sets of screening plates. The first set is prepared the same way as the previous week (NAs added and the media pH adjusted prior to autoclaving); the second set is the same except the NAs are added by spreading 100uL of a 50mg/L sterile stock NA solution in NaOH (pH12) prior to replica plating. The 1/1000 dilution plate of the mating reaction is replica plated on both plates (with the same controls as before). However, the same results are obtained as before, where no growth is observed on the M9+0.2% lactose+X-GAL or the M9+0.2% lactose+X-GAL+50mg/L NAs plates. Lastly, we had an interesting observation where overnight growth of the lactose screening plates showed no growth, if they are left for up to 48hr, some colonies can be observed; however, none of the colonies are blue. The problem may be that the M9 media conditions are not suitable for selecting the Tn mutants, or that the replica plating approach we used cannot effectively transfer all colonies. At this point, since no NA-sensitive strains are isolated, a new approach needs to be taken in order to find a tranposon insertion mutant that has an NA-sensitive promoter upstream. Since all possible approaches have been taken using the replica plating strategy, another mass screening method is needed. </p><br />
<br />
<h2>Week 13 (July 23-July 27)</h2><br />
<p> Even though, the previous screening method is quick and convenient, the waiting period is too long, and no real time data in terms of growth or lactose utilization can be observed/discerned. Therefore, a 96-well mass screening method is devised, where the conjugation mating reaction, and the selection on PIA+Tet50 are the same, but the screening step is different. The screening media is a M9 minimal media with 50 µg/mL tetracycline to maintain the genomic Tn insertions, 0.2% lactose to select for lactose utilization due to Tn insertions, and 50 mg/L NAs to maintain the strain's ability to use/degrade NAs (in short the media is M9+0.2%lactose+50µg/mL Tet+50mg/L NAs). On each 96-well plate, 94 colonies are inoculated and screened; the other two wells are used for negative and positive controls. The negative control well has the same media (M9+0.2%lactose+50µg/mL Tet+50mg/L NAs), with no colonies inoculated. The positive control has M9+0.2%glucose+50µg/mL Tet+50mg/L NAs, and is inoculated with a random colony.<br />
<br />
</p><p><br />
<br />
Using this new method, any inoculated colony that grows in the liquid media can utilize lactose, and this should be because of a transposon insertion downstream of a promoter. To select for a NA sensitive promoter, each colony is inoculated into two plates with the same media components (as described above), except one has NAs, and the other does not. If a promoter is NA-sensitive, then it should not grow if NAs are absent from the media, as the NA mixture would activate the promoter. When NAs are present however, the NA-sensitive promoter would turn on, allowing it to utilize lactose, survive, and grow. <br />
<br />
As a first run of this method, a plate is set up using mutant colonies from a previous mating reaction. This plate contained M9+0.2%lactose+50µg/mL Tet+50mg/L NAs. The appropriate control plate with no NAs was not included as this is only to test feasibility of this approach. The plate is incubated over the weekend for 60hrs in a plate reader at 30&deg;C. </p><br />
<br />
<h2>Week 14 (July 30-August 3)</h2><br />
<p>The data from the plate incubated from last week was collected. First, since the lid of the 96-well plate was kept on the plate to prevent dehydration of the media, there was significant condensation on the middle of the lid (covering about 30 wells), which prevented proper absorbance measurements of those wells (data from these wells were omitted). However, six wells/colonies demonstrated growth from a baseline. The following table is a display of the 96-well plate at the end point, with the colonies/wells that grow were highlighted.</p><br />
<br />
</html>[[File:Ucalgary2012 0801data1.png|center|thumb|400px|Figure 2: A 60hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media]]<html><br />
<br />
<p>The cultures from these six wells are washed with M9+0.2%lactose+50µg/mL Tet (no NAs) three times, and resuspended in 1 mL of M9+0.2%lactose+50µg/mL Tet. 500µl of each colony was inoculated into 5mL cultures with M9+0.2%lactose+50µg/mL Tet or M9+0.2%lactose+50µg/mL Tet+50mg/L NAs. These cultures are grown over 48 hours to observe growth (any strain that grows only when NAs are added would be noted). Also, the colonies are restreaked on PIA+Tet50 plates to ensure that the absorbance increase actually indicated growth (since the increase was so small at around +0.200). The results were not promising, on the restreaked plates, only 2 colonies (from H12 and B1) showed growth, this suggests some of the measurements were not very accurate. Also, in the culturing experiment, only 1 set of cultures (from 1 colony, H12) showed growth, but in both media conditions, which is not desirable.</p><br />
<br />
<p>Even though, no colonies were shown as NA sensitive, this result is still very promising, as this experiment shows that some colonies from the transposon mutagenesis can actually utilize lacZ, which demonstrates that the transposable element approach is an appropriate and feasible approach for screen promoters sensitive to environmental stimuli. (Please note, that mating reactions and mutant selection are conducted regularly every week, usually twice per week, with two mating spots each time, to provide mutants for these screens.)</p><br />
<br />
<h2>Week 15 (August 6-August 10)</h2><br />
<p><br />
The same experiment from the previous week was repeated. However, the plate was not incubated in the plate reader, but incubated in a 30&deg;C shaker, at 140rpm. At the baseline (beginning of incubation), 24hr, and 48hr, timepoint measurements were made. The results are as follows.<br />
</p><p><br />
</html>[[File:Ucalgary2012_080312.png|center|thumb|400px|Figure 3: A 48hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media at time points (baseline, 24hr, and 48hr)]]<html><br />
</p> <br />
<br />
<p><br />
However, these measurements were made with the lid of the plate on. Even though there was no clear trend in terms of the growth in the absorbance data, it is possible to observe some wells become cloudy by eye. For instance, well F9 was cloudy at the end of the 48hr incubation, but the data showed no change in absorbance. The lid should be taken off in the future when making timepoint measurements. Also, the absorbance data can support the visual data.<br />
</p><br />
<br />
<p><br />
Also, this week, two runs of the screening experiment was conducted with two plates run in parallel, one with NAs and the other without (the proper screening setup). However, it was found that the lactose solution (which was filter sterilized) used to make the M9 media for the screen was contaminated with yeast. Therefore, new media solutions and lactose solutions needed to be made. These data are not used because of this.<br />
</p><br />
<br />
<h2>Week 16 (August 13-August 17)</h2><br />
<p><br />
New lactose and glucose stock solutions as well as new M9 screening media were prepared. The experiments from last week were repeated using proper solutions. Also, to prevent fogging of the plate lid and cross-contamination, all media solutions and plates were pre-warmed in a 30&deg;C incubator before dispensing the media into the plates and colony inoculation. The new trial, again, has two plates, with the same wells (e.g. A1) inoculated with the same colony, where one plate has M9 screening media (with Tet and lactose) alone and the other plate containing 50mg/L NAs. Two runs were completed this week. The data from one of those trials are shown below.<br />
</p><p><br />
</html>[[File:Ucalgary2012 081612.png|center|thumb|800px| Figure 4: A 48hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media at time points (baseline, 24hr, and 48hr), comparing the same colonies in M9 screening media with/without 50mg/L NAs]]<html><br />
<br />
<p><br />
In this particular run, only two colonies showed growth (D8 and H11), these were subcultured in 5mL of M9 screening media with NAs. An interesting observation is that there is more growth in the lactose+NAs plate than the lactose alone plate. This suggests that the NAs may play a role in activating the lacZ gene, and improving the survival rates. These results are consistently observed in the other trials conducted. <br />
<br />
<p><br />
At this point, since time is limited, the entire mutant selection process needs to be more efficient. Therefore, the mating spots are diluted, and plated on a more selective media, containing PIA+50ug/mL tetracycline+50mg/L NAs+20 μg/mL X-Gal. This way, the NA-responsive and/or lactose-utilizing Pseudomonas transconjugants can be selected as they would appear blue on the media. This reduces the number of colonies to be inoculated, and improves the efficiency of the whole process. (Note: Wildtype Pseudomonas fluorescens pf-5 are always plated as positive control to observe the natural forward mutation rates, and to ensure that the amount of spontaneous tetracycline resistant Pf-5 mutants are at a reasonable level.<br />
</p><br />
<br />
<p><br />
At this point, a large number of matings should be conducted and mutants selected, in order to provide enough of lactose-utilizing and NA-sensitive Tn-insertion mutants, and to cover as much of the Pseudomonas genome as possible<br />
</p><br />
<br />
<h2>Week 17 (August 20-August 24)</h2><br />
<p>No work on the transposon library was done this week, other than planning for the upcoming screen.</p><br />
<br />
<h2>Week 18 (August 27-August 31)</h2><br />
<p>This week, a large screen was accomplished. First, cultures of <i>P. fluorescens</i> Pf-5 and <i>E. coli</i> SM10 were grown up overnight at 30&deg;C and 37&deg;C respectively to a high optical density. The following morning, the <i>E. coli</i> was subcultured in a 1/4 dilution to dilute out the tetracycline, and grown for approximately 2 more hours. After this, 100 &micro;L of each culture were mixed, spun down, and resuspended in 20 &micro;L of media before being spotted onto LB plates.</p><br />
<p>In total, 500 separate mating spots were plated. These spots were allowed to grow at 37&deg;C overnight. The following day, 2 mating spots were scraped up, combined, and resuspended in 500 &micro;L PBS. 1/400 dilutions of these resuspensions were made, and 100 &micro;L of these was plated onto selective plates consisting of PIA, 100mg/L NAs, and tetracycline. These plates had 40 &micro;L of 20 mg/mL X-gal spread on their surface in order to allow for blue-white screening. 250 plates were made in total.</p><br />
<br />
<p></html>[[File:2012-08-29 21-32-07 236 Calgary.jpg|center|500px|thumb|Figure 5: Plates in incubator]]<html> <p><br />
<br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<br />
<p>Plates were left to grow for 2 days, after which blue colonies (24 in total) were selected and pinned in duplicate into 96-well plates for response testing. Initially, minimal media + lactose with and without NAs was used, however no growth in any of the wells was observed. Because of this, the screening protocol was altered such that LB with or without NAs was used instead of minimal media, and X-gal was used instead of lactose for screening- the idea being that if naphthenic acids were sensed, a blue color change would be observed relative to the negative LB control.</p><br />
<br />
<p></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 6: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16h. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><br />
<br />
<p>When results were observed it was found that 4 colonies showed clear differential regulation in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. Therefore, these colonies will be used in further screening to test the specificity of the response.</p><br />
<br />
<br />
<h2>Week 19 (September 10- September 14)</h2><br />
<p>This week, further screens on the previously identified four hits were performed. These involved the use of different toxins at environmentally relevant concentrations to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used in one of the media samples in order to attempt to rule out a general stress response by the cell.<br />
<br />
<p></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 7: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:170-1data.png|thumb|600px|center|Figure 8: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 9: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 10: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p>Due to these results, further screens on these two colonies will be performed, using lower hydrogen peroxide concentrations to rule out a general stress response, and decanoic acid to rule out a response to fatty acids.</p><br />
<br />
<h2>Week 20 (September 17- September 21)</h2><p><br />
Further screens were conducted, however due to cells drying out in the plate the results were invalidated. Because of this, the screen will be repeated, and explained in further detail when this is done.</p><br />
<br />
<h2>Week 21 (September 24- September 28)</h2><p><br />
Genomes of 66-1 and 170-1 were isolated, digested with BglII and with XbaI, and religated before being transformed into <i>E.coli</i>. Cells were plated onto tetracycline plates to isolate cells containing a ligation product with the transposon present. These colonies have been miniprepped, and are awaiting being sent for sequencing to determine which genes the transposon has been inserted into. </p><br />
<br />
<h2>Week 22 (October 1-October 5)</h2><p><br />
Minipreps from previous weeks of the transposon self-cloned plasmid were nanodropped, and it was determined that no DNA had been isolated. Therefore, colonies were re-prepped, and sent for sequencing (as sequencing primers have arrived from Dr. Hynes).</p><p><br />
In addition, the assay to test the specificity of the response of the transposon clones was repeated. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The assay was carried out according to the <b>protocol</b> on our protocols section. The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure 8:Stress response screen on <i>P. fluorescens</i>Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 %micro&L of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.]]<html></p><br />
<p><br />
These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.</p><br />
<h2>Week 23 (October 8-October 11)</h2><p><br />
Unfortunately, no transposon work was accomplished this week.<br />
<br />
<h2>Week 24 (October 15-October 19)</h2><p><br />
Sequencing reactions came back as having no reads, so sequencing samples were resent. This happened multiple times this week, and it is unclear why reactions continue to fail.<br />
<br />
<h2>Week 25 (October 22-October 26)</h2><p><br />
This week, data from the previously run assay was analyzed.<br />
<br />
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}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/FRED/DetectingTeam:Calgary/Project/FRED/Detecting2012-10-26T21:24:00Z<p>Lisa.O: </p>
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This year, our team wanted to identify a novel responsive element capable of detecting and quantifying different tailings ponds toxins (e.g. naphthenic acids, NAs) in solution. While numerous studies have begun to identify species of bacteria capable of surviving and sensing a variety of toxic compounds (e.g. NAs), the degradation pathways have not yet been fully characterized. Therefore, we needed to design and implement novel approaches to efficiently isolate the genetic elements that detect and potentially lead to the breakdown of these toxins.<br />
</p><br />
<h2>Transposons: What, How, Why?</h2><br />
<p align="justify"><br />
The transposable element (TE), Tn5, is a conservative transposon that can insert a segment of genes bordered by specific 19bp insertion sequences from one part of the genome (e.g. plasmid vector) randomly to another location like a chromosome (Reznikoff, 2008). The transposition event is catalyzed by a transposase enzyme encoded by <i>tnp</i> gene included in the TE. Using the appropriate selective pressure, the insertion can be maintained permanently in the genome.</p><br />
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</html>[[File:Transposon.jpg|thumb|700px|center|Figure 1: "Transposition reaction from plasmid entry into the recipient cell to integration of the transposon into the genome. Modified from Transposons: Shifting Segments of the Genome" by McGraw Hill]]<html><br />
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<br />
<p align="justify">By inserting a vector construct containing the TE with selectable markers (such as tetracyclin resistance and lacZ) into an organism with a desirable phenotype, we can find out what genetic elements (e.g. genes and promoters) are responsible for that particular function. This can happen via a random insertion of a TE containing a promoterless reporter gene downstream of promoter elements that creates a transcriptional fusion, providing activity in response to specific environmental stimuli. Using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">bipartite-mating (conjugation) method</a> to transfer the TE vector into the organism of choice is an efficient method for creating the massive number of mutants required.</p><br />
<p align="justify"><br />
Due to the complexity of biological systems, our team focused our efforts on utilizing a system for identification of promoter elements that respond specifically in the presence of environmental stimuli. Our hypothesis requires that the organisms we use respond specifically to particular toxins and result in upregulation of metabolic genes with little background effect in the cell. We recognize that any number of biological molecules may play a role in toxin sensing, such as enzymes, transcription factors, and even RNA elements (e.g. riboswitches). However, the identification of a promoter sequence takes us further in that we can better understand the degradation mechanism by elucidating the genes involved.<br />
</p><br />
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<br><br />
<h2>Toxin-Degrading Organism Used</h2><br />
<p align="justify"><br />
<i>Pseudomonas spp. </i>have been isolated from oil sands tailings ponds and shown to biodegrade model and tailings-associated NAs and nitrogen- and sulfur-containing heterocyclic aromatic compounds (Ramos-Padrón <i>et al</i>. 2010; Herman <i>et al</i>., 1994; Del Rio <i>et al</i>., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins.</p><p> We wanted to use a commercially available strain of <i>Pseudomonas fluorescens</i> characterized for a response to toxins found in tailings pond water (TPW). The <i>P. fluorescens </i>PF-5 strain (Paulsen <i>et al</i>., 2005) is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, the genome sequence is available for this strain with annotations (Pseudomonas Genome Database V2, http://pseudomonas.com/). This allows us to use sequencing data from the mutants and identify where in the genome the TE insertion occurred, and what genes (if present) are located downstream of it.<br />
</p><br />
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<br><br />
<br />
<br />
<a name="library"></a><h2>Method Design</h2><br />
<h3>Mutant Library Generation</h3><br />
<p align="justify"><br />
To construct the promoter library, a pOT182 vector construct (containing a IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR transposable element) is introduced into commercially purchased <i>E. coli SM10</i> donor strain.</p><br />
<br />
<p align="justify"><br />
</html>[[File:Transposonproject Tn5OT182constructucalgary.png|thumb|750px|center|Figure 2: The transposable Tn5 element used in the pOT182 plasmid, containing a lacZ reporter gene, ampicillin and tetracycline resistance, an<br />
<i> E. coli</i> origin of replication for use during downstream sequencing protocols, and transposase. The genes are flanked by the transposon insertion elements]]<html><br />
</p><br />
<br />
<p align="justify">The plasmid contains a RP4 mob conjugation region and a p15A origin of replication (ori), and is engineered to only replicate in <i>E. coli</i>. The TE construct is transferred from the <i>E. coli</i> donor strain to the recipient <i>P. fluorescens </i> PF-5 using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in <i>P. fluorescens</i>, and selected by isolating the recipients that have a genomic TE insertion on Pseudomonas Isolation Agar/PIA with tetracycline. If a promoter element is fused upstream of the TE construct, then promoter activation will turn on the expression of lacZ, which can be detected by the degradation of a colorless compound, X-Gal, to an insoluble blue pigment product (an indoxyl compound) (Juers <i>et al</i>., 2012). If the fused promoter is activated in response to a stimulus, then the lacZ enzyme will be produced in response. Mutant strains sensitive to the particular toxic stimulus will appear as blue colonies on the selective plate.</p><br />
<br />
<h3>Mutant Strain Characterization</h3><br />
<p align="justify">Mutants generated are characterized for their roles in the response to toxins with dose response experiments, and compared to general stress-inducing agents (e.g. H<font style="text-transform: lowercase;">2</font>O<font style="text-transform: lowercase;">2</font>) and compounds such as fatty acids to ensure the specificity of the response. These measurements help to determine thresholds of detection, robustness of the signal, and specificity of response. The dose response curves will also assess the usefulness of correlating the concentration of NA to the level of response, and the possibility of measuring NA concentrations in a sample, rather than simply by presence/absence.</p><br />
</p><br />
<h3>Self-Cloning and Sequencing</h3><br />
<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman and Lamont, 1993). The TE used is a self-cloning construct because it contains all the elements required for plasmid replication (i.e. origin of replication) and selection (Tet resistance). Genomic DNA from a desirable mutant is isolated, and restriction digested with BglII (a restriction enzyme that does not cut within the TE but numerous times within the genome). The resulting fragments may contain the TE construct with flanking sequences. The genomic fragments are circularized by self-ligation and transformed into <i>E. coli</i>. Plasmids from the transformed cells contain the TE construct with the upstream and downstream flanking sequencing connected by the BglII restriction site. Sequencing primers designed against the 19 bp recognition sequence in the TE to sequence the isolated plasmids.</p><br />
<br />
<p align="justify">For a detailed protocol, please consult our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">methods section</a>.</p><br />
<br />
<h2>Results</h2><br />
<h3>Detection by Mutant <i>Pseudomonas fluorescens</i> PF-5</h3><br />
<br />
<br />
<p align="justify">After mating experiments and plating on selective media (Pseudomonas isolation agar, with tetracycline and naphthenic acids), 24 responsive (blue) colonies were found. Screens were conducted on these blue colonies found on selective plates comparing a response in LB and LB with 100mg/L naphthenic acids (both with X-Gal). When results were observed it was found that 4 mutant strains are differentially regulated in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. These colonies were further screened to test the specificity of their responses.</p><br />
<br />
<p align="justify"></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 3: Transposons: Shifting Segments of the Genome: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16hr. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><p align="justify"><br />
<br />
<br />
Screens involving the use of different toxins at environmentally relevant concentrations were performed to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used as one testing condition to determine if the response is simply stress-induced.<br />
</p><br />
<p align="justify"></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 4: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:170-1data.png|thumb|650px|center|Figure 5: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 6: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 7: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<p align="justify"><br />
From these screens, it was seen that both colony 66-1 and colony 170-1 appear to respond to toxins when compared to a response in LB media. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure 8:Stress response screen on <i>P. fluorescens</i>Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 %micro&L of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.]]<html></p><br />
<p><br />
These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.</p><br />
<br />
<h2>Promoter Constructs Isolated</h2><br />
<p align="justify">To determine the location of the transposon insertion, we utilized the self-cloning properties of the transposon. By digesting the genome, religating, and transforming the ligated genomic fragments into <i>E. coli</i>, plasmids containing the transposon and flanking gene sequences were isolated. These plasmids have been isolated and sent for sequencing. However, we are having difficulty with getting sequencing reactions to produce a read. The results so far are a promising step towards finding a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds. In tandem as we await sequencing results, our next steps will be to test these strains in conjunction with our electrochemical detector.</p><br />
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}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/OSCAR/DesulfurizationTeam:Calgary/Project/OSCAR/Desulfurization2012-10-26T21:19:56Z<p>Lisa.O: </p>
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<br />
<h2>Why Remove Sulfur?</h2><br />
<br />
<p align="justify"><br />
Sulfur is the third most abundant element in crude oil (Ma, 2010), and when sulfur containing hydrocarbons are burned they release S0<sub>2</sub> and S0<sub>3</sub> gasses into the atmosphere. Not only does this reduce the efficiency and value of our product, but it also contributes to global warming, acid rain, and various health issues due to the pollution (Reichmuth <i>et al</i>., 2000). Strict regulation on sulfur in fuels are now in place and low-sulfur gasoline is mandated across all of Canada (Source: Environment Canada). To upgrade the quality of our fuel we need to remove the sulfur but keep the hydrocarbon backbone.</p><br />
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<h2>Our Vision</h2><p align="justify"><br />
Though a few pathways for biodesulfurization exist in the microbial world, most involve the destruction of part of the carbon skeleton (an example would be the Kodama pathway)(Soleimani <i>et al</i>., 2007). This would effectively reduce the quality of our product. With this in mind the pathway we have chosen is the 4S pathway found in <i>Rhodococcus spp</i>. It has been characterized and shown to remove sulfur from the model substrate dibenzothiophene (DBT) and convert it to 2-hydroxybiphenyl (2-HBP) in a non-destructive manner. DBT and its derivatives make up 70% of the organic sulfur compounds found in crude oil (Ma 2010), and are also some of the most difficult to remove through chemical means. By using the 4S pathway we will be able to upgrade our fuel and remove recalcitrant compounds at the same time. <br />
</p><br />
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</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|750px|thumb|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD.]]<html></p><br />
<br />
<h2>4S pathway</h2><br />
<p align="justify"><br />
Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO<sub>2</sub>) through the addition of 2 oxygen atoms to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the sulfur, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-HBP. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.</p><p align="justify"><br />
The first three steps of the 4S pathway require FMNH<sub>2</sub> and subsequently reduces the reductive power of the cell. WIn order to regain this power an oxidoreductase (DszD) uses NADH to recycle the FMNH<sub>2</sub>, allowing the reaction to proceed. Without DszD the desulfurization pathway would grind to a halt.</p><p align="justify"><br />
The <i>dszA</i>,<i>B</i>, and <i>C</i> genes form an operon on the pSOX plasmid of <i>R. erythropolis</i>, while <i>dszD</i> is found in the chromosome. Naturally this pathway is slow, however using synthetic biology approaches this process can be optimized.</p><br />
<br />
<h2>Our Approach</h2><br />
<a name="Degradation"></a><h3>1) Find the genes!</h3><br />
<p align="justify">We isolated the plasmid containing the <i>dsz</i> genes from a desulfurising environmental isolate of <i>Rhodococcus</i> using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">modified miniprep procedure</a>. As the native promoter has been shown to be repressed by various sulfur-containing compounds (Li <i>et al</i>., 1996), we designed primers for just the coding sequences of the <i>A, B, </i> and <i>C</i> genes. As these genes all have some illegal cutsites in them we constructed them into the PSB1C3 vector and started our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis protocol</a>.</p><br />
<p align="justify"> We performed an experiment to measure the desulfurization rate of select organosulfur compounds by our <i>Rhodococcus</i> strain (Figures 4-6 below). These experiments monitored the degradation of the compounds by our strain over time. We discovered that the <i>dsz</i> operon is capable of desulfurizing a wider range of compounds than just the commonly studied DBT. This shows that this pathway could be a promising solution for degradation of a wide variety of sulfur containing toxins, including those that resemble naphthenic acids. </p> <br />
<br />
<p align="justify"></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 2: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through the GC/MS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GC/MS after 6 days of incubation. ]]<html></p><br />
<br />
<p align="justify"></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 3: The peak in this mass spectrum demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<p align="justify"><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound (A: dibenzothiophene, B: tetrahydro-4h-thiopyran-4-one, and C: benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdownn. Degradation is seen for DBT (the model compound) as well as other sulfur containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<h3>2) Mutagenesis: Biobrick Compatability and Increasing DszB Activity </h3><br />
<p align="justify">In total the <i>dszABC</i> genes had 7 PstI sites and 1 NotI site that needed to be mutated for the biobrick standard. The primers were designed such that the site was removed without the amino acid being changed. In addition, a point mutation of Y63F in DszB increased the activity of the protein (Oshiro <i>et al</i>., 2007), and was included in the mass mutagenesis we undertook. Mutagenesis was performed as described in <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">this protocol.</a></p><br />
<br />
<a name="catalase"></a><h3>3) Replacing DszD with HpaC & Introducing Catalase </h3><br />
<p align="justify"><br />
As FMNH<sub>2</sub> is consumed in the first three steps of the pathway it needs to be regenerated or the process will grind to a halt. This usually falls to the <i>dszD</i> gene, however it has been shown that the <i>hpaC</i> gene from <i>E. coli</i> performs the same function more efficiently (Gala´n <i>et al</i>., 2000). One problem arises from this though, as high levels of FMNH<sub>2</sub> cause the production of toxic hydrogen peroxide inside the cell (Gala´n <i>et al</i>. 2000). To address this issue we have included a catalase gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"> <i>P<sub>lacI</sub>-katG-LAA</i></a>) that will remove the peroxide that would be toxic to the cell.</p><br />
<br />
<p align="justify"></html>[[File:Ucalgary_sulfur_constructs_KatandHpaC.PNG|center|250px|thumb|Figure 7: Diagrammatic representation of the full "optimization circuit", consisting of the oxidoreductase HpaC and a catalase (KatG).]]<html></p><br />
<br />
<br />
<h3>Results</h3><br />
<p align="justify">To show that catalase activity increased <i>E. coli</i> survivability in peroxide we cultured the inducible catalase against a catalase-free control with varying levels of peroxide. After growing overnight the negative didn't grow in any culture except in the absence of peroxide, while the catalase cultures could tolerate peroxide. This is shown below.</p><p align="justify"><br />
<br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 8: Catalase Assay. Overnight cultures of P<sub>lacI</sub> and P<sub>lacI</sub>-KatGLAA were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbidity was observed. It was found that at 1 mM of peroxide, cultures with just the lacI promotor perished, however when KatG-LAA was expressed, the cells survived.]]<html></p><br />
<br />
<br />
<p align="justify">To test the action of HpaC to use NADH to recycle FMN into FMNH<sub>2</sub> cell lysates were exposed to NADH and it's absorbance at 340nm (Kamali <i>et al</i>., 2010) was measured over time. Both native HpaC expression and an induced <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> system were tested as well as a negative control. The results are shown below.</p><br />
<br />
<p align="justify"> </html> <br />
[[File:Ucalgary2012 HpaC assaycumulativeforthedatapage.png|center|850px|thumb|Figure 9: HpaC Assay with '''A)''' 2 mL cell lysate and '''B)''' 100 &micro;L cell lysate. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, various amounts of cell lysate were transferred to a cuvette, and a spectrophotometer was blanked at 340 nm with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p align="justify">The assay showed that NADH does not abiotically convert into NAD+, however the native expression of HpaC did show a steady decrease in the levels of NADH. The induced overexpression of HpaC caused extremely rapid conversion into NAD+ as reflected by a sharp drop in the absorbance of NADH (see figure B). This drop was much sharper than what was seen when native levels of oxidoreductases were tested, showing that the <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> was functional and that it would effectively recycle FMN.</p><br />
<br />
<br />
<a name="UBC"></a><h3>4) Optimizing Gene Order</h3><br />
<br />
<p align="justify">Further optimization of the system was achieved through reorganization of the reconstructed operon. Natively the genes are arranged ABC, however the catalytic efficiency of the protein products are 25:1:5 for A:B:C respectively (Li <i>et al</i>., 2008). By rearranging the genes into BCA there is stronger transcription of the weaker proteins, giving a more balanced system overall. These would all be constructed with the same strong ribosomal binding site, <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>.</p><p align="justify"><br />
<br />
</html>[[File:DszOperonOptimize.png|center|400px|thumb|Figure 10: Method of optimizing gene order. The top circuit represents that found natively in the organism, with the bottom circuit representing our modified version.]]<html><br />
</p><br />
<br />
<a name="sulfur"></a><h2>Final Constructs</h2><br />
<p align="justify">After all of the above considerations are met, four final constructs for our system will be made to allow us to test desulfurization under different conditions.</p><p align="justify"><br />
<br />
</html>[[File:WikiConstructs_ucalgary_sulfur_2012_final_systems.png|center|700px|thumb|Figure 11: First set of final constructs for the desulfurization operon, with constitutive Dsz expression and inducible expression of the optimization proteins; either HpaC on its own or coexpressed with KatG]]<html></p><br />
<br />
<p align="justify"><br />
The first two constructs have the modified <i>dsz</i> operon (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902052"><i>dszB</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><i>dszC</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902050"><i>dszA</i></a>) under the control of a constitutive TetR promotor (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>) This is to allow for the testing of the optimization circuit, which is under the control of a lacI promotor inducible by IPTG (<a href="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</a>). The set-up of these two constructs will therefore allow for the expression of the <i>dsz</i> genes with the ability to test and compare their desulfurization rates <br> A) On their own <br> B) With the addition of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> <br> C) With the addition of both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K137068"><i>katG-LAA</i></a></p><br />
<br />
<p align="justify">This will allow us to determine what the optimal construct and expression levels of the additional genes must be in order to have the most effective sulfur removal system.</p><br />
<br />
</html>[[File:WikiConstructs2 sulfur ucalgary induciblesytems.PNG|center|700px||thumb|Figure 12: Second set of final constructs for the desulfurization operon, with all genes under an IPTG inducible promotor.]]<html><br />
<br />
<p align="justify"><br />
Due to the large number of proteins being expressed in this system, the possibility of forming inclusion bodies is present. As such, a backup system was built where both the optimization circuit and the <i>dsz</i> operon were under the control of the inducible lacI promoter. This system would allow us to tune the expression of the genes, and determine which expression level is optimal for desulfurization in our bioreactor.</p> <br />
<br />
<p align="justify">Currently the final steps of construction of these constructs is underway, following which functionality tests will begin.</p><br />
<br />
<br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/OSCAR/DesulfurizationTeam:Calgary/Project/OSCAR/Desulfurization2012-10-26T21:13:13Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Desulfurization|<br />
<br />
CONTENT=<html><br />
<img src="https://static.igem.org/mediawiki/2012/5/5e/UCalgary2012_OSCAR_Desulfurization_Low-Res.png" style="float: right; padding: 10px;"></img><br />
<br />
<h2>Why Remove Sulfur?</h2><br />
<br />
<p align="justify"><br />
Sulfur is the third most abundant element in crude oil (Ma, 2010), and when sulfur containing hydrocarbons are burned they release S0<sub>2</sub> and S0<sub>3</sub> gasses into the atmosphere. Not only does this reduce the efficiency and value of our product, but it also contributes to global warming, acid rain, and various health issues due to the pollution (Reichmuth <i>et al</i>., 2000). Strict regulation on sulfur in fuels are now in place and low-sulfur gasoline is mandated across all of Canada (Source: Environment Canada). To upgrade the quality of our fuel we need to remove the sulfur but keep the hydrocarbon backbone.</p><br />
<br />
<h2>Our Vision</h2><p><br />
Though a few pathways for biodesulfurization exist in the microbial world, most involve the destruction of part of the carbon skeleton (an example would be the Kodama pathway)(Soleimani <i>et al</i>., 2007). This would effectively reduce the quality of our product. With this in mind the pathway we have chosen is the 4S pathway found in <i>Rhodococcus spp</i>. It has been characterized and shown to remove sulfur from the model substrate dibenzothiophene (DBT) and convert it to 2-hydroxybiphenyl (2-HBP) in a non-destructive manner. DBT and its derivatives make up 70% of the organic sulfur compounds found in crude oil (Ma 2010), and are also some of the most difficult to remove through chemical means. By using the 4S pathway we will be able to upgrade our fuel and remove recalcitrant compounds at the same time. <br />
</p><br />
<br />
</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|750px|thumb|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD.]]<html></p><br />
<br />
<h2>4S pathway</h2><br />
<p><br />
Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO<sub>2</sub>) through the addition of 2 oxygen atoms to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the sulfur, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-HBP. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.</p><p> <br />
The first three steps of the 4S pathway require FMNH<sub>2</sub> and subsequently reduces the reductive power of the cell. WIn order to regain this power an oxidoreductase (DszD) uses NADH to recycle the FMNH<sub>2</sub>, allowing the reaction to proceed. Without DszD the desulfurization pathway would grind to a halt.</p><p align="justify"><br />
The <i>dszA</i>,<i>B</i>, and <i>C</i> genes form an operon on the pSOX plasmid of <i>R. erythropolis</i>, while <i>dszD</i> is found in the chromosome. Naturally this pathway is slow, however using synthetic biology approaches this process can be optimized.</p><br />
<br />
<h2>Our Approach</h2><br />
<a name="Degradation"></a><h3>1) Find the genes!</h3><br />
<p>We isolated the plasmid containing the <i>dsz</i> genes from a desulfurising environmental isolate of <i>Rhodococcus</i> using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">modified miniprep procedure</a>. As the native promoter has been shown to be repressed by various sulfur-containing compounds (Li <i>et al</i>., 1996), we designed primers for just the coding sequences of the <i>A, B, </i> and <i>C</i> genes. As these genes all have some illegal cutsites in them we constructed them into the PSB1C3 vector and started our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis protocol</a>.</p><br />
<p> We performed an experiment to measure the desulfurization rate of select organosulfur compounds by our <i>Rhodococcus</i> strain (Figures 4-6 below). These experiments monitored the degradation of the compounds by our strain over time. We discovered that the <i>dsz</i> operon is capable of desulfurizing a wider range of compounds than just the commonly studied DBT. This shows that this pathway could be a promising solution for degradation of a wide variety of sulfur containing toxins, including those that resemble naphthenic acids. </p> <br />
<br />
<p></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 2: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through the GC/MS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GC/MS after 6 days of incubation. ]]<html></p<br />
<br />
<p></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 3: The peak in this mass spectrum demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound (A: dibenzothiophene, B: tetrahydro-4h-thiopyran-4-one, and C: benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdownn. Degradation is seen for DBT (the model compound) as well as other sulfur containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<h3>2) Mutagenesis: Biobrick Compatability and Increasing DszB Activity </h3><br />
<p>In total the <i>dszABC</i> genes had 7 PstI sites and 1 NotI site that needed to be mutated for the biobrick standard. The primers were designed such that the site was removed without the amino acid being changed. In addition, a point mutation of Y63F in DszB increased the activity of the protein (Oshiro <i>et al</i>., 2007), and was included in the mass mutagenesis we undertook. Mutagenesis was performed as described in <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">this protocol.</a></p><br />
<br />
<a name="catalase"></a><h3>3) Replacing DszD with HpaC & Introducing Catalase </h3><br />
<p><br />
As FMNH<sub>2</sub> is consumed in the first three steps of the pathway it needs to be regenerated or the process will grind to a halt. This usually falls to the <i>dszD</i> gene, however it has been shown that the <i>hpaC</i> gene from <i>E. coli</i> performs the same function more efficiently (Gala´n <i>et al</i>., 2000). One problem arises from this though, as high levels of FMNH<sub>2</sub> cause the production of toxic hydrogen peroxide inside the cell (Gala´n <i>et al</i>. 2000). To address this issue we have included a catalase gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"> <i>P<sub>lacI</sub>-katG-LAA</i></a>) that will remove the peroxide that would be toxic to the cell.</p><br />
<br />
<p></html>[[File:Ucalgary_sulfur_constructs_KatandHpaC.PNG|center|250px|thumb|Figure 7: Diagrammatic representation of the full "optimization circuit", consisting of the oxidoreductase HpaC and a catalase (KatG).]]<html></p><br />
<br />
<br />
<h3>Results</h3><br />
<p>To show that catalase activity increased <i>E. coli</i> survivability in peroxide we cultured the inducible catalase against a catalase-free control with varying levels of peroxide. After growing overnight the negative didn't grow in any culture except in the absence of peroxide, while the catalase cultures could tolerate peroxide. This is shown below.</p><p><br />
<br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 8: Catalase Assay. Overnight cultures of P<sub>lacI</sub> and P<sub>lacI</sub>-KatGLAA were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbidity was observed. It was found that at 1 mM of peroxide, cultures with just the lacI promotor perished, however when KatG-LAA was expressed, the cells survived.]]<html></p><br />
<br />
<br />
<p>To test the action of HpaC to use NADH to recycle FMN into FMNH<sub>2</sub> cell lysates were exposed to NADH and it's absorbance at 340nm (Kamali <i>et al</i>., 2010) was measured over time. Both native HpaC expression and an induced <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> system were tested as well as a negative control. The results are shown below.</p><br />
<br />
<p> </html> <br />
[[File:Ucalgary2012 HpaC assaycumulativeforthedatapage.png|center|850px|thumb|Figure 9: HpaC Assay with '''A)''' 2 mL cell lysate and '''B)''' 100 &micro;L cell lysate. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, various amounts of cell lysate were transferred to a cuvette, and a spectrophotometer was blanked at 340 nm with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p>The assay showed that NADH does not abiotically convert into NAD+, however the native expression of HpaC did show a steady decrease in the levels of NADH. The induced overexpression of HpaC caused extremely rapid conversion into NAD+ as reflected by a sharp drop in the absorbance of NADH (see figure B). This drop was much sharper than what was seen when native levels of oxidoreductases were tested, showing that the <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> was functional and that it would effectively recycle FMN.</p><br />
<br />
<br />
<a name="UBC"></a><h3>4) Optimizing Gene Order</h3><br />
<br />
<p>Further optimization of the system was achieved through reorganization of the reconstructed operon. Natively the genes are arranged ABC, however the catalytic efficiency of the protein products are 25:1:5 for A:B:C respectively (Li <i>et al</i>., 2008). By rearranging the genes into BCA there is stronger transcription of the weaker proteins, giving a more balanced system overall. These would all be constructed with the same strong ribosomal binding site, <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>.</p><br />
<br />
</html>[[File:DszOperonOptimize.png|center|400px|thumb|Figure 10: Method of optimizing gene order. The top circuit represents that found natively in the organism, with the bottom circuit representing our modified version.]]<html><br />
</p><br />
<br />
<a name="sulfur"></a><h2>Final Constructs</h2><br />
<p>After all of the above considerations are met, four final constructs for our system will be made to allow us to test desulfurization under different conditions.</p><p><br />
<br />
</html>[[File:WikiConstructs_ucalgary_sulfur_2012_final_systems.png|center|700px|thumb|Figure 11: First set of final constructs for the desulfurization operon, with constitutive Dsz expression and inducible expression of the optimization proteins; either HpaC on its own or coexpressed with KatG]]<html></p><br />
<br />
<p><br />
The first two constructs have the modified <i>dsz</i> operon (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902052"><i>dszB</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><i>dszC</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902050"><i>dszA</i></a>) under the control of a constitutive TetR promotor (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>) This is to allow for the testing of the optimization circuit, which is under the control of a lacI promotor inducible by IPTG (<a href="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</a>). The set-up of these two constructs will therefore allow for the expression of the <i>dsz</i> genes with the ability to test and compare their desulfurization rates <br> A) On their own <br> B) With the addition of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> <br> C) With the addition of both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K137068"><i>katG-LAA</i></a></p><br />
<br />
<p>This will allow us to determine what the optimal construct and expression levels of the additional genes must be in order to have the most effective sulfur removal system.</p><br />
<br />
</html>[[File:WikiConstructs2 sulfur ucalgary induciblesytems.PNG|center|700px||thumb|Figure 12: Second set of final constructs for the desulfurization operon, with all genes under an IPTG inducible promotor.]]<html><br />
<br />
<p><br />
Due to the large number of proteins being expressed in this system, the possibility of forming inclusion bodies is present. As such, a backup system was built where both the optimization circuit and the <i>dsz</i> operon were under the control of the inducible lacI promoter. This system would allow us to tune the expression of the genes, and determine which expression level is optimal for desulfurization in our bioreactor.</p> <br />
<br />
<p>Currently the final steps of construction of these constructs is underway, following which functionality tests will begin.</p><br />
<br />
<br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/OSCAR/DesulfurizationTeam:Calgary/Project/OSCAR/Desulfurization2012-10-26T21:12:54Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Desulfurization|<br />
<br />
CONTENT=<html><br />
<img src="https://static.igem.org/mediawiki/2012/5/5e/UCalgary2012_OSCAR_Desulfurization_Low-Res.png" style="float: right; padding: 10px;"></img><br />
<br />
<h2>Why Remove Sulfur?</h2><br />
<br />
<p align="justify"><br />
Sulfur is the third most abundant element in crude oil (Ma, 2010), and when sulfur containing hydrocarbons are burned they release S0<sub>2</sub> and S0<sub>3</sub> gasses into the atmosphere. Not only does this reduce the efficiency and value of our product, but it also contributes to global warming, acid rain, and various health issues due to the pollution (Reichmuth <i>et al</i>., 2000). Strict regulation on sulfur in fuels are now in place and low-sulfur gasoline is mandated across all of Canada (Source: Environment Canada). To upgrade the quality of our fuel we need to remove the sulfur but keep the hydrocarbon backbone for combustion.</p><br />
<br />
<h2>Our Vision</h2><p><br />
Though a few pathways for biodesulfurization exist in the microbial world, most involve the destruction of part of the carbon skeleton (an example would be the Kodama pathway)(Soleimani <i>et al</i>., 2007). This would effectively reduce the quality of our product. With this in mind the pathway we have chosen is the 4S pathway found in <i>Rhodococcus spp</i>. It has been characterized and shown to remove sulfur from the model substrate dibenzothiophene (DBT) and convert it to 2-hydroxybiphenyl (2-HBP) in a non-destructive manner. DBT and its derivatives make up 70% of the organic sulfur compounds found in crude oil (Ma 2010), and are also some of the most difficult to remove through chemical means. By using the 4S pathway we will be able to upgrade our fuel and remove recalcitrant compounds at the same time. <br />
</p><br />
<br />
</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|750px|thumb|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD.]]<html></p><br />
<br />
<h2>4S pathway</h2><br />
<p><br />
Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO<sub>2</sub>) through the addition of 2 oxygen atoms to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the sulfur, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-HBP. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.</p><p> <br />
The first three steps of the 4S pathway require FMNH<sub>2</sub> and subsequently reduces the reductive power of the cell. WIn order to regain this power an oxidoreductase (DszD) uses NADH to recycle the FMNH<sub>2</sub>, allowing the reaction to proceed. Without DszD the desulfurization pathway would grind to a halt.</p><p align="justify"><br />
The <i>dszA</i>,<i>B</i>, and <i>C</i> genes form an operon on the pSOX plasmid of <i>R. erythropolis</i>, while <i>dszD</i> is found in the chromosome. Naturally this pathway is slow, however using synthetic biology approaches this process can be optimized.</p><br />
<br />
<h2>Our Approach</h2><br />
<a name="Degradation"></a><h3>1) Find the genes!</h3><br />
<p>We isolated the plasmid containing the <i>dsz</i> genes from a desulfurising environmental isolate of <i>Rhodococcus</i> using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">modified miniprep procedure</a>. As the native promoter has been shown to be repressed by various sulfur-containing compounds (Li <i>et al</i>., 1996), we designed primers for just the coding sequences of the <i>A, B, </i> and <i>C</i> genes. As these genes all have some illegal cutsites in them we constructed them into the PSB1C3 vector and started our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis protocol</a>.</p><br />
<p> We performed an experiment to measure the desulfurization rate of select organosulfur compounds by our <i>Rhodococcus</i> strain (Figures 4-6 below). These experiments monitored the degradation of the compounds by our strain over time. We discovered that the <i>dsz</i> operon is capable of desulfurizing a wider range of compounds than just the commonly studied DBT. This shows that this pathway could be a promising solution for degradation of a wide variety of sulfur containing toxins, including those that resemble naphthenic acids. </p> <br />
<br />
<p></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 2: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through the GC/MS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GC/MS after 6 days of incubation. ]]<html></p<br />
<br />
<p></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 3: The peak in this mass spectrum demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound (A: dibenzothiophene, B: tetrahydro-4h-thiopyran-4-one, and C: benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdownn. Degradation is seen for DBT (the model compound) as well as other sulfur containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<h3>2) Mutagenesis: Biobrick Compatability and Increasing DszB Activity </h3><br />
<p>In total the <i>dszABC</i> genes had 7 PstI sites and 1 NotI site that needed to be mutated for the biobrick standard. The primers were designed such that the site was removed without the amino acid being changed. In addition, a point mutation of Y63F in DszB increased the activity of the protein (Oshiro <i>et al</i>., 2007), and was included in the mass mutagenesis we undertook. Mutagenesis was performed as described in <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">this protocol.</a></p><br />
<br />
<a name="catalase"></a><h3>3) Replacing DszD with HpaC & Introducing Catalase </h3><br />
<p><br />
As FMNH<sub>2</sub> is consumed in the first three steps of the pathway it needs to be regenerated or the process will grind to a halt. This usually falls to the <i>dszD</i> gene, however it has been shown that the <i>hpaC</i> gene from <i>E. coli</i> performs the same function more efficiently (Gala´n <i>et al</i>., 2000). One problem arises from this though, as high levels of FMNH<sub>2</sub> cause the production of toxic hydrogen peroxide inside the cell (Gala´n <i>et al</i>. 2000). To address this issue we have included a catalase gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"> <i>P<sub>lacI</sub>-katG-LAA</i></a>) that will remove the peroxide that would be toxic to the cell.</p><br />
<br />
<p></html>[[File:Ucalgary_sulfur_constructs_KatandHpaC.PNG|center|250px|thumb|Figure 7: Diagrammatic representation of the full "optimization circuit", consisting of the oxidoreductase HpaC and a catalase (KatG).]]<html></p><br />
<br />
<br />
<h3>Results</h3><br />
<p>To show that catalase activity increased <i>E. coli</i> survivability in peroxide we cultured the inducible catalase against a catalase-free control with varying levels of peroxide. After growing overnight the negative didn't grow in any culture except in the absence of peroxide, while the catalase cultures could tolerate peroxide. This is shown below.</p><p><br />
<br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 8: Catalase Assay. Overnight cultures of P<sub>lacI</sub> and P<sub>lacI</sub>-KatGLAA were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbidity was observed. It was found that at 1 mM of peroxide, cultures with just the lacI promotor perished, however when KatG-LAA was expressed, the cells survived.]]<html></p><br />
<br />
<br />
<p>To test the action of HpaC to use NADH to recycle FMN into FMNH<sub>2</sub> cell lysates were exposed to NADH and it's absorbance at 340nm (Kamali <i>et al</i>., 2010) was measured over time. Both native HpaC expression and an induced <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> system were tested as well as a negative control. The results are shown below.</p><br />
<br />
<p> </html> <br />
[[File:Ucalgary2012 HpaC assaycumulativeforthedatapage.png|center|850px|thumb|Figure 9: HpaC Assay with '''A)''' 2 mL cell lysate and '''B)''' 100 &micro;L cell lysate. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, various amounts of cell lysate were transferred to a cuvette, and a spectrophotometer was blanked at 340 nm with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p>The assay showed that NADH does not abiotically convert into NAD+, however the native expression of HpaC did show a steady decrease in the levels of NADH. The induced overexpression of HpaC caused extremely rapid conversion into NAD+ as reflected by a sharp drop in the absorbance of NADH (see figure B). This drop was much sharper than what was seen when native levels of oxidoreductases were tested, showing that the <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> was functional and that it would effectively recycle FMN.</p><br />
<br />
<br />
<a name="UBC"></a><h3>4) Optimizing Gene Order</h3><br />
<br />
<p>Further optimization of the system was achieved through reorganization of the reconstructed operon. Natively the genes are arranged ABC, however the catalytic efficiency of the protein products are 25:1:5 for A:B:C respectively (Li <i>et al</i>., 2008). By rearranging the genes into BCA there is stronger transcription of the weaker proteins, giving a more balanced system overall. These would all be constructed with the same strong ribosomal binding site, <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>.</p><br />
<br />
</html>[[File:DszOperonOptimize.png|center|400px|thumb|Figure 10: Method of optimizing gene order. The top circuit represents that found natively in the organism, with the bottom circuit representing our modified version.]]<html><br />
</p><br />
<br />
<a name="sulfur"></a><h2>Final Constructs</h2><br />
<p>After all of the above considerations are met, four final constructs for our system will be made to allow us to test desulfurization under different conditions.</p><p><br />
<br />
</html>[[File:WikiConstructs_ucalgary_sulfur_2012_final_systems.png|center|700px|thumb|Figure 11: First set of final constructs for the desulfurization operon, with constitutive Dsz expression and inducible expression of the optimization proteins; either HpaC on its own or coexpressed with KatG]]<html></p><br />
<br />
<p><br />
The first two constructs have the modified <i>dsz</i> operon (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902052"><i>dszB</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><i>dszC</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902050"><i>dszA</i></a>) under the control of a constitutive TetR promotor (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>) This is to allow for the testing of the optimization circuit, which is under the control of a lacI promotor inducible by IPTG (<a href="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</a>). The set-up of these two constructs will therefore allow for the expression of the <i>dsz</i> genes with the ability to test and compare their desulfurization rates <br> A) On their own <br> B) With the addition of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> <br> C) With the addition of both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K137068"><i>katG-LAA</i></a></p><br />
<br />
<p>This will allow us to determine what the optimal construct and expression levels of the additional genes must be in order to have the most effective sulfur removal system.</p><br />
<br />
</html>[[File:WikiConstructs2 sulfur ucalgary induciblesytems.PNG|center|700px||thumb|Figure 12: Second set of final constructs for the desulfurization operon, with all genes under an IPTG inducible promotor.]]<html><br />
<br />
<p><br />
Due to the large number of proteins being expressed in this system, the possibility of forming inclusion bodies is present. As such, a backup system was built where both the optimization circuit and the <i>dsz</i> operon were under the control of the inducible lacI promoter. This system would allow us to tune the expression of the genes, and determine which expression level is optimal for desulfurization in our bioreactor.</p> <br />
<br />
<p>Currently the final steps of construction of these constructs is underway, following which functionality tests will begin.</p><br />
<br />
<br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/FRED/DetectingTeam:Calgary/Project/FRED/Detecting2012-10-26T20:42:26Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Transposon-Mediated Mutant Library for Toxin Detection|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/5/52/UCalgary2012_FRED_Detecting.png" style="float: right; padding: 10px; height: 280px;"></img><br />
<p align="justify"><br />
This year, our team wanted to identify a novel responsive element capable of detecting and quantifying different tailings ponds toxins (e.g. naphthenic acids, NAs) in solution. While numerous studies have begun to identify species of bacteria capable of surviving and sensing a variety of toxic compounds (e.g. NAs), the degradation pathways have not yet been fully characterized. Therefore, we needed to design and implement novel approaches to efficiently isolate the genetic elements that detect and potentially lead to the breakdown of these toxins.<br />
</p><br />
<h2>Transposons: What, How, Why?</h2><br />
<p align="justify"><br />
The transposable element (TE), Tn5, is a conservative transposon that can insert a segment of genes bordered by specific 19bp insertion sequences from one part of the genome (e.g. plasmid vector) randomly to another location like a chromosome (Reznikoff, 2008). The transposition event is catalyzed by a transposase enzyme encoded by <i>tnp</i> gene included in the TE. Using the appropriate selective pressure, the insertion can be maintained permanently in the genome.</p><br />
<br />
</html>[[File:Transposon.jpg|thumb|700px|center|Figure 1: "Transposition reaction from plasmid entry into the recipient cell to integration of the transposon into the genome. Modified from Transposons: Shifting Segments of the Genome" by McGraw Hill]]<html><br />
<br />
<br />
<p align="justify">By inserting a vector construct containing the TE with selectable markers (such as tetracyclin resistance and lacZ) into an organism with a desirable phenotype, we can find out what genetic elements (e.g. genes and promoters) are responsible for that particular function. This can happen via a random insertion of a TE containing a promoterless reporter gene downstream of promoter elements that creates a transcriptional fusion, providing activity in response to specific environmental stimuli. Using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">bipartite-mating (conjugation) method</a> to transfer the TE vector into the organism of choice is an efficient method for creating the massive number of mutants required.</p><br />
<p align="justify"><br />
Due to the complexity of biological systems, our team focused our efforts on utilizing a system for identification of promoter elements that respond specifically in the presence of environmental stimuli. Our hypothesis requires that the organisms we use respond specifically to particular toxins and result in upregulation of metabolic genes with little background effect in the cell. We recognize that any number of biological molecules may play a role in toxin sensing, such as enzymes, transcription factors, and even RNA elements (e.g. riboswitches). However, the identification of a promoter sequence takes us further in that we can better understand the degradation mechanism by elucidating the genes involved.<br />
</p><br />
<br />
<br />
<br><br />
<h2>Toxin-Degrading Organism Used</h2><br />
<p align="justify"><br />
<i>Pseudomonas spp. </i>have been isolated from oil sands tailings ponds and shown to biodegrade model and tailings-associated NAs and nitrogen- and sulfur-containing heterocyclic aromatic compounds (Ramos-Padrón <i>et al</i>. 2010; Herman <i>et al</i>., 1994; Del Rio <i>et al</i>., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins.</p><p> We wanted to use a commercially available strain of <i>Pseudomonas fluorescens</i> characterized for a response to toxins found in tailings pond water (TPW). The <i>P. fluorescens </i>PF-5 strain (Paulsen <i>et al</i>., 2005) is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, the genome sequence is available for this strain with annotations (Pseudomonas Genome Database V2, http://pseudomonas.com/). This allows us to use sequencing data from the mutants and identify where in the genome the TE insertion occurred, and what genes (if present) are located downstream of it.<br />
</p><br />
<br />
<br><br />
<br />
<br />
<a name="library"></a><h2>Method Design</h2><br />
<h3>Mutant Library Generation</h3><br />
<p align="justify"><br />
To construct the promoter library, a pOT182 vector construct (containing a IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR transposable element) is introduced into commercially purchased <i>E. coli SM10</i> donor strain.</p><br />
<br />
<p align="justify"><br />
</html>[[File:Transposonproject Tn5OT182constructucalgary.png|thumb|750px|center|Figure 2: The transposable Tn5 element used in the pOT182 plasmid, containing a lacZ reporter gene, ampicillin and tetracycline resistance, an<br />
<i> E. coli</i> origin of replication for use during downstream sequencing protocols, and transposase. The genes are flanked by the transposon insertion elements]]<html><br />
</p><br />
<br />
<p align="justify">The plasmid contains a RP4 mob conjugation region and a p15A origin of replication (ori), and is engineered to only replicate in <i>E. coli</i>. The TE construct is transferred from the <i>E. coli</i> donor strain to the recipient <i>P. fluorescens </i> PF-5 using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in <i>P. fluorescens</i>, and selected by isolating the recipients that have a genomic TE insertion on Pseudomonas Isolation Agar/PIA with tetracycline. If a promoter element is fused upstream of the TE construct, then promoter activation will turn on the expression of lacZ, which can be detected by the degradation of a colorless compound, X-Gal, to an insoluble blue pigment product (an indoxyl compound) (Juers <i>et al</i>., 2012). If the fused promoter is activated in response to a stimulus, then the lacZ enzyme will be produced in response. Mutant strains sensitive to the particular toxic stimulus will appear as blue colonies on the selective plate.</p><br />
<br />
<h3>Mutant Strain Characterization</h3><br />
<p align="justify">Mutants generated are characterized for their roles in the response to toxins with dose response experiments, and compared to general stress-inducing agents (e.g. H<font style="text-transform: lowercase;">2</font>O<font style="text-transform: lowercase;">2</font>) and compounds such as fatty acids to ensure the specificity of the response. These measurements help to determine thresholds of detection, robustness of the signal, and specificity of response. The dose response curves will also assess the usefulness of correlating the concentration of NA to the level of response, and the possibility of measuring NA concentrations in a sample, rather than simply by presence/absence.</p><br />
</p><br />
<h3>Self-Cloning and Sequencing</h3><br />
<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman and Lamont, 1993). The TE used is a self-cloning construct because it contains all the elements required for plasmid replication (i.e. origin of replication) and selection (Tet resistance). Genomic DNA from a desirable mutant is isolated, and restriction digested with BglII (a restriction enzyme that does not cut within the TE but numerous times within the genome). The resulting fragments may contain the TE construct with flanking sequences. The genomic fragments are circularized by self-ligation and transformed into <i>E. coli</i>. Plasmids from the transformed cells contain the TE construct with the upstream and downstream flanking sequencing connected by the BglII restriction site. Sequencing primers designed against the 19 bp recognition sequence in the TE to sequence the isolated plasmids.</p><br />
<br />
<p align="justify">For a detailed protocol, please consult our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">methods section</a>.</p><br />
<br />
<h2>Results</h2><br />
<h3>Detection by Mutant <i>Pseudomonas fluorescens</i> PF-5</h3><br />
<br />
<br />
<p align="justify">After mating experiments and plating on selective media (Pseudomonas isolation agar, with tetracycline and naphthenic acids), 24 responsive (blue) colonies were found. Screens were conducted on these blue colonies found on selective plates comparing a response in LB and LB with 100mg/L naphthenic acids (both with X-Gal). When results were observed it was found that 4 mutant strains are differentially regulated in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. These colonies were further screened to test the specificity of their responses.</p><br />
<br />
<p align="justify"></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 3: Transposons: Shifting Segments of the Genome: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16hr. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><p align="justify"><br />
<br />
<br />
Screens involving the use of different toxins at environmentally relevant concentrations were performed to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used as one testing condition to determine if the response is simply stress-induced.<br />
</p><br />
<p align="justify"></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 4: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:170-1data.png|thumb|650px|center|Figure 5: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 6: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 7: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<p align="justify"><br />
From these screens, it was seen that both colony 66-1 and colony 170-1 appear to respond to toxins when compared to a response in LB media. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure 8:Stress response screen on <i>P. fluorescens</i>Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 %micro&L of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.]]<html></p><br />
<p><br />
These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.</p><br />
<br />
<h2>Promoter Constructs Isolated</h2><br />
<p align="justify">To determine the location of the transposon insertion, we utilized the self-cloning properties of the transposon. By digesting the genome, religating, and transforming the ligated genomic fragments into <i>E. coli</i>, plasmids containing the transposon and flanking gene sequences were isolated. These plasmids have been isolated and sent for sequencing. However, we are still waiting for the sequencing the results. The results so far are a promising step towards finding a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds. In tandem as we await sequencing results, our next steps will be to test these strains in conjunction with our electrochemical detector.</p><br />
<br><br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
</html><br />
<br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/FRED/DetectingTeam:Calgary/Project/FRED/Detecting2012-10-26T20:21:38Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Transposon-Mediated Mutant Library for Toxin Detection|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/5/52/UCalgary2012_FRED_Detecting.png" style="float: right; padding: 10px; height: 280px;"></img><br />
<p align="justify"><br />
This year, our team wanted to identify a novel responsive element capable of detecting and quantifying different tailings ponds toxins (e.g. naphthenic acids, NAs) in solution. While numerous studies have begun to identify species of bacteria capable of surviving and sensing a variety of toxic compounds (e.g. NAs), the degradation pathways have not yet been fully characterized. Therefore, we needed to design and implement novel approaches to efficiently isolate the genetic elements that detect and potentially lead to the breakdown of these toxins.<br />
</p><br />
<h2>Transposons: What, How, Why?</h2><br />
<p align="justify"><br />
The transposable element (TE), Tn5, is a conservative transposon that can insert a segment of genes bordered by specific 19bp insertion sequences from one part of the genome (e.g. plasmid vector) randomly to another location like a chromosome (Reznikoff, 2008). The transposition event is catalyzed by a transposase enzyme encoded by <i>tnp</i> gene included in the TE. Using the appropriate selective pressure, the insertion can be maintained permanently in the genome.</p><br />
<br />
</html>[[File:Transposon.jpg|thumb|700px|center|Figure 1: "Transposition reaction from plasmid entry into the recipient cell to integration of the transposon into the genome. Modified from Transposons: Shifting Segments of the Genome" by McGraw Hill]]<html><br />
<br />
<br />
<p align="justify">By inserting a vector construct containing the TE with selectable markers (such as tetracyclin resistance and lacZ) into an organism with a desirable phenotype, we can find out what genetic elements (e.g. genes and promoters) are responsible for that particular function. This can happen via a random insertion of a TE containing a promoterless reporter gene downstream of promoter elements that creates a transcriptional fusion, providing activity in response to specific environmental stimuli. Using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">bipartite-mating (conjugation) method</a> to transfer the TE vector into the organism of choice is an efficient method for creating the massive number of mutants required.</p><br />
<p align="justify"><br />
Due to the complexity of biological systems, our team focused our efforts on utilizing a system for identification of promoter elements that respond specifically in the presence of environmental stimuli. Our hypothesis requires that the organisms we use respond specifically to particular toxins and result in upregulation of metabolic genes with little background effect in the cell. We recognize that any number of biological molecules may play a role in toxin sensing, such as enzymes, transcription factors, and even RNA elements (e.g. riboswitches). However, the identification of a promoter sequence takes us further in that we can better understand the degradation mechanism by elucidating the genes involved.<br />
</p><br />
<br />
<br />
<br><br />
<h2>Toxin-Degrading Organism Used</h2><br />
<p align="justify"><br />
<i>Pseudomonas spp. </i>have been isolated from oil sands tailings ponds and shown to biodegrade model and tailings-associated NAs and nitrogen- and sulfur-containing heterocyclic aromatic compounds (Ramos-Padrón <i>et al</i>. 2010; Herman <i>et al</i>., 1994; Del Rio <i>et al</i>., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins.</p><p> We wanted to use a commercially available strain of <i>Pseudomonas fluorescens</i> characterized for a response to toxins found in tailings pond water (TPW). The <i>P. fluorescens </i>PF-5 strain (Paulsen <i>et al</i>., 2005) is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, the genome sequence is available for this strain with annotations (Pseudomonas Genome Database V2, http://pseudomonas.com/). This allows us to use sequencing data from the mutants and identify where in the genome the TE insertion occurred, and what genes (if present) are located downstream of it.<br />
</p><br />
<br />
<br><br />
<br />
<br />
<a name="library"></a><h2>Method Design</h2><br />
<h3>Mutant Library Generation</h3><br />
<p align="justify"><br />
To construct the promoter library, a pOT182 vector construct (containing a IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR transposable element) is introduced into commercially purchased <i>E. coli SM10</i> donor strain.</p><br />
<br />
<p align="justify"><br />
</html>[[File:Transposonproject Tn5OT182constructucalgary.png|thumb|750px|center|Figure 2: The transposable Tn5 element used in the pOT182 plasmid, containing a lacZ reporter gene, ampicillin and tetracycline resistance, an<br />
<i> E. coli</i> origin of replication for use during downstream sequencing protocols, and transposase. The genes are flanked by the transposon insertion elements]]<html><br />
</p><br />
<br />
<p align="justify">The plasmid contains a RP4 mob conjugation region and a p15A origin of replication (ori), and is engineered to only replicate in <i>E. coli</i>. The TE construct is transferred from the <i>E. coli</i> donor strain to the recipient <i>P. fluorescens </i> PF-5 using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in <i>P. fluorescens</i>, and selected by isolating the recipients that have a genomic TE insertion on Pseudomonas Isolation Agar/PIA with tetracycline. If a promoter element is fused upstream of the TE construct, then promoter activation will turn on the expression of lacZ, which can be detected by the degradation of a colorless compound, X-Gal, to an insoluble blue pigment product (an indoxyl compound) (Juers <i>et al</i>., 2012). If the fused promoter is activated in response to a stimulus, then the lacZ enzyme will be produced in response. Mutant strains sensitive to the particular toxic stimulus will appear as blue colonies on the selective plate.</p><br />
<br />
<h3>Mutant Strain Characterization</h3><br />
<p align="justify">Mutants generated are characterized for their roles in the response to toxins with dose response experiments, and compared to general stress-inducing agents (e.g. H<font style="text-transform: lowercase;">2</font>O<font style="text-transform: lowercase;">2</font>) and compounds such as fatty acids to ensure the specificity of the response. These measurements help to determine thresholds of detection, robustness of the signal, and specificity of response. The dose response curves will also assess the usefulness of correlating the concentration of NA to the level of response, and the possibility of measuring NA concentrations in a sample, rather than simply by presence/absence.</p><br />
</p><br />
<h3>Self-Cloning and Sequencing</h3><br />
<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman and Lamont, 1993). The TE used is a self-cloning construct because it contains all the elements required for plasmid replication (i.e. origin of replication) and selection (Tet resistance). Genomic DNA from a desirable mutant is isolated, and restriction digested with BglII (a restriction enzyme that does not cut within the TE but numerous times within the genome). The resulting fragments may contain the TE construct with flanking sequences. The genomic fragments are circularized by self-ligation and transformed into <i>E. coli</i>. Plasmids from the transformed cells contain the TE construct with the upstream and downstream flanking sequencing connected by the BglII restriction site. Sequencing primers designed against the 19 bp recognition sequence in the TE to sequence the isolated plasmids.</p><br />
<br />
<p align="justify">For a detailed protocol, please consult our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">methods section</a>.</p><br />
<br />
<h2>Results</h2><br />
<h3>Detection by Mutant <i>Pseudomonas fluorescens</i> PF-5</h3><br />
<br />
<br />
<p align="justify">After mating experiments and plating on selective media (Pseudomonas isolation agar, with tetracycline and naphthenic acids), 24 responsive (blue) colonies were found. Screens were conducted on these blue colonies found on selective plates comparing a response in LB and LB with 100mg/L naphthenic acids (both with X-Gal). When results were observed it was found that 4 mutant strains are differentially regulated in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. These colonies were further screened to test the specificity of their responses.</p><br />
<br />
<p align="justify"></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 3: Transposons: Shifting Segments of the Genome: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16hr. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><p align="justify"><br />
<br />
<br />
Screens involving the use of different toxins at environmentally relevant concentrations were performed to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used as one testing condition to determine if the response is simply stress-induced.<br />
</p><br />
<p align="justify"></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 4: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:170-1data.png|thumb|650px|center|Figure 5: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 6: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 7: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<p align="justify"><br />
From these screens, it was seen that both colony 66-1 and colony 170-1 appear to respond to toxins when compared to a response in LB media. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure x: ]]<html></p><br />
<br />
<h2>Promoter Constructs Isolated</h2><br />
<p align="justify">To determine the location of the transposon insertion, we utilized the self-cloning properties of the transposon. By digesting the genome, religating, and transforming the ligated genomic fragments into <i>E. coli</i>, plasmids containing the transposon and flanking gene sequences were isolated. These plasmids have been isolated and sent for sequencing. However, we are still waiting for the sequencing the results. The results so far are a promising step towards finding a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds. In tandem as we await sequencing results, our next steps will be to test these strains in conjunction with our electrochemical detector.</p><br />
<br><br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
</html><br />
<br />
}}</div>Lisa.Ohttp://2012.igem.org/File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.pngFile:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png2012-10-26T20:21:07Z<p>Lisa.O: uploaded a new version of &quot;File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png&quot;</p>
<hr />
<div></div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/FRED/DetectingTeam:Calgary/Project/FRED/Detecting2012-10-26T20:17:29Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Transposon-Mediated Mutant Library for Toxin Detection|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/5/52/UCalgary2012_FRED_Detecting.png" style="float: right; padding: 10px; height: 280px;"></img><br />
<p align="justify"><br />
This year, our team wanted to identify a novel responsive element capable of detecting and quantifying different tailings ponds toxins (e.g. naphthenic acids, NAs) in solution. While numerous studies have begun to identify species of bacteria capable of surviving and sensing a variety of toxic compounds (e.g. NAs), the degradation pathways have not yet been fully characterized. Therefore, we needed to design and implement novel approaches to efficiently isolate the genetic elements that detect and potentially lead to the breakdown of these toxins.<br />
</p><br />
<h2>Transposons: What, How, Why?</h2><br />
<p align="justify"><br />
The transposable element (TE), Tn5, is a conservative transposon that can insert a segment of genes bordered by specific 19bp insertion sequences from one part of the genome (e.g. plasmid vector) randomly to another location like a chromosome (Reznikoff, 2008). The transposition event is catalyzed by a transposase enzyme encoded by <i>tnp</i> gene included in the TE. Using the appropriate selective pressure, the insertion can be maintained permanently in the genome.</p><br />
<br />
</html>[[File:Transposon.jpg|thumb|700px|center|Figure 1: "Transposition reaction from plasmid entry into the recipient cell to integration of the transposon into the genome. Modified from Transposons: Shifting Segments of the Genome" by McGraw Hill]]<html><br />
<br />
<br />
<p align="justify">By inserting a vector construct containing the TE with selectable markers (such as tetracyclin resistance and lacZ) into an organism with a desirable phenotype, we can find out what genetic elements (e.g. genes and promoters) are responsible for that particular function. This can happen via a random insertion of a TE containing a promoterless reporter gene downstream of promoter elements that creates a transcriptional fusion, providing activity in response to specific environmental stimuli. Using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">bipartite-mating (conjugation) method</a> to transfer the TE vector into the organism of choice is an efficient method for creating the massive number of mutants required.</p><br />
<p align="justify"><br />
Due to the complexity of biological systems, our team focused our efforts on utilizing a system for identification of promoter elements that respond specifically in the presence of environmental stimuli. Our hypothesis requires that the organisms we use respond specifically to particular toxins and result in upregulation of metabolic genes with little background effect in the cell. We recognize that any number of biological molecules may play a role in toxin sensing, such as enzymes, transcription factors, and even RNA elements (e.g. riboswitches). However, the identification of a promoter sequence takes us further in that we can better understand the degradation mechanism by elucidating the genes involved.<br />
</p><br />
<br />
<br />
<br><br />
<h2>Toxin-Degrading Organism Used</h2><br />
<p align="justify"><br />
<i>Pseudomonas spp. </i>have been isolated from oil sands tailings ponds and shown to biodegrade model and tailings-associated NAs and nitrogen- and sulfur-containing heterocyclic aromatic compounds (Ramos-Padrón <i>et al</i>. 2010; Herman <i>et al</i>., 1994; Del Rio <i>et al</i>., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins.</p><p> We wanted to use a commercially available strain of <i>Pseudomonas fluorescens</i> characterized for a response to toxins found in tailings pond water (TPW). The <i>P. fluorescens </i>PF-5 strain (Paulsen <i>et al</i>., 2005) is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, the genome sequence is available for this strain with annotations (Pseudomonas Genome Database V2, http://pseudomonas.com/). This allows us to use sequencing data from the mutants and identify where in the genome the TE insertion occurred, and what genes (if present) are located downstream of it.<br />
</p><br />
<br />
<br><br />
<br />
<br />
<a name="library"></a><h2>Method Design</h2><br />
<h3>Mutant Library Generation</h3><br />
<p align="justify"><br />
To construct the promoter library, a pOT182 vector construct (containing a IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR transposable element) is introduced into commercially purchased <i>E. coli SM10</i> donor strain.</p><br />
<br />
<p align="justify"><br />
</html>[[File:Transposonproject Tn5OT182constructucalgary.png|thumb|750px|center|Figure 2: The transposable Tn5 element used in the pOT182 plasmid, containing a lacZ reporter gene, ampicillin and tetracycline resistance, an<br />
<i> E. coli</i> origin of replication for use during downstream sequencing protocols, and transposase. The genes are flanked by the transposon insertion elements]]<html><br />
</p><br />
<br />
<p align="justify">The plasmid contains a RP4 mob conjugation region and a p15A origin of replication (ori), and is engineered to only replicate in <i>E. coli</i>. The TE construct is transferred from the <i>E. coli</i> donor strain to the recipient <i>P. fluorescens </i> PF-5 using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in <i>P. fluorescens</i>, and selected by isolating the recipients that have a genomic TE insertion on Pseudomonas Isolation Agar/PIA with tetracycline. If a promoter element is fused upstream of the TE construct, then promoter activation will turn on the expression of lacZ, which can be detected by the degradation of a colorless compound, X-Gal, to an insoluble blue pigment product (an indoxyl compound) (Juers <i>et al</i>., 2012). If the fused promoter is activated in response to a stimulus, then the lacZ enzyme will be produced in response. Mutant strains sensitive to the particular toxic stimulus will appear as blue colonies on the selective plate.</p><br />
<br />
<h3>Mutant Strain Characterization</h3><br />
<p align="justify">Mutants generated are characterized for their roles in the response to toxins with dose response experiments, and compared to general stress-inducing agents (e.g. H<font style="text-transform: lowercase;">2</font>O<font style="text-transform: lowercase;">2</font>) and compounds such as fatty acids to ensure the specificity of the response. These measurements help to determine thresholds of detection, robustness of the signal, and specificity of response. The dose response curves will also assess the usefulness of correlating the concentration of NA to the level of response, and the possibility of measuring NA concentrations in a sample, rather than simply by presence/absence.</p><br />
</p><br />
<h3>Self-Cloning and Sequencing</h3><br />
<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman and Lamont, 1993). The TE used is a self-cloning construct because it contains all the elements required for plasmid replication (i.e. origin of replication) and selection (Tet resistance). Genomic DNA from a desirable mutant is isolated, and restriction digested with BglII (a restriction enzyme that does not cut within the TE but numerous times within the genome). The resulting fragments may contain the TE construct with flanking sequences. The genomic fragments are circularized by self-ligation and transformed into <i>E. coli</i>. Plasmids from the transformed cells contain the TE construct with the upstream and downstream flanking sequencing connected by the BglII restriction site. Sequencing primers designed against the 19 bp recognition sequence in the TE to sequence the isolated plasmids.</p><br />
<br />
<p align="justify">For a detailed protocol, please consult our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">methods section</a>.</p><br />
<br />
<h2>Results</h2><br />
<h3>Detection by Mutant <i>Pseudomonas fluorescens</i> PF-5</h3><br />
<br />
<br />
<p align="justify">After mating experiments and plating on selective media (Pseudomonas isolation agar, with tetracycline and naphthenic acids), 24 responsive (blue) colonies were found. Screens were conducted on these blue colonies found on selective plates comparing a response in LB and LB with 100mg/L naphthenic acids (both with X-Gal). When results were observed it was found that 4 mutant strains are differentially regulated in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. These colonies were further screened to test the specificity of their responses.</p><br />
<br />
<p align="justify"></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 3: Transposons: Shifting Segments of the Genome: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16hr. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><p align="justify"><br />
<br />
<br />
Screens involving the use of different toxins at environmentally relevant concentrations were performed to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used as one testing condition to determine if the response is simply stress-induced.<br />
</p><br />
<p align="justify"></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 4: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:170-1data.png|thumb|650px|center|Figure 5: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 6: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 7: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<p align="justify"><br />
From these screens, it was seen that both colony 66-1 and colony 170-1 appear to respond to toxins when compared to a response in LB media. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|900px|center|Figure x: ]]<html></p><br />
<br />
<h2>Promoter Constructs Isolated</h2><br />
<p align="justify">To determine the location of the transposon insertion, we utilized the self-cloning properties of the transposon. By digesting the genome, religating, and transforming the ligated genomic fragments into <i>E. coli</i>, plasmids containing the transposon and flanking gene sequences were isolated. These plasmids have been isolated and sent for sequencing. However, we are still waiting for the sequencing the results. The results so far are a promising step towards finding a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds. In tandem as we await sequencing results, our next steps will be to test these strains in conjunction with our electrochemical detector.</p><br />
<br><br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
</html><br />
<br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/FRED/DetectingTeam:Calgary/Project/FRED/Detecting2012-10-26T20:16:57Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Transposon-Mediated Mutant Library for Toxin Detection|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/5/52/UCalgary2012_FRED_Detecting.png" style="float: right; padding: 10px; height: 280px;"></img><br />
<p align="justify"><br />
This year, our team wanted to identify a novel responsive element capable of detecting and quantifying different tailings ponds toxins (e.g. naphthenic acids, NAs) in solution. While numerous studies have begun to identify species of bacteria capable of surviving and sensing a variety of toxic compounds (e.g. NAs), the degradation pathways have not yet been fully characterized. Therefore, we needed to design and implement novel approaches to efficiently isolate the genetic elements that detect and potentially lead to the breakdown of these toxins.<br />
</p><br />
<h2>Transposons: What, How, Why?</h2><br />
<p align="justify"><br />
The transposable element (TE), Tn5, is a conservative transposon that can insert a segment of genes bordered by specific 19bp insertion sequences from one part of the genome (e.g. plasmid vector) randomly to another location like a chromosome (Reznikoff, 2008). The transposition event is catalyzed by a transposase enzyme encoded by <i>tnp</i> gene included in the TE. Using the appropriate selective pressure, the insertion can be maintained permanently in the genome.</p><br />
<br />
</html>[[File:Transposon.jpg|thumb|700px|center|Figure 1: "Transposition reaction from plasmid entry into the recipient cell to integration of the transposon into the genome. Modified from Transposons: Shifting Segments of the Genome" by McGraw Hill]]<html><br />
<br />
<br />
<p align="justify">By inserting a vector construct containing the TE with selectable markers (such as tetracyclin resistance and lacZ) into an organism with a desirable phenotype, we can find out what genetic elements (e.g. genes and promoters) are responsible for that particular function. This can happen via a random insertion of a TE containing a promoterless reporter gene downstream of promoter elements that creates a transcriptional fusion, providing activity in response to specific environmental stimuli. Using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">bipartite-mating (conjugation) method</a> to transfer the TE vector into the organism of choice is an efficient method for creating the massive number of mutants required.</p><br />
<p align="justify"><br />
Due to the complexity of biological systems, our team focused our efforts on utilizing a system for identification of promoter elements that respond specifically in the presence of environmental stimuli. Our hypothesis requires that the organisms we use respond specifically to particular toxins and result in upregulation of metabolic genes with little background effect in the cell. We recognize that any number of biological molecules may play a role in toxin sensing, such as enzymes, transcription factors, and even RNA elements (e.g. riboswitches). However, the identification of a promoter sequence takes us further in that we can better understand the degradation mechanism by elucidating the genes involved.<br />
</p><br />
<br />
<br />
<br><br />
<h2>Toxin-Degrading Organism Used</h2><br />
<p align="justify"><br />
<i>Pseudomonas spp. </i>have been isolated from oil sands tailings ponds and shown to biodegrade model and tailings-associated NAs and nitrogen- and sulfur-containing heterocyclic aromatic compounds (Ramos-Padrón <i>et al</i>. 2010; Herman <i>et al</i>., 1994; Del Rio <i>et al</i>., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins.</p><p> We wanted to use a commercially available strain of <i>Pseudomonas fluorescens</i> characterized for a response to toxins found in tailings pond water (TPW). The <i>P. fluorescens </i>PF-5 strain (Paulsen <i>et al</i>., 2005) is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, the genome sequence is available for this strain with annotations (Pseudomonas Genome Database V2, http://pseudomonas.com/). This allows us to use sequencing data from the mutants and identify where in the genome the TE insertion occurred, and what genes (if present) are located downstream of it.<br />
</p><br />
<br />
<br><br />
<br />
<br />
<a name="library"></a><h2>Method Design</h2><br />
<h3>Mutant Library Generation</h3><br />
<p align="justify"><br />
To construct the promoter library, a pOT182 vector construct (containing a IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR transposable element) is introduced into commercially purchased <i>E. coli SM10</i> donor strain.</p><br />
<br />
<p align="justify"><br />
</html>[[File:Transposonproject Tn5OT182constructucalgary.png|thumb|750px|center|Figure 2: The transposable Tn5 element used in the pOT182 plasmid, containing a lacZ reporter gene, ampicillin and tetracycline resistance, an<br />
<i> E. coli</i> origin of replication for use during downstream sequencing protocols, and transposase. The genes are flanked by the transposon insertion elements]]<html><br />
</p><br />
<br />
<p align="justify">The plasmid contains a RP4 mob conjugation region and a p15A origin of replication (ori), and is engineered to only replicate in <i>E. coli</i>. The TE construct is transferred from the <i>E. coli</i> donor strain to the recipient <i>P. fluorescens </i> PF-5 using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in <i>P. fluorescens</i>, and selected by isolating the recipients that have a genomic TE insertion on Pseudomonas Isolation Agar/PIA with tetracycline. If a promoter element is fused upstream of the TE construct, then promoter activation will turn on the expression of lacZ, which can be detected by the degradation of a colorless compound, X-Gal, to an insoluble blue pigment product (an indoxyl compound) (Juers <i>et al</i>., 2012). If the fused promoter is activated in response to a stimulus, then the lacZ enzyme will be produced in response. Mutant strains sensitive to the particular toxic stimulus will appear as blue colonies on the selective plate.</p><br />
<br />
<h3>Mutant Strain Characterization</h3><br />
<p align="justify">Mutants generated are characterized for their roles in the response to toxins with dose response experiments, and compared to general stress-inducing agents (e.g. H<font style="text-transform: lowercase;">2</font>O<font style="text-transform: lowercase;">2</font>) and compounds such as fatty acids to ensure the specificity of the response. These measurements help to determine thresholds of detection, robustness of the signal, and specificity of response. The dose response curves will also assess the usefulness of correlating the concentration of NA to the level of response, and the possibility of measuring NA concentrations in a sample, rather than simply by presence/absence.</p><br />
</p><br />
<h3>Self-Cloning and Sequencing</h3><br />
<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman and Lamont, 1993). The TE used is a self-cloning construct because it contains all the elements required for plasmid replication (i.e. origin of replication) and selection (Tet resistance). Genomic DNA from a desirable mutant is isolated, and restriction digested with BglII (a restriction enzyme that does not cut within the TE but numerous times within the genome). The resulting fragments may contain the TE construct with flanking sequences. The genomic fragments are circularized by self-ligation and transformed into <i>E. coli</i>. Plasmids from the transformed cells contain the TE construct with the upstream and downstream flanking sequencing connected by the BglII restriction site. Sequencing primers designed against the 19 bp recognition sequence in the TE to sequence the isolated plasmids.</p><br />
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<p align="justify">For a detailed protocol, please consult our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">methods section</a>.</p><br />
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<h2>Results</h2><br />
<h3>Detection by Mutant <i>Pseudomonas fluorescens</i> PF-5</h3><br />
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<p align="justify">After mating experiments and plating on selective media (Pseudomonas isolation agar, with tetracycline and naphthenic acids), 24 responsive (blue) colonies were found. Screens were conducted on these blue colonies found on selective plates comparing a response in LB and LB with 100mg/L naphthenic acids (both with X-Gal). When results were observed it was found that 4 mutant strains are differentially regulated in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. These colonies were further screened to test the specificity of their responses.</p><br />
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<p align="justify"></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 3: Transposons: Shifting Segments of the Genome: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16hr. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><p align="justify"><br />
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Screens involving the use of different toxins at environmentally relevant concentrations were performed to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used as one testing condition to determine if the response is simply stress-induced.<br />
</p><br />
<p align="justify"></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 4: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
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<p align="justify"></html>[[File:170-1data.png|thumb|650px|center|Figure 5: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
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<p align="justify"></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 6: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 7: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<p align="justify"><br />
From these screens, it was seen that both colony 66-1 and colony 170-1 appear to respond to toxins when compared to a response in LB media. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure x: ]]<html></p><br />
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<h2>Promoter Constructs Isolated</h2><br />
<p align="justify">To determine the location of the transposon insertion, we utilized the self-cloning properties of the transposon. By digesting the genome, religating, and transforming the ligated genomic fragments into <i>E. coli</i>, plasmids containing the transposon and flanking gene sequences were isolated. These plasmids have been isolated and sent for sequencing. However, we are still waiting for the sequencing the results. The results so far are a promising step towards finding a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds. In tandem as we await sequencing results, our next steps will be to test these strains in conjunction with our electrochemical detector.</p><br />
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}}</div>Lisa.Ohttp://2012.igem.org/File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.pngFile:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png2012-10-26T20:08:28Z<p>Lisa.O: </p>
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<div></div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/DesulfurizationTeam:Calgary/Notebook/Desulfurization2012-10-26T18:17:12Z<p>Lisa.O: </p>
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<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Desulfurization Journal|<br />
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CONTENT=<br />
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<h2>Week 1 (May 1-4)</h2><br />
<p>During this week, literature search was performed.</p><br />
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<h2>Week 2 (May 7-11)</h2><br />
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<p>Along with the rest of the team, this week was dedicated to familiarizing ourselves on the protocols that will be utilized during this years project; specifically the polymerase chain reaction, gel verification, preparation of overnight cultures, as well as developing a procedural flowchart to transform competent cells with registry biobricks. With regards to our sub-group specific goals, we reviewed the current available literature around various industrial and laboratory approaches to desulfurization of organic groups, especially in the petroleum industry. This included a comparison of non-biological processes such as conventional hydrodesulfurization, which is currently employed in petroleum product refinery stages, and how a biological approach would supplement and perhaps even offer several advantages over these methods. Current limitations to biological desulfurization, however, include such factors as biocatalyst stability, enzyme specificity and desulfurization rate. We also identified the enzyme desulfinase (DszB) as being one of the bottlenecks in the desulfurization 4S pathway. Overall, our goals moving forward involve determining the specific pathways involved in the desulfurization process as well as the reaction conditions we would want to employ, and identifying specific model compounds in addition to dibenzothiophene (DBT) that we could use to test the effectivity of our biosystem in order to determine its functionality in the conversion of naphthenic acids to economically valuable hydrocarbons.</p><br />
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<h2>Week 3 (May 14-18)</h2><br />
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<p> Building on the previous week's literature review, the 4S pathway was recognized as the preferred biological mechanism that we would explore in devising a desulfurization biosystem. Of specific interest is the dsz operon consisting of the genes for <i> dszA, dszB, </i>and<i> dszC</i> which selectively and non-destructively remove the sulfur from the hydrocarbon structure, and therefore preserves the carbon skeleton. In addition to these, another dsz gene exists.<i> dszD</i>, which codes for a FMN:NADH reductase, is an essential component of the pathway, but not part of the operon. Instead, it is on the genome. The enzyme produced by this gene is required to regenerate the FMNH2 consumed by the reactions carried out by DszA and DszC. <i>Rhodococcus erythropolis</i> IGTS8 is the most studied model organism in investigations of the 4S pathway, and has been shown in many different research endeavors to be capable of converting DBT to 2-HBP.</p><p><br />
</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|thumb|700px|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD. Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO2) through the addition of oxygen to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the heteroatom, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-hydroxybiphenyl. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.]]<html></p><p><br />
An alternative to the DszD gene is HpaC, an oxidoreductase encoded in the <i>E. coli</i> W genome. This enzyme has been shown to increase the rate of desulfurization. Following this, other protocols added to our growing lab methods 'toolkit' were a restriction digest protocol, PCR purification, and finally, DNA construction digest. Aims moving forward include obtaining strains of the <i> R. erythropolis </i>, while also executing a timeline devised to biobrick, test, and incorporate the genes necessary in the above processes in a biobrick circuit.</p><br />
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<h2>Week 4 (May 22-25)</h2><br />
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<p>This week was kicked off with a project development meeting with Emily and David, and we devised a protocol for biobricking the <i> hpaC </i> gene. Additionally, methods to place the genes coding for the 4 enzymes, DszA,B,C and HpaC into a single construct were explored. Within the lab, the PCR performed on the resuspended pUC18-hpaC was not successful initially. <br />
Furthermore, we ordered the substrates/compounds that we intend to use for desulfurization tests. Once the substrates and the <i>Rhodococcus</i> strain arrive we are going to test how effectively the bacteria can desulfurize different sulphur-containing compounds that resemble naphthenic acids. Finally, we came across a paper where a team had developed an improved efficiency DszB through site-directed mutagenesis in 2007. This was through a point mutation to the gene, converting a tyrosine at position 63 to a phenylalanine residue. A member of this team was contacted to request the plasmid that contains the mutated gene. The conversion step carried out by DszB is the major bottleneck in the 4S pathway and if a strain or sample containing this mutation was obtained, it would significantly bolster our later testing efforts on DBT, as well as other compounds such as thiophane.<br />
</p><br />
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<h2>Week 5 (May 28 - June 1)</h2><br />
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<p>Since we wanted to make sure we would not run out of pUC18(plasmid containing the hpaC gene), we transformed some E.coli cells with it. We grew them on plates containing ampicillin (A), kanamycin (K), tetracycline (T) and chloramphenicol (C) antibiotics and they only grew on A. Therefore pUC18 has A resistance. We did a three sets of PCR with primers designed against <i>hpaC</i>, one using 1/10 dilution of pUC18, the other using 1/100 dilution of pUC18 and one with the colonies we had just obtained by transforming the <i>E.coli</i> cells. </p><br />
<p>The PCR worked and we saw bands of the same size for all three sets of PCR. (Unfortunately, the picture we saved is not a good one since some of the bands faded away under UV due to prolonged exposure. Following this, PCR purification was performed to obtain the pure <i>hpaC</i> with biobrick prefix and suffix attatched to gene, which would allow us to insert the sequence into a biobrick standard backbone. 3 sets of digestion, ligation, and transformation (using pairs of X&P enzymes, E&S enzymes and E&P enzymes) were carried out in order to insert the <i>hpaC </i> gene into the <a href=" http://partsregistry.org/Part:pSB1C3">pSB1C3</a><br />
vector. All the sets grew successfully.<br />
Following the above successes with<i> hpaC</i>, the arrival of our <i>Rhodococcus</i> strain afforded us the opportunity to begin investigation of the Dsz operon using the primers currently in our possession. This strain is an environmental isolate that has been shown to be an active desulfurizer. The gram-positive nature of the strain also dictated we explore various lysing strategies before the genes encoding the Dsz enzymes could be amplified for further purification and biobrick construction steps. PCR was carried out using <i>dszA</i> primers on three different treatments {microwave, lysate buffer, and a control} which yielded banding pattern around 1200 base pairs for the lysate treatment (2%SDS and 10% tritonX-100, plus heat for 5mins at 98C).<br />
</p> <br />
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<h2>Week 6 (June 4 - June 8)</h2><br />
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<p> In order to confirm the <i>hpaC</i> biobrick construction, two sets of colony PCR were performed, choosing white colonies from the 3 plates we grew last week (white colonies indicate a loss of the RFP generator in the <a href=" http://partsregistry.org/Part:pSB1C3">pSB1C3</a><br />
backbone, and therefore allow for weeding out of the colonies which are simply the original plasmid vector). These reactions were carried out both with <i>hpaC</i> primers and with standard biobrick primers designed against the plasmid backbone. After running them on the gel we saw equal bands for the PCR reactions performed using <i>hpaC</i> primers (However, a PCR using biobrick primers was performed later and the same results were obtained). Colonies 1(-) and 5(-) were used to make overnight cultures, which were then miniprepped the following day to obtain the plasmid DNA of the putative <i>hpaC</i> biobrick. Digestions were performed on the miniprep products using EcoRI and PstI to look for part size as further verification for the genes presence in the plasmid. The results were good and two bands were observed on each column (one for vector and the other for <i>hpaC</i>)). <i>hpaC</i> was sent in for sequencing. </p><br />
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</html>[[File:UCalgary2012_04.06.2012-desulfurisation_hpacverification.jpg|thumb|700px|center|Figure 2: HpaC verification cPCR. HpaC gene was inserted into the pSB1C33 vector and E. coli cells were transformed. In order to confirm that pSB1C3 contains the hpaC gene, two sets of colony PCR's were conducted. One with biobrick primers, and the other with hpaC primers. Bands indicate successful amplification at the approximate size of the hpaC gene (517 bp)]]<br />
[[File:Ucalgary2012 06.06.2012-digestion of hpaC with E and P.jpg|thumb|500px|center|Figure 3: HpaC confirmation digest. Plasmid was miniprepped and digested for 2h with EcoRI and SpeI before being run on a 1% gel for 1h and 120V. Bands between 500 and 700 bp indicate the hpaC part is present as an insert. Bands of about 2000bp show the size of the psb1c3 vector.]]<html></p><br />
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<p> PCR reagents were prepared to re-test/confirm previous results of <i>dszA</i> amplification following two different lysing treatments (microwave + lysate buffer). This time, all three genes were amplified and gel verification showed clear banding patterns around 500bp range for all three genes for the microwave treatment. Remaining PCR products were run on a gel and extracted for further purification steps; however, presence of any genetic material were not confirmed through nanodropping which raised concerns about the composition of the purified products, the success of the initial amplification step, or perhaps even the lysis treatment. Further experimentation will have to be carried out to troubleshoot.</p><br />
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<h2>Week 7 (June 11 - June 15)</h2><br />
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<p> This week, we focused on amplifying <i>dsz</i> genes from our <i>Rhodococcus</i> strain for construction into biobricks. We also wanted to purify the <a href=" http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"> BBa_K902057 <i>hpaC</i></a> and pUC18-<i>hpaC</i> plasmids to replenish our current stocks. For the <i>dsz</i> aspect, we were able to successfully grow extra plates of <i>Rhodococcus</i> strain which was used to inoculate PCR tubes. The PCR did not go well, with significant streaking and false positives with similar banding pattern to previous gels run in the previous week. A final gel verification of a random sample of a tube of PCR products from dszA,B,C respectively and two negative control treatments involving master mix only and the lysed cells only illustrated the lack of discrepancy between the supposed successful amplification and the lysed cells (with lysate buffer) alone. Because of this we decided to take a different approach involving plasmid isolation carried out before PCR, rather than applying the PCR reagents directly to a lysed culture sample.</p><br />
<p> <a href=" http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> verification through sequencing was successful, confirming the construction of our first biobrick. Subsequently, O/N cultures of the plasmid containing cultures were prepared and stored in glycerol at -80C. Furthermore, verification of catalase gene part (<a href=" http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a>), which was sent as a culture stab from the parts registry was initiated. Our newly identified biobricked-hpaC was used as a positive control, but the banding pattern was not very conclusive. </p><br />
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<h2>Week 8 (June 18 - June 22)</h2><br />
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<p>PCR was reattempted on <i>Rhodoccocus</i> that was lysed using two different dilutions of the lysate buffer, but the gel verification confirmed the previous failure in using this approach. An alternative that involved preparation of an overnight culture of the <i>Rhodococcus</i> cells followed by a plasmid purification was followed. The plasmid purification eventually yielded plasmid samples with concentrations of 98.6ng/μL to 182.7ng/μL (4 samples obtained overall). Additionally, the catalase biobrick was used to transform some stock competent cells, and samples of some colonies were subsequently PCR'ed. Although, the gel verification showed some potential contamination, and the required banding patterns at around 2200bp was not obtained. </p><br />
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<h2>Week 9 (June 25 - June 29)</h2><br />
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<p> PCR was attempted to amplify the genes of the <I>dsz</i> operon utilising an adapted PCR protocol with purified <i>Taq</i> polymerase that had been isolated from the host organism. Eventually, some banding pattern was obtained between 1200 and 1500 base pairs when a gradient thermocycler was used with melting temperatures ranging betweeen 55°C to 65°C. This was assumed to be indicative of successful amplification of <i>dszB</i>; however, further purification and gel verification results were inconclusive and no yield was obtained when placed tested using a nanodrop machine. </p><br />
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<h2>Week 10 (July 2-July 6)</h2><br />
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<p>Top 10 E.coli cells were transformed with <a href="http://partsregistry.org/Part:BBa_R0011">BBa_R0011</a><br />
(IPTG inducible promoter in psb1C3 backbone), and resulting colonies were tested using cPCR. <br />
Colony PCR was performed on cells containing the catalase biobrick. Catalase is 2217bp long but since biobrick primers add about 200bp, bands of 2400 bp were expected if the part was present in the biobrick. These bands were observed, indicating that the <a href=" http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a> gene was most likely present.</p><br />
<p></html>[[File:Ucalgary2012 4.7.2012 catalase colony pcr 2.jpg|thumb|500px|center|Figure 4: Colony PCR on potential colonies containing katG-LAA. Biobrick primers were used. Bands at 2200 indicate successful amplification of the catalase part within the biobrick backbone. Smaller bands are indicative of primer degradation and are nonspecific amplification. +C indicates the positive control, and RFP generator, which should amplify at around 1000 bp.]]<html></p> <p><br />
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PCR using Phusion high fidelity polymerase was carried out on <i>dszA</i>, <i>dszB</i>, and <i>dszC</i> in a gradient thermocycler. Amplification of non-specific bands was present for <i>dszA</i> and <i>dszB</i>, however strong banding for the desired size of the gene was observed for both (around 1500 for <i>dszA</i>, 1100 for <i>dszB</i> </p><br />
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<p></html>[[File:Ucalgary2012 6.7.2012.dszABphusionPCR.jpg|thumb|500px|center|Figure 5:DszA and DszB PCR from <i>Rhodococcus</i>. DszA amplicon is around 1300 bp, and is observed to run higher on the gel. DszB amplicon is expected to be 1098 bp, which is observed in addition to multiple nonspecific banding. +C indicates the positive control, and RFP generator, which when PCRed with biobrick primers should be around 1000 bp. As this control is running high as well, it is believed that both amplicons for DszA and DszB have been obtained. No contamination is observed in the NTC (no template control).]]<html><br />
</p> <br />
<p></html>[[File:Ucalgary2012 7.5.2012 dszC.png|thumb|500px|center|Figure 6:DszC PCR from <i>Rhodococcus</i>. The numbers above the figure show the colony number. DszC amplicon is around 1450 bp, and is observed to run at almost the right size on the gel. No contamination is observed in the NTC (no template control).]]<html><br />
<p><br />
Examining the sequences of the <i>dszABC</i> genes led to the discovery that all 4 had multiple illegal enzyme cut-sites in them that we have to eliminate before biobrick composite part construction can occur. <i>dszA</i> has four PstI cut sites, <i>dszB</i> has a PstI and a NotI and <i>dszC</i> has a PstI cut site. The Stratagene QuickChange mutagenesis procedure is going to be used to eliminate illegal cut sites with the only alteration being that Kapa HiFi polymerase would be used during the process. Primers needed for the mutagenesis were designed based on the procedure mentioned above. </p><br />
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<h2>Week 11 (July 9-July 13)</h2><br />
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<p> Following successful amplification of the <i>dsz</i> operon genes in the previous week, the genes were constructed into the <a href="http://partsregistry.org/Part:pSB1C3"> pSB1C3</a> vector. Colony PCR verifications were observed to be positive. Furthermore, the insertion of part <a href="http://partsregistry.org/Part:BBa_J13002"> BBa_J13002, (P<sub>tetR</sub>-RBS)</a><br />
in front of the previously biobricked <i>hpaC</i> was attempted. Overnight cultures were also prepared using two colonies each for <a href="http://partsregistry.org/Part:BBa_J13002"> <i>P<sub>tetR</sub>-RBS</i></a> and <a href="http://partsregistry.org/Part:BBa_R0011"> <i>P<sub>lacI</sub></i> </a> (an IPTG inducible promoter that we hope to build in front of <ahref="http://partsregistry.org/Part:BBa_B0034">an RBS site, BBa_B0034</a>). These cultures were then miniprepped to yield the respective parts.</p> <br />
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<p>Additionally, <a href="http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a> was built into a <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> backbone. The construction and availability of all these parts will be critical in the construction of our overall circuit for biodesulfurization. Colonies which looked good on cPCR were used to prepare overnight cultures, and were miniprepped and sent in for sequencing verification the following day.<br />
On the side, M9 minimal media was also prepared to carry out growth experimentation and overall desulfurization capability of <i>Rhodococcus</i> when exposed to DBT. The various growth treatments were M9 Media and glucose only, M9+glucose+DBT, M9+glucose+MgSO4+/-DBT, M9+glucose+MgCl2+/-DBT. 0.008g of FeCl2.4H2O was also added to each of the tubes. Samples were then inoculated with colonies of the <i>Rhodococcus</i>.</p><br />
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<h2>Week 12 (July 16 -July 20)</h2><br />
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<p>This week, while awaiting sequencing verification results which were required before we could begin the construction process, the desulfurization team initially aided in some of the tasks related to the other hydrocarbon groups. The success of the construction of <a href="http://partsregistry.org/Part:BBa_J13002"><i>P<sub>tetR</sub></i>-RBS</a><br />
with <a href="http://partsregistry.org/Part:BBa_K902057"><i>hpaC</i></a> was also explored by using forward and reverse primers of <a href="http://partsregistry.org/Part:BBa_R0040">BBa_R0040</a> (the promoter component of the composite part BBa_J13002). However, the eventual gel verification was inconclusive and sequencing results finally indicated an unsuccessful ligation. Additionally, the minimal media M9 preparation had been contaminated in the previous effort so this process was repeated to create tubes of each of the growth condition treatments detailed previously, and two repeats, one with an extra filtration step and one without was used to prepare the cultures.</p><br />
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<h2>Week 13 (July 23 - July 27)</h2><br />
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<p> Mutagenic primers were redesigned after the initial ones were found to have premature stop codons. As part of the redesign process in constructing our overall gene circuits for desulfurization, a backbone switch of <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> into a chloramphenicol (Chlor) resistant vector was necessary. The subsequent transformed products were plated on a Chlor plate and selected colonies were used to prepare O/N cultures, then minipreped before finally being digested with enzymes EcoRI and PstI. The resulting gel verification images were inconclusive as they did not show the required banding pattern around 50bp. Meanwhile, colony PCR was run on colonies transformed with <i>katG-LAA</i> constructed into a <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> backbone, as well as the <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> +<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a> construct. <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></a> was shown to have been successfully amplified, so overnight cultures were prepared and subsequently miniprepped. On the other hand, the construct was not successful so a third attempt was carried out. Colony PCR treatments that used either <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> forward primers or <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
primers were used and the overall constructs were made either on a chlor-resistant, or ampicillin-resistant vectors. Preliminary images of the gel verification appeared to have confirmed the construct, although sequencing verification will be the final indicator of overall success. </p><br />
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<h2>Week 14 (July 30 - August 3)</h2><br />
<br />
<br />
<p>Sequencing results from the previous week's constructs were available confirming that we constructed KatGLAA in a chlor-resistant backbone. However, switching the plasmid backbone of <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> to <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> was not successful. The construction of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>+<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> was finally sent in for sequencing. Site-directed mutagenesis of the <i>dsz</i> operon was also initiated: <i>dszA</i> has four PstI cut sites; <i>dszB</i> has a PstI and a NotI site; <i>dszC</i> has two PstI cut sites. Site directed mutagenesis was started this week to change a single base pair in these genes in a way that eliminates the cut site but preserves the amino acid codons, so as to not mutate the protein coding sequence. Ohshiro 2007 demonstrated that replacing the Tyr residue at position 63 of <i>dszB</i> gene with a Phe increases the activity of the enzyme. Therefore we want to introduce the same mutation into our <i>dszB</i>.</p><p><br />
For the first attempt at mutagenesis we chose to mutate the second PstI site in <i>dszC</i> (PstI2). As a positive control for the procedure, we also performed the mutagenic PCR on a plasmid containing the β-galactosidase gene with a point mutation where the PCR would cause it to regain its function. For both mutagenesis protocols we used the Kappa Hifi kit. After confirming that the PCR worked by running some produce on a gel, the PCR products were DpnI digested, the purpose of which is to degrade the unmodified parental DNA (DpnI degrades methylated DNA only). Control PCR products were plated on an ampicillin plate containing IPTG and X-gal. The colonies that grew on the control plates were blue indicating that the mutagenesis had worked for the β-galactosidase gene. Minipreps of the O/N culture of <i>dszC</i> mutants were digested with PstI enzyme and the results indicated that the mutagenesis was successful.</p><p> <br />
Attempts to simultaneously perform all the mutations in <i>dszC</i> genes in one step using the Knight procedure failed (<a href="http://openwetware.org/wiki/Knight:Site-directed_mutagenesis/Multi_site">Knight Multi-site Mutagenesis Procedure</a>). What enables simultaneous mutations is that Taq ligase closes the gaps in PCR products after each cycle. In the protocol it instructs to use Taq ligase buffer only for the PCR/ligation protocol. We suspected that the reason this procedure did not work might be that the Kappa polymerase is not functional in Taq ligase buffer. Therefore we did some experiments on the controls in Taq ligase kit and kappa polymerase kit to find out which buffer that Kappa polymerase and Taq ligase both work best in. The result was that both enzymes work best in a buffer made of half Taq ligase buffer and half Kappa polymerase buffer. </p><br />
<br />
<br />
<br />
</html>[[File:Ucalgary2012 31.7.2012 dszc mutagenesis 5 20 and 50ng.jpg|thumb|500px|center|Figure 7: DszC PstI2 mutagenesis PCR with varying concentrations of template plasmid was performed. The gel shows the PCR products that were run on a gel. +C lanes show the PCR products of the control. The control was pWhitescript™ 4.5-kb control plasmid from Stratagene mutagenesis kit which was mutated with primers also from the kit. Bands indicate successful amplification of the plasmid.]]<br />
[[File:UCalgary 02.08.12 dszC psti digest mutagenesis.jpg|thumb|700px|center|Figure 8: The control and the mutated plasmids were digested with PstI restriction enzyme. The control is dszC biobrick. Other lanes show the plasmids purified from the cells transformed with PstI2 mutagenesis PCR products followed by digestion. The control shows three bands since it has three PstI cut sites. The other lanes show two bands which indicates one of the cut sites has been eliminated through mutagenesis. ]]<html><br />
<br />
</html>[[File:Ucalgary2012 2.8.2012 finding the right buffer for multisite mutagenesis.png|thumb|800px|center|Figure 9: The multisite mutagenesis using the Knight procedure was tried in different buffers to find out the buffer that Kappa works optimally at. All the PCRs were performed on the pWhitescript™ 4.5-kb control plasmid from Stratagene mutagenesis kit which contains the beta-galactosidase gene. Based on this gel, the optimal buffer is composed of 50% kappa buffer and 50% Taq ligase buffer.]]<html><br />
<br />
</html>[[File:Ucalgary 3.8.2012 optimal buffer for Taq ligase.png|thumb|800px|center|Figure 10: Using the control provided in the NEB Taq ligase buffer (BsteII digested lambda DNA), we tried to find if the Taq ligase enzyme can function in combinations of Kappa Hifi buffer and Taq ligase buffer. Lane two is the control which is only the digested lambda DNA. Lanes 1 and 3 show that some of the bands compared to control have been ligated together. Therefore, Taq ligase functions just as good in the buffer composed of 50% kappa hifi buffer and 50% Taq ligase as it would in its own buffer.]]<html><br />
<br />
<h2>Week 15 (August 6 - August 11)</h2><br />
<br />
<p>Sequencing results for <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A> <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> returned negative, so a 3-part ligation method was used to retry this construction. The following parts were ligated with the restriction enzymes indicated in brackets after each: <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>(EcoRI/SpeI) + <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> (XbaI/PstI) + <A HREF=" http://partsregistry.org/Part:PSB1K3">BBa_PSB1K3</A> (EcoRI/PstI). Also, the more conventional construction (only 1 insert) of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>(SpeI/PstI) + <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> (XbaI/PstI) was reattempted. Furthermore, 3-way ligations were also attempted for <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
+<A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A>+<A HREF="http://partsregistry.org/Part:PSB1K3">BBa_PSB1K3</A>, and <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a>+<a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
+<A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A>, as well as the two-way contruction of just <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> after the <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a>. After plating these transformations, colony PCRs were carried out and samples that gave an indication of being successful on the gels were used to prepare O/N cultures followed by miniprep. With regards to the site-directed mutagenesis side of the experimentation, <i>dszA</i>-PstI1 (the first PstI cut site in <i>dszA</i>) ,<i> dszB</i>-PstI and <i>dszC</i>(PstI2 mutated)-PstI1 mutagenesis were performed following the procedure explained in the previous week. The gel below shows the successful result of digest confirmation (Fig. 8). <br />
Multisite mutagenesis (Knight method) was repeated using the modified buffer (half Taq ligase buffer and half Kappa buffer). However it was not successful again. We also tried doing multisite mutagenesis using Pfu Turbo polymerase and following the Knight procedure without any buffer modifications. No successful results were observed. </p><br />
<br />
<br />
<br />
</html>[[File:Ucalgary2012 Digestion confirmation of mutagenesis in dszAPstI1, dszBPstI and dszCPstI1 (PstI2 site mutated)..jpg|thumb|800px|center|Figure 11: Confirmation of site-directed mutagensis to eliminate PstI cut sites in: <i>dszA</i>PstI1 cut site, <i>dszB</i>PstI cut site and <i>dszC</i>PstI1 (PstI2 site mutated). Note that in each case, the unmutated controls for each gene are to the left of each set of plasmids derived from mutagenic PCRs. Plasmids from cloned <i>dsz</i> genes are smaller than the bands to the right of them (which have undergone mutagenesis), indicating that in the plasmids on the right, the PstI cut site was lost. ]]<html><br />
<br />
<br />
<h2>Week 16 (August 12 - August 18)</h2><br />
<br />
<br />
<p>The progress in mutagenesis of <i>dsz</i> genes was continued from the previous week: <i>dszB</i>(PstI mutated)-Y63F and <i>dszA</i>(PstI1 mutated)-PstI3 mutagenesis. The gel below shows the digest confirmation.</p><br />
<br />
<br />
</html><br />
[[File:Ucalgary2012 15.08.2012 dszAPstI1&amp;3 dszB Psti y63f muta diges-1.jpg|thumb|800px|center|Figure 12: Digestion confirmation of biobricks of a) dszA (PstI1 mutated) PstI3 mutagenesis and b) dszB (PstI mutated) Y63F mutagenesis. Cutting dszA (PstI1 mutated) with PstI is expected to produce fragments of 2790, 333, 255, and 114bp. Cutting dszA (PstI1 and PstI3 mutated) is expected to produce fragments of 2730, 588, and 114bp. The primers for dszB-Y63F mutagenesis introduce an HpyAV cut site. pSB1C3 inteslf has two more HpyAV cut sites. Before the mutation bands of 2858 abd 310bp are expected, and after successful mutation bands of 1502, 1356, 310 are expected. Lane legend: 1- dszA (PstI1 mutated). 2-dszA (PstI1 mutated) digested with PstI. 3-dszA (PstI1 mutated) PstI3 mutagenesis c1 Digested with PstI. 4- dszA (PstI1 mutated) PstI3 mutagenesis c2 Digested with PstI. 5-dszA (PstI1 mutated) PstI3 mutagenesis c3 Digested with PstI. 6- dszA (PstI1 mutated) PstI3 mutagenesis c4 Digested with PstI. 7- Fermentas 1kb Plus Ladder. 8- Empty. 9- dszB (PstI mutated). 10-dszB (PstI mutated) digested with HpyAV. 11- dszB (PstI mutated) Y63F mutagenesis c1 digested with HpyAV. 12- dszB (PstI mutated) Y63F mutagenesis c2 digested with HpyAV. 13- dszB (PstI mutated) Y63F mutagenesis c3 digested with HpyAV. 14-dszB (PstI mutated) Y63F mutagenesis c4 digested with HpyAV.]]<html></p><br />
<br />
<p>We attempted a different approach to speed up the turnover time of the mutagenesis PCR. Briefly, after the PCR mutagenesis the PCR products were purified and then incubated with T4 polynucleotide kinase (PNK) and ligase. After heat inactivating the ligase and T4 PNK, the products were DpnI digested. Subsequently another round of DNA purification was performed. However, the results were unsatisfactory after the digest confirmation.</p><p><br />
Sequencing results came back. <i>dszA</i> (PstI1 and PstI3 mutated) and <i>dszB</i>(PstI and Y63F mutated) were good. However <i>dszC</i> (PstI1 and PstI2 mutated) had an insertion next to the PstI1 cut site. Mutagenesis was repeated on the <i>dszC</i>(PstI2 mutated).<br />
<i>dszB</i>(PstI and Y63F mutated)-NotI and <i>dszA</i>(PstI1 and PstI3 mutated)-PstI4 mutagenesis were also performed.</p><br />
<p> To investigate the desulfurisation capability of the <i>Rhodococcus</i> sp. from which we cloned the <i>dsz</i> operon, a desulfurization assay was prepared by inoculating different treatments of M9 media. We also prepared some solutions that will be needed for analysis in the following week: a conditioning agent composed of 100ml of 95% ethanol, 50ml glycerol, 30ml of 12M HCl (aq) and 70g of NaCl(s) was prepared. The assay relies on the turbidity of a sample containing sulphate ions which are precipitated (hence the turbidometric nature of the assay) upon adding BaCl2(s), therefore if the <i>dsz</i> pathway is active, we expect a more turbid solution to form than in control samples. </p><br />
<br />
<br />
<br />
<h2>Week 17 (August 19 - August 25)</h2><br />
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<br />
<p>This week, progress was made in determining the desulfurization activity of our <i>Rhodococcus</i> strain as measured by the sulfate release using a turbidometric assay. We encountered several challenges in our prescribed protocol as the concentrations that we used to prepare the standard curve may have been too dilute, or the composition of out conditioning agent may have been flawed. Additionally, steps were taken to determine the decomposition of DBT to 2-HBP through Gas Chromatograph-Mass Spectroscopy (GC-MS) analysis, but due to a preparation error, the DBT was added to a growth solution of M9 media prematurely and the autoclaving process decomposed the DBT releasing a yellow colouration into the solution. These two approaches in determining the desulfurization capability of the <i>dsz</i> operon will be further investigated. </p><br />
<br />
<p>Since the <i>dszC</i> second mutagenesis had proven to be unsuccessful last week, the <i>dszC</i>(PstI2 mutated)PstI1 mutagenesis was repeated. Also <i>dszA</i>(PstI1,3,4 mutated) PstI2 mutagenesis was performed. <i>dszA</i> and <i>dszC</i> were sent for sequencing on Wednesday. <i>dszB</i> was sent for sequencing on Friday. Sequencing results of <i>dszA</i> and <i>dszC</i> were back by Friday. <i>dszC</i> was successful. However, <i>dszA</i> contained an insertion next to the binding site of PstI4 cut sit, so the last two mutations must be redone. <i>dszB</i>(PstI and Y63F mutated)-NotI-mutagenesis was also repeated in case the result of the sequencing was not successful. These constructions were repeated. <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>-<A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
-<i>dszC</i> constructions were attempted, however they were not successful as indicated by colony PCR. Constructions of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>/<i>hpaC</i> were carried out and also came back negative in sequencing, however <a href="http://partsregistry.org/Part:BBa_K902059">BBa_B0034<br />
/<i>katG-LAA</i> (BBa_K902059)</a> was sequence confirmed.</p><br />
</html>[[File:Ucalgary2012 20.8.2012 dszC(psti2)psti1 mutagenesis.png|thumb|700px|center|Figure 13: Another attempt to mutagenize dszC(PstI2 mutated)at PstI1 site. The gel shows the confirmation digestion. C is the control which is dszC(PstI2 mutated). Since the control has two PstI cut sites, two bands are observed on the gel. The mutagenesis has been successful since only one band is observed on the other lanes indicating that one of the cut sites has been eliminated during mutagenesis.]]<html><br />
<br />
</html>[[File:Ucalgary2012 21.8.2012 dszA final mutagenesis digest.png|thumb|700px|center|Figure 14: DszA (PstI1, PstI2, PstI3 mutated)PstI4 mutagenesis digestion confirmation gel. All the lanes show the results of the plasmids being cut with PstI enzyme. C indicates the lane containing the control which is dszA(PstI1, PstI3 and PstI4 mutated). The other lanes are the digestions of the plasmids that are mutagenized, from different colonies. Control plasmid has two PstI cut sites and therefore the two bands observed on the gel were expected. The other lanes show only one band of about 3500bp which is about the right size for dszA in a psb1c3 vector. Therefore one of the cutsites has been eliminated during mutagenesis.]]<html><br />
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<br />
<br />
<br />
<h2>Week 18 (August 26 - September 1)</h2><br />
<br />
<p> <A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i> (BBa_K902052)</i></A> sequencing results came back as successful. <i>dszA</i>(PstI1,3 mutated)-PstI2-mutagenesis was performed and sent for sequencing. Also <i>dszA</i>(PstI1,2,3 mutated)-PstI4-mutagenesis was performed, and this was also sent for sequencing. </p><br />
<p>Constructions of <A HREF="http://partsregistry.org/Part:BBa_K902053">P<sub>tetR</sub>-RBS/<i>dszB</i> (BBa_K902053)</a> and <a href="http://partsregistry.org/Part:BBa_K902056">BBa_B0034<br />
/<i>dszC</i> (BBa_K902056)</a> were attempted, verification digested, and sent for sequencing. Sequencing results for these constructs came back as positive, along with successful mutagenesis of <A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i> (BBa_K902050)</A>.</p><br />
<p>At this point, all of the <i>dsz</i> genes have been successfully made biobrick compatible, and <i>hpaC</i> has been biobricked. We have also successfully constructed <a href="http://partsregistry.org/Part:BBa_K902059">BBa_B0034 with <i>katG-LAA</i></a> to be used in the optimization circuit, as well as <A HREF="http://partsregistry.org/Part:BBa_K902053"><i>P<sub>tetR</sub>-RBS</i>/<i>dszB</i> (BBa_K902053) </a>and <a href="http://partsregistry.org/Part:BBa_K902056">BBa_B0034/<i>dszC</i> (BBa_K902056)</a>.<br />
<p> Constructions of <A HREF="http://partsregistry.org/Part:BBa_J04500">P<sub>lacI</sub>-RBS</A>with <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> were performed. As well, attempts to construct <A HREF="http://partsregistry.org/Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></a>/<a href="http://partsregistry.org/Part:BBa_K902056">RBS-<i>dszC</i></a> as well as <A HREF="http://partsregistry.org/Part:BBa_J13002">P<sub>tetR</sub>-RBS</A>/<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
/<A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i></A>, and <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>/<A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> were also carried out. These parts are intended as construction intermediates towards building the final systems, as well as providing a way of testing the genes functionality (namely, to test HpaC for oxidoreductase activity and to test if over-expression of KatG in the cell will increase its ability to survive H<sub>2</sub>O<sub>2</sub> stress). Transformations of all these constructions were carried out at the end of the week.</p><br />
<br />
<h2>Week 19 (September 2- September 8)</h2><br />
<p>Confirmation digests on colonies of the previous constructions that gave bands of the expected size with cPCR were performed. Positive results were found for colonies of <A HREF="http://partsregistry.org/Part:BBa_J04500"><i>P<sub>lacI</sub>-RBS</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <A HREF="http://partsregistry.org/Part:BBa_J04500"><i>P<sub>lacI</sub>-RBS</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
/<A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i></A>. Sequencing was sent, and results indicated that the constructions of <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS/hpaC</i> (BBa_K902058)</a> were successful, meaning that after many months of trying we FINALLY have a promoter in front of the <i>hpaC</i> gene and can proceed to test the parts functionality. Attempts to construct <i>hpaC</i> with the (<A HREF="http://partsregistry.org/Part:BBa_J13002">evil TetR promotor, BBa_J13002</A>) were abandoned, as it was believed that this construction was failing due to toxicity of over-expressing the protein, and it was determined that this part was not necessary after all. <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub>-RBS/dszB</i>, (BBa_K902054)</a> also worked, though this was less exciting. <a href="http://partsregistry.org/Part:BBa_K902051">BBa_B0034<br />
/<i>dszA</i></a> came back as a bad read despite looking very good on the confirmation digest gel, so this part will be resent for sequencing. Constructions of <A HREF="http://partsregistry.org/Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> were reattempted, and confirmation digests for this part looked good, and so samples were sent for sequencing.</p><br />
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<br />
<h2>Week 20 (September 9- September 15)</h2><br />
<p>Construction attempts on <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> were performed. Colonies grew for the constructions, however further confirmation results were dissapointing (only 2 clones of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a> appeared to have been successful). These clones were sent for sequencing, and constructions were reattempted.</p><p> However, when sequencing came back, somehow reads indicated that these clones were in fact a gene from the Denitrogenation project (which is 990bp and a completely different band then what we saw on the gel). We believe, somewhere, something has gone very wrong- further investigation into this will be carried out. In the meantime, the above constructions were reattempted, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902051">BBa_B0034/<i>dszA</i></A> was re-prepped in case a contaminant in the plasmid stock was to blame for the bad reads found in this batch of sequencing as well as the last. In addition, plasmid switches of multiple sequence confirmed parts into a <a href=" http://partsregistry.org/Part:pSB1C3"> pSB1C3</a> backbone were carried out. </p><br />
<br />
<h2>Week 21 (September 16- September 22)</h2><br />
<p>Colonies for the transforms of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> have been few and far between, and cPCR results are always discouraging. Sequencing results for other sections of the project have once again come back very confusing, and further research continues into the source of this madness. </p><br />
<p> The sulfur compound degradation assay was set up to test the desulfurization rate of the original <i>Rhodococcus baikonurensis </i> (refer to the protocol page).</p><br />
<br />
<br />
<h2>Week 22 (September 23- September 29)</h2><br />
<p>Attempts to construct <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, , and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> continue. In the meantime, <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> was tested for functionality. In order to do this, cultures of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> were grown up overnight in LB media. A strain carrying <A HREF="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</A> only was used as a negative control. The following morning, 20 &micro;L of each culture was inoculated into 3 mL of LB with various concentrations of hydrogen peroxide; 0 mM, 1 mM, 5 mM, and 10 mM. These cultures were then allowed to grow overnight, and culture turbidity was observed. It was found that the negative control exhibited no growth after 12h at 1 mM peroxide, however cultures with induced expression of catalase were turbid after 12 h of growth at this concentration (Fig. 10). This demonstrated the ability of the catalase to protect the cells from excessive peroxide concentrations.</p><p><br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 15: Catalase Assay. Overnight cultures of J04500 and J04500-<i>KatGLAA</i> were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbitity was observed.]]<html></p><br />
</p><br />
<p>In addition to this, activity of <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-hpaC</i></a> was tested. In order to do this, cultures of <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-hpaC</i></a> and <a href="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub>-dszB</i></a> were grown up overnight in LB with appropriate antibiotics. Following this, protein expression was induced with IPTG, after which the assay was carried out as described in the following figure and on the protocols page.</p><br />
<p> </html> <br />
[[File:Ucalgary2012 DesulfurizationGroup HpaC assayTake1.png|center|600px|thumb|Figure 16: HpaC Assay. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 2 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, 1 mL of cell lysate was transferred to a cuvette, and a spectrophotometer was blanked at 340 nm (maximal absorbance of NADH) with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p>When the assay was run, it was found that NADH does not convert readily to NAD+ on its own. When cell lysate containing the naturally expressed amounts of oxidoreductase was added, a decrease in absorbance could quickly be observed as the NADH was converted to NAD+. When cultures over-expressing HpaC were tested, the absorbance levels were found to start much lower than the control. We believe that this is because with the amount of cell lysate tested, when the HpaC protein is overexpressed the NADH is consumed almost immediately and therefore the data reflecting the drop in absorbance is missed. Further tests will use differing amounts of cell lysate in order to try to capture data that shows the drop in absorbance for HpaC cultures.</p><br />
<br />
<h2>Week 23 (September 30-October 3): Wiki-Freeze PANIC!!</h2><br />
<br />
<p>The GCMS results of the sulfur assay were received. DBT desulfurization was successful (figures below). Some compounds were not detected by GCMS since they were too polar. However, 2 additional compounds showed degradation in addition to DBT, indicating that the pathway has a wider substrate specificity!</p><br />
<p></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 17: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol in the protocols section for details). Samples were taken out at different time points and were run through GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria, run through the GCMS after 6 days of being in the incubator to account for abiotic degradation. ]]<html></p<br />
<br />
<p></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 18: The peak in this mass spectra demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound (A: dibenzothiophene, B: tetrahydro-4h-thiopyran-4-one, and C: benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdownn. Degradation is seen for DBT (the model compound) as well as other sulfur containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<p>The HpaC assay was repeated using 100 &micro;L of cell lysate instead of 1 mL of 2x concentrated cell lysate, following the previously used protocol. With this smaller amount, a clear sharp decrease in the absorbance of NADH can be observed, indicating a very fast conversion by HpaC, and further confirming that the part was functional.</p><br />
<br />
<p></html>[[File:Ucalgary2012Desulfurization-Hpacasay2.PNG|center|550px|thumb|Figure 20: HpaC Assay. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, 100 &micro;L of cell lysate was transferred to a cuvette, and a spectrophotometer was blanked at 340 nm (maximal absorbance of NADH) with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<h2>Week 24 (October 4- October 11)</h2><br />
<p>This week, before the Americas West Regional Competition, attempts to construct the previously mentioned parts continued. Unfortunately, they were unsuccessful.<br />
<br />
<h2>Week 25 (October 16- October 20)</h2><br />
<p>Traditional attempts to build the sulfur operon and its sub-parts were repeated, however attempts failed once more. During this week, it was decided that due to time consstraints, alternative approaches to construct the operon would be undertaken. Due to this, splice-overlap-extention (SOE) PCR primers (containing scar-sites where appropriate) were designed according to <b>protocol</b> and ordered.</p><br />
<p><br><br />
<br />
<br />
Primer: 1a (BBK)-J13/DszB F - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGTCCCTATCAGTGATAGAGATTGACATCCC-3'<br />
<br><br><br />
Primer: 1b (BBK)-J04/DszB F - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 2 Jxx/B-(RBS/C) R - 5'-GGTGACAGTGTCATCTAGTATTTCTCCTCTTTCTAGTACTATCGGTGGCGATTGAGGCTGTTG-3'<br />
<br><br><br />
Primer: 3 (Jxx/B)-RBS/C F - 5'-CAACAGCCTCAATCGCCACCGATAGTACTAGAAAGAGGAGAAATACTAGATGACACTGTCACC-3'<br />
<br><br><br />
Primer: 4 RBC/C-(RBS/A) R - 5'-GCCAGATGCATTTGTCGTTGTTGAGTCATCTAGTATTTCTCCTCTTTCTAGTATCAGGAGGTGAAGCCGGGAATCG-3'<br />
<br><br><br />
Primer: 5 (RBS/C)-RBS/A F - 5'-CGATTCCCGGCTTCACCTCCTGATACTAGAAAGAGGAGAAATACTAGATGACTCAACAACGACAAATGCATCTGGC-3'<br />
<br><br><br />
Primer: 6 RBS/A-(J04/HpaC) R - 5'-GGGGAGAGGCGGTTTGCGTATTGCTAGTATCATGAAGGTTGTCCTTGCAGTTGTGG-3'<br />
<br><br><br />
Primer: 7 RBS/A-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATCATGAAGGTTGTCCTTGCAGTTGTGG-3'<br />
<br><br><br />
Primer: 8 (BBK)-J04/HpaC F - 5'-CGAATTCGCGGCCGCTTCTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 9 (RBS/A)-J04/HpaC F - 5'-CCACAACTGCAAGGACAACCTTCATGATACTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 10 J04/HpaC-(RBS/Kat) R - 5'-CTGACGTGCTCATCTAGTATTTCTCCTCTTTCTAGTATTAAATCGCAGCTTCCATTTCCAGCATCAC-3'<br />
<br><br><br />
Primer: 11 J04/HpaC-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTAAATCGCAGCTTCCATTTCCAGCATCAC-3'<br />
<br><br><br />
Primer: 12 (J04/HpaC)-RBS/Kat F - 5'-GTGATGCTGGAAATGGAAGCTGCGATTTAATACTAGAAAGAGGAGAAATACTAGATGAGCACGTCAG-3'<br />
<br><br><br />
Primer: 13 RBS/Kat-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTATTAAGCAGCCAGAGCGTAGTTTTCGTC-3'<br />
</p><br><br />
<p><br />
Colony PCR was done on potential colonies of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, , and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A>. Faint bands around the expected size were seen, and these were stabbed with a pipette tip and stirred into water in order to be used as template in a further round of PCR to get amplification of the part. The plan was that these amplicons could then be used as an insert in further constructions, however no amplification was seen. Further optimization of this procedure could possibly result in a short-cut to construction, however the time needed to do this is a luxury that we do not have.</p><p>Additional attempts to create inserts by doing PCR on ligation reactions in addition to gel extraction of colony PCR bands were attempted as well, however these attempts were unsuccessful as well. SOE PCR and possible Gibson Assembly appear to be the Sulfur Teams last hope at this point, and we are eagerly awaiting our primers.<br />
</p><br />
<br />
<br />
<h2>Week 26 (October 21- October 26)</h2><br />
<p><br />
Much to our delight, SOE PCR primers were recieved this week. Therefore, alongside traditional construction of the aforementioned parts, SOE PCR was carried out according to <b>protocol</b> and these parameters:</p><br />
<br><br />
<h4>Round 1:</h4><p><br />
<ul><br />
<li>Primers 1a and 2 with <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A> as a template = Product L1 (J13002/DszB with BBk prefix and B0034/DszC overlap)<br />
<br></li><li><br />
Primers 1b and 2 with <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A> as a template = Product L2 (J04500/<i>dszB</i> with BBk prefix and B0034/<i>dszC</i> overlap)<br />
<br></li><li><br />
Primers 3 and 4 with <A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a> as a template = Product L3 (B0034/<i>dszC</i> with <i>dszB</i> and B0034/<i>dszA</i> overlap)<br />
<br></li><li><br />
Primers 5 and 6 with <A HREF="http://partsregistry.org/Part:BBa_K902050"><i>dszA</i></a> as a template = Product L4 (B0034/<i>dszA</i> with <i>dszC</i> and J04500/<i>hpaC</i> overlap)<br />
<br></li><li><br />
Primers 5 and 7 with <A HREF="http://partsregistry.org/Part:BBa_K902050"><i>dszA</i></a> as a template = Product L5 (B0034/<i>dszA</i> with <i>dszC</i> overlap and BBk suffix)<br />
<br></li><li><br />
Primers 8 and 10 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L6 (J04500/<i>hpaC</i> with BBk prefix and B0034/<i>katG-LAA</i> overlap)<br />
<br></li><li><br />
Primers 9 and 10 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L7 (J04500/<i>hpaC</i> with <i>dszA</i> and B0034/<i>katG-LAA</i> overlap)<br />
<br></li><li><br />
Primers 9 and 11 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L8 (J04500/<i>hpaC</i> with <i>dszA</i> overlap and BBk suffix)<br />
<br></li><li><br />
Primers 12 and 13 with <A HREF="http://partsregistry.org/Part:BBa_K902059"><i>RBS-katG-LAA</i></a> as a template = Product L9 (B0034/<i>katG-LAA</i> with J04500/<i>hpaC</i> overlap and BBk suffix)<br />
</li></ul><br />
</p><br />
<p><br />
</html>[[File:Ucalgary2012 Spliceoverlapextentionpcr1.png|center|thumb|800px|Figure 21: Round 1 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with varying amounts of DNA template as indicated above the wells. Cycling conditions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 55%deg;C for 1 min, 72%deg;C for 2 min. 30 s.)x 31, Stage 3 (72%deg;C for 10 min.). Bands seen at ~1500 bp for L1 through L5, ~800 bp for L6-L8, and ~2200 bp for L9 indicate successful amplification of the desired product.]]<html><br />
</p><br />
<h4>Round 2</h4><br />
<p><br />
<ul><br />
<li>Primers 1a and 4 with templates L1 and L3 = Product X1 (J13002/<i>dszB</i>/B0034/<i>dszC</i> with BBk prefix and B0034/<i>dszA</i> overlap)</li><br />
<li>Primers 1b and 4 with templates L2 and L3 = Product X2 (J04500/<i>dszB</i>/B0034/<i>dszC</i> with BBk prefix and B0034/<i>dszA</i> overlap)</li><br />
<li>Primers 3 and 6 with templates L3 and L4 = Product X3 (B0034/<i>dszC</i>/B0034/<i>dszA</i> with <i>dszB</i> and J04500/<i>hpaC</i> overlap)</li><br />
<li>Primers 3 and 7 with templates L3 and L5 = Product X4 (B0034/<i>dszC</i>/B0034/<i>dszA</i> with <i>dszB</i> overlap and BBk suffix)</li><br />
<li>Primers 5 and 10 with templates L4 and L7 = Product X5 (B0034/<i>dszA</i>/J04500/<i>hpaC</i> with <i>dszC</i> and B0034/<i>katG-LAA</i> overlap)</li><br />
<li>Primers 5 and 11 with templates L4 and L8 = Product X6 (B0034/<i>dszA</i>/J04500/<i>hpaC</i> with <i>dszC</i> overlap and BBk suffix)</li><br />
<li>Primers 8 and 13 with templates L6 and L9 = Product X7 (J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with BBk prefix and suffix)</li><br />
<li>Primers 9 and 13 with templates L7 and L9 = Product X8 (J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with <i>dszA</i> overlap and BBk suffix)</li><br />
</ul></p><p><br />
</html>[[File:Ucalgary2012 Spliceoverlapextentionpcr2.png|center|thumb|800px|Figure 22: Round 2 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with varying amounts of gel-extracted DNA template from the previous PCR round as indicated above the wells. Bands were extracted using Qiagen QIAquick Gel Extraction Kit. Cycling conditions for the PCR reactions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 65%deg;C for 1 min, 72%deg;C for 3 min. 30 s.)x 31, Stage 3 (72%deg;C for 10 min.). Faint bands seen at ~3000bp indicate amplification of the desired product.]]<html> <br />
</p><br />
<h4>Round 3</h4><br />
<p><br />
<ul><br />
<li>Primers 1a and 10 with templates X1 and X5 = Product Z1 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with B0034/<i>katG-LAA</i> overlap and BBk prefix)</li><br />
<li>Primers 1a and 11 with templates X1 and X6 = Product Z2 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with BBk prefix and suffix)</li><br />
<li>Primers 1a and 7 with templates X1 and L5 = Product Z3 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 10 with templates X2 and X5 = Product Z4 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with B0034/<i>katG-LAA</i> overlap and BBk prefix)</li><br />
<li>Primers 1b and 7 with templates X2 and L5 = Product Z5 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 11 with templates X2 and X6 = Product Z6 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 6 with templates L2 and X3 = Product Z7(J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with J04500/<i>hpaC</i> overlap and BBk prefix)</li><br />
<li>Primers 1b and 7 with templates L2 and X4 = Product Z8 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1a and 6 with templates L1 and X3 = Product Z9 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with J04500/<i>hpaC</i> overlap and BBk prefix)</li><br />
<li>Primers 1a and 7 with templates L1 and X4 = Product Z10 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 5 and 13 with templates X5 and X8 = Product Z11 (B0034/<i>dszA</i>/J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with <i>dszC</i> overlap and BBk suffix)</li><br />
</ul></p><p><br />
</html>[[File:Ucalgary2012 sulfurSpliceoverlapextentionpcr3.png|center|thumb|800px|Figure 23: Round 3 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with 50 ng of gel-extracted DNA template from the previous PCR round. Bands were extracted using Qiagen QIAquick Gel Extraction Kit. Cycling conditions for the PCR reactions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 65%deg;C for 1 min, 72%deg;C for 6 min.)x 31, Stage 3 (72%deg;C for 10 min.). A faint band (~6000bp) can be seen in lane 4, indicating possible amplification of the desired product. Other reactions appear to have failed, as this bands are not present elsewhere.]]<html> <br />
</p><br />
<p><br />
Though it appears that S.O.E. PCR consists of crushed dreams and lies (or that extensive optimization would have to be performed in order to amplify the correct product), extraction was carried out on the single band seen, in the hopes that a subsequent round of PCR with the terminal primers for this product would possibly amplify it to a concentration that can be used in construction in the next week to assemble and test the sulfur operon constructs before competition. Conveniently, the first round of PCR creates <b>Gibson Assembly</b> compatible products. Because a kit is available in the lab, this will be attempted in the following days as well. Meanwhile, a round of traditional construction appears to have finally created the parts <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902079"><i>P<sub>tetR</sub></i>-<i>dszB</i>-B0034-<i>dszC</i></A>, <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902080"><i>P<sub>lacI</sub></i>-<i>dszB</i>-B0034-<i>dszC</i></A>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902081"><i>P<sub>lacI</sub></i>-<i>hpaC</i>-B0034-<i>katG</i></A>. Though sequence confirmation could not be obtained before Wiki-Freeze, we are confident that these parts are correct due to the clear size difference seen between these parts and their sub-parts. A plasmid switch was attempted the night before DNA submission had to be sent, however due to technical issues the incubator stayed at a low temperature overnight, and cultures did not grow enough to obtain the first two parts in a pSB1C3 backbone. Because of the difficulty assembling these parts, the deadline for DNA submission, and the fact that having them in an alternate backbone in the registry would allow for easier and quicker assembly of the sulfur operon by others (no plasmid switches of large parts would have to be performed, as we have found that plasmid switching the larger constructs is quite difficult.) we decided to submit these parts in an ampicillin backbone. Further attempts to switch the backbone for these parts will continue, and hopefully we will be able to submit them in the standard backbone at a later date before the competition. In addition, a reconstruction of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902051">B0034-<i>dszA</i></A> was obtained and finally, at long last, sequence confirmed. Because of the verification of this part and the excellent sequence read when compared to those obtained for the previously submitted version, we decided to also resubmit this part to the registry.</p><p>Construction attempts of the operon will continue into the following week, as only 1 additional construction is needed to assemble a testable version of the operon. Hopefully, this data will be obtained before the competition, and documented on the respective parts pages.</p><br />
<p><br><br>This is the Desulfurization Team, signing off.</p><br />
</h><br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Notebook/DesulfurizationTeam:Calgary/Notebook/Desulfurization2012-10-26T18:14:58Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Desulfurization Journal|<br />
<br />
CONTENT=<br />
<html><br />
<h2>Week 1 (May 1-4)</h2><br />
<p>During this week, literature search was performed.</p><br />
<br />
<br />
<br />
<br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<br />
<br />
<p>Along with the rest of the team, this week was dedicated to familiarizing ourselves on the protocols that will be utilized during this years project; specifically the polymerase chain reaction, gel verification, preparation of overnight cultures, as well as developing a procedural flowchart to transform competent cells with registry biobricks. With regards to our sub-group specific goals, we reviewed the current available literature around various industrial and laboratory approaches to desulfurization of organic groups, especially in the petroleum industry. This included a comparison of non-biological processes such as conventional hydrodesulfurization, which is currently employed in petroleum product refinery stages, and how a biological approach would supplement and perhaps even offer several advantages over these methods. Current limitations to biological desulfurization, however, include such factors as biocatalyst stability, enzyme specificity and desulfurization rate. We also identified the enzyme desulfinase (DszB) as being one of the bottlenecks in the desulfurization 4S pathway. Overall, our goals moving forward involve determining the specific pathways involved in the desulfurization process as well as the reaction conditions we would want to employ, and identifying specific model compounds in addition to dibenzothiophene (DBT) that we could use to test the effectivity of our biosystem in order to determine its functionality in the conversion of naphthenic acids to economically valuable hydrocarbons.</p><br />
<br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<br />
<br />
<p> Building on the previous week's literature review, the 4S pathway was recognized as the preferred biological mechanism that we would explore in devising a desulfurization biosystem. Of specific interest is the dsz operon consisting of the genes for <i> dszA, dszB, </i>and<i> dszC</i> which selectively and non-destructively remove the sulfur from the hydrocarbon structure, and therefore preserves the carbon skeleton. In addition to these, another dsz gene exists.<i> dszD</i>, which codes for a FMN:NADH reductase, is an essential component of the pathway, but not part of the operon. Instead, it is on the genome. The enzyme produced by this gene is required to regenerate the FMNH2 consumed by the reactions carried out by DszA and DszC. <i>Rhodococcus erythropolis</i> IGTS8 is the most studied model organism in investigations of the 4S pathway, and has been shown in many different research endeavors to be capable of converting DBT to 2-HBP.</p><p><br />
</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|thumb|700px|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD. Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO2) through the addition of oxygen to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the heteroatom, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-hydroxybiphenyl. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.]]<html></p><p><br />
An alternative to the DszD gene is HpaC, an oxidoreductase encoded in the <i>E. coli</i> W genome. This enzyme has been shown to increase the rate of desulfurization. Following this, other protocols added to our growing lab methods 'toolkit' were a restriction digest protocol, PCR purification, and finally, DNA construction digest. Aims moving forward include obtaining strains of the <i> R. erythropolis </i>, while also executing a timeline devised to biobrick, test, and incorporate the genes necessary in the above processes in a biobrick circuit.</p><br />
<br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<br />
<br />
<p>This week was kicked off with a project development meeting with Emily and David, and we devised a protocol for biobricking the <i> hpaC </i> gene. Additionally, methods to place the genes coding for the 4 enzymes, DszA,B,C and HpaC into a single construct were explored. Within the lab, the PCR performed on the resuspended pUC18-hpaC was not successful initially. <br />
Furthermore, we ordered the substrates/compounds that we intend to use for desulfurization tests. Once the substrates and the <i>Rhodococcus</i> strain arrive we are going to test how effectively the bacteria can desulfurize different sulphur-containing compounds that resemble naphthenic acids. Finally, we came across a paper where a team had developed an improved efficiency DszB through site-directed mutagenesis in 2007. This was through a point mutation to the gene, converting a tyrosine at position 63 to a phenylalanine residue. A member of this team was contacted to request the plasmid that contains the mutated gene. The conversion step carried out by DszB is the major bottleneck in the 4S pathway and if a strain or sample containing this mutation was obtained, it would significantly bolster our later testing efforts on DBT, as well as other compounds such as thiophane.<br />
</p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<br />
<br />
<p>Since we wanted to make sure we would not run out of pUC18(plasmid containing the hpaC gene), we transformed some E.coli cells with it. We grew them on plates containing ampicillin (A), kanamycin (K), tetracycline (T) and chloramphenicol (C) antibiotics and they only grew on A. Therefore pUC18 has A resistance. We did a three sets of PCR with primers designed against <i>hpaC</i>, one using 1/10 dilution of pUC18, the other using 1/100 dilution of pUC18 and one with the colonies we had just obtained by transforming the <i>E.coli</i> cells. </p><br />
<p>The PCR worked and we saw bands of the same size for all three sets of PCR. (Unfortunately, the picture we saved is not a good one since some of the bands faded away under UV due to prolonged exposure. Following this, PCR purification was performed to obtain the pure <i>hpaC</i> with biobrick prefix and suffix attatched to gene, which would allow us to insert the sequence into a biobrick standard backbone. 3 sets of digestion, ligation, and transformation (using pairs of X&P enzymes, E&S enzymes and E&P enzymes) were carried out in order to insert the <i>hpaC </i> gene into the <a href=" http://partsregistry.org/Part:pSB1C3">pSB1C3</a><br />
vector. All the sets grew successfully.<br />
Following the above successes with<i> hpaC</i>, the arrival of our <i>Rhodococcus</i> strain afforded us the opportunity to begin investigation of the Dsz operon using the primers currently in our possession. This strain is an environmental isolate that has been shown to be an active desulfurizer. The gram-positive nature of the strain also dictated we explore various lysing strategies before the genes encoding the Dsz enzymes could be amplified for further purification and biobrick construction steps. PCR was carried out using <i>dszA</i> primers on three different treatments {microwave, lysate buffer, and a control} which yielded banding pattern around 1200 base pairs for the lysate treatment (2%SDS and 10% tritonX-100, plus heat for 5mins at 98C).<br />
</p> <br />
<br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<br />
<br />
<p> In order to confirm the <i>hpaC</i> biobrick construction, two sets of colony PCR were performed, choosing white colonies from the 3 plates we grew last week (white colonies indicate a loss of the RFP generator in the <a href=" http://partsregistry.org/Part:pSB1C3">pSB1C3</a><br />
backbone, and therefore allow for weeding out of the colonies which are simply the original plasmid vector). These reactions were carried out both with <i>hpaC</i> primers and with standard biobrick primers designed against the plasmid backbone. After running them on the gel we saw equal bands for the PCR reactions performed using <i>hpaC</i> primers (However, a PCR using biobrick primers was performed later and the same results were obtained). Colonies 1(-) and 5(-) were used to make overnight cultures, which were then miniprepped the following day to obtain the plasmid DNA of the putative <i>hpaC</i> biobrick. Digestions were performed on the miniprep products using EcoRI and PstI to look for part size as further verification for the genes presence in the plasmid. The results were good and two bands were observed on each column (one for vector and the other for <i>hpaC</i>)). <i>hpaC</i> was sent in for sequencing. </p><br />
<p><br />
<br />
<br />
<br />
</html>[[File:UCalgary2012_04.06.2012-desulfurisation_hpacverification.jpg|thumb|700px|center|Figure 2: HpaC verification cPCR. HpaC gene was inserted into the pSB1C33 vector and E. coli cells were transformed. In order to confirm that pSB1C3 contains the hpaC gene, two sets of colony PCR's were conducted. One with biobrick primers, and the other with hpaC primers. Bands indicate successful amplification at the approximate size of the hpaC gene (517 bp)]]<br />
[[File:Ucalgary2012 06.06.2012-digestion of hpaC with E and P.jpg|thumb|500px|center|Figure 3: HpaC confirmation digest. Plasmid was miniprepped and digested for 2h with EcoRI and SpeI before being run on a 1% gel for 1h and 120V. Bands between 500 and 700 bp indicate the hpaC part is present as an insert. Bands of about 2000bp show the size of the psb1c3 vector.]]<html></p><br />
<br />
<br />
<br />
<p> PCR reagents were prepared to re-test/confirm previous results of <i>dszA</i> amplification following two different lysing treatments (microwave + lysate buffer). This time, all three genes were amplified and gel verification showed clear banding patterns around 500bp range for all three genes for the microwave treatment. Remaining PCR products were run on a gel and extracted for further purification steps; however, presence of any genetic material were not confirmed through nanodropping which raised concerns about the composition of the purified products, the success of the initial amplification step, or perhaps even the lysis treatment. Further experimentation will have to be carried out to troubleshoot.</p><br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<br />
<br />
<p> This week, we focused on amplifying <i>dsz</i> genes from our <i>Rhodococcus</i> strain for construction into biobricks. We also wanted to purify the <a href=" http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"> BBa_K902057 <i>hpaC</i></a> and pUC18-<i>hpaC</i> plasmids to replenish our current stocks. For the <i>dsz</i> aspect, we were able to successfully grow extra plates of <i>Rhodococcus</i> strain which was used to inoculate PCR tubes. The PCR did not go well, with significant streaking and false positives with similar banding pattern to previous gels run in the previous week. A final gel verification of a random sample of a tube of PCR products from dszA,B,C respectively and two negative control treatments involving master mix only and the lysed cells only illustrated the lack of discrepancy between the supposed successful amplification and the lysed cells (with lysate buffer) alone. Because of this we decided to take a different approach involving plasmid isolation carried out before PCR, rather than applying the PCR reagents directly to a lysed culture sample.</p><br />
<p> <a href=" http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> verification through sequencing was successful, confirming the construction of our first biobrick. Subsequently, O/N cultures of the plasmid containing cultures were prepared and stored in glycerol at -80C. Furthermore, verification of catalase gene part (<a href=" http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a>), which was sent as a culture stab from the parts registry was initiated. Our newly identified biobricked-hpaC was used as a positive control, but the banding pattern was not very conclusive. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<br />
<br />
<p>PCR was reattempted on <i>Rhodoccocus</i> that was lysed using two different dilutions of the lysate buffer, but the gel verification confirmed the previous failure in using this approach. An alternative that involved preparation of an overnight culture of the <i>Rhodococcus</i> cells followed by a plasmid purification was followed. The plasmid purification eventually yielded plasmid samples with concentrations of 98.6ng/μL to 182.7ng/μL (4 samples obtained overall). Additionally, the catalase biobrick was used to transform some stock competent cells, and samples of some colonies were subsequently PCR'ed. Although, the gel verification showed some potential contamination, and the required banding patterns at around 2200bp was not obtained. </p><br />
<br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<br />
<br />
<p> PCR was attempted to amplify the genes of the <I>dsz</i> operon utilising an adapted PCR protocol with purified <i>Taq</i> polymerase that had been isolated from the host organism. Eventually, some banding pattern was obtained between 1200 and 1500 base pairs when a gradient thermocycler was used with melting temperatures ranging betweeen 55°C to 65°C. This was assumed to be indicative of successful amplification of <i>dszB</i>; however, further purification and gel verification results were inconclusive and no yield was obtained when placed tested using a nanodrop machine. </p><br />
<br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<br />
<br />
<p>Top 10 E.coli cells were transformed with <a href="http://partsregistry.org/Part:BBa_R0011">BBa_R0011</a><br />
(IPTG inducible promoter in psb1C3 backbone), and resulting colonies were tested using cPCR. <br />
Colony PCR was performed on cells containing the catalase biobrick. Catalase is 2217bp long but since biobrick primers add about 200bp, bands of 2400 bp were expected if the part was present in the biobrick. These bands were observed, indicating that the <a href=" http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a> gene was most likely present.</p><br />
<p></html>[[File:Ucalgary2012 4.7.2012 catalase colony pcr 2.jpg|thumb|500px|center|Figure 4: Colony PCR on potential colonies containing katG-LAA. Biobrick primers were used. Bands at 2200 indicate successful amplification of the catalase part within the biobrick backbone. Smaller bands are indicative of primer degradation and are nonspecific amplification. +C indicates the positive control, and RFP generator, which should amplify at around 1000 bp.]]<html></p> <p><br />
<br />
PCR using Phusion high fidelity polymerase was carried out on <i>dszA</i>, <i>dszB</i>, and <i>dszC</i> in a gradient thermocycler. Amplification of non-specific bands was present for <i>dszA</i> and <i>dszB</i>, however strong banding for the desired size of the gene was observed for both (around 1500 for <i>dszA</i>, 1100 for <i>dszB</i> </p><br />
<br />
<p></html>[[File:Ucalgary2012 6.7.2012.dszABphusionPCR.jpg|thumb|500px|center|Figure 5:DszA and DszB PCR from <i>Rhodococcus</i>. DszA amplicon is around 1300 bp, and is observed to run higher on the gel. DszB amplicon is expected to be 1098 bp, which is observed in addition to multiple nonspecific banding. +C indicates the positive control, and RFP generator, which when PCRed with biobrick primers should be around 1000 bp. As this control is running high as well, it is believed that both amplicons for DszA and DszB have been obtained. No contamination is observed in the NTC (no template control).]]<html><br />
</p> <br />
<p></html>[[File:Ucalgary2012 7.5.2012 dszC.png|thumb|500px|center|Figure 6:DszC PCR from <i>Rhodococcus</i>. The numbers above the figure show the colony number. DszC amplicon is around 1450 bp, and is observed to run at almost the right size on the gel. No contamination is observed in the NTC (no template control).]]<html><br />
<p><br />
Examining the sequences of the <i>dszABC</i> genes led to the discovery that all 4 had multiple illegal enzyme cut-sites in them that we have to eliminate before biobrick composite part construction can occur. <i>dszA</i> has four PstI cut sites, <i>dszB</i> has a PstI and a NotI and <i>dszC</i> has a PstI cut site. The Stratagene QuickChange mutagenesis procedure is going to be used to eliminate illegal cut sites with the only alteration being that Kapa HiFi polymerase would be used during the process. Primers needed for the mutagenesis were designed based on the procedure mentioned above. </p><br />
<br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<br />
<p> Following successful amplification of the <i>dsz</i> operon genes in the previous week, the genes were constructed into the <a href="http://partsregistry.org/Part:pSB1C3"> pSB1C3</a> vector. Colony PCR verifications were observed to be positive. Furthermore, the insertion of part <a href="http://partsregistry.org/Part:BBa_J13002"> BBa_J13002, (P<sub>tetR</sub>-RBS)</a><br />
in front of the previously biobricked <i>hpaC</i> was attempted. Overnight cultures were also prepared using two colonies each for <a href="http://partsregistry.org/Part:BBa_J13002"> <i>P<sub>tetR</sub>-RBS</i></a> and <a href="http://partsregistry.org/Part:BBa_R0011"> <i>P<sub>lacI</sub></i> </a> (an IPTG inducible promoter that we hope to build in front of <ahref="http://partsregistry.org/Part:BBa_B0034">an RBS site, BBa_B0034</a>). These cultures were then miniprepped to yield the respective parts.</p> <br />
<br />
<p>Additionally, <a href="http://partsregistry.org/Part:BBa_K137068"><i>katG-LAA</i></a> was built into a <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> backbone. The construction and availability of all these parts will be critical in the construction of our overall circuit for biodesulfurization. Colonies which looked good on cPCR were used to prepare overnight cultures, and were miniprepped and sent in for sequencing verification the following day.<br />
On the side, M9 minimal media was also prepared to carry out growth experimentation and overall desulfurization capability of <i>Rhodococcus</i> when exposed to DBT. The various growth treatments were M9 Media and glucose only, M9+glucose+DBT, M9+glucose+MgSO4+/-DBT, M9+glucose+MgCl2+/-DBT. 0.008g of FeCl2.4H2O was also added to each of the tubes. Samples were then inoculated with colonies of the <i>Rhodococcus</i>.</p><br />
<br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<br />
<p>This week, while awaiting sequencing verification results which were required before we could begin the construction process, the desulfurization team initially aided in some of the tasks related to the other hydrocarbon groups. The success of the construction of <a href="http://partsregistry.org/Part:BBa_J13002"><i>P<sub>tetR</sub></i>-RBS</a><br />
with <a href="http://partsregistry.org/Part:BBa_K902057"><i>hpaC</i></a> was also explored by using forward and reverse primers of <a href="http://partsregistry.org/Part:BBa_R0040">BBa_R0040</a> (the promoter component of the composite part BBa_J13002). However, the eventual gel verification was inconclusive and sequencing results finally indicated an unsuccessful ligation. Additionally, the minimal media M9 preparation had been contaminated in the previous effort so this process was repeated to create tubes of each of the growth condition treatments detailed previously, and two repeats, one with an extra filtration step and one without was used to prepare the cultures.</p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<br />
<p> Mutagenic primers were redesigned after the initial ones were found to have premature stop codons. As part of the redesign process in constructing our overall gene circuits for desulfurization, a backbone switch of <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> into a chloramphenicol (Chlor) resistant vector was necessary. The subsequent transformed products were plated on a Chlor plate and selected colonies were used to prepare O/N cultures, then minipreped before finally being digested with enzymes EcoRI and PstI. The resulting gel verification images were inconclusive as they did not show the required banding pattern around 50bp. Meanwhile, colony PCR was run on colonies transformed with <i>katG-LAA</i> constructed into a <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> backbone, as well as the <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> +<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a> construct. <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></a> was shown to have been successfully amplified, so overnight cultures were prepared and subsequently miniprepped. On the other hand, the construct was not successful so a third attempt was carried out. Colony PCR treatments that used either <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> forward primers or <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
primers were used and the overall constructs were made either on a chlor-resistant, or ampicillin-resistant vectors. Preliminary images of the gel verification appeared to have confirmed the construct, although sequencing verification will be the final indicator of overall success. </p><br />
<br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<br />
<br />
<p>Sequencing results from the previous week's constructs were available confirming that we constructed KatGLAA in a chlor-resistant backbone. However, switching the plasmid backbone of <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a> to <A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A> was not successful. The construction of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>+<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> was finally sent in for sequencing. Site-directed mutagenesis of the <i>dsz</i> operon was also initiated: <i>dszA</i> has four PstI cut sites; <i>dszB</i> has a PstI and a NotI site; <i>dszC</i> has two PstI cut sites. Site directed mutagenesis was started this week to change a single base pair in these genes in a way that eliminates the cut site but preserves the amino acid codons, so as to not mutate the protein coding sequence. Ohshiro 2007 demonstrated that reP<sub>lacI</sub>ng the Tyr residue at position 63 of <i>dszB</i> gene with a Phe increases the activity of the enzyme. Therefore we want to introduce the same mutation into our <i>dszB</i>.</p><p><br />
For the first attempt at mutagenesis we chose to mutate the second PstI site in <i>dszC</i> (PstI2). As a positive control for the procedure, we also performed the mutagenic PCR on a plasmid containing the β-galactosidase gene with a point mutation where the PCR would cause it to regain its function. For both mutagenesis protocols we used the Kappa Hifi kit. After confirming that the PCR worked by running some produce on a gel, the PCR products were DpnI digested, the purpose of which is to degrade the unmodified parental DNA (DpnI degrades methylated DNA only). Control PCR products were plated on an ampicillin plate containing IPTG and X-gal. The colonies that grew on the control plates were blue indicating that the mutagenesis had worked for the β-galactosidase gene. Minipreps of the O/N culture of <i>dszC</i> mutants were digested with PstI enzyme and the results indicated that the mutagenesis was successful.</p><p> <br />
Attempts to simultaneously perform all the mutations in <i>dszC</i> genes in one step using the Knight procedure failed (<a href="http://openwetware.org/wiki/Knight:Site-directed_mutagenesis/Multi_site">Knight Multi-site Mutagenesis Procedure</a>). What enables simultaneous mutations is that Taq ligase closes the gaps in PCR products after each cycle. In the protocol it instructs to use Taq ligase buffer only for the PCR/ligation protocol. We suspected that the reason this procedure did not work might be that the Kappa polymerase is not functional in Taq ligase buffer. Therefore we did some experiments on the controls in Taq ligase kit and kappa polymerase kit to find out which buffer that Kappa polymerase and Taq ligase both work best in. The result was that both enzymes work best in a buffer made of half Taq ligase buffer and half Kappa polymerase buffer. </p><br />
<br />
<br />
<br />
</html>[[File:Ucalgary2012 31.7.2012 dszc mutagenesis 5 20 and 50ng.jpg|thumb|500px|center|Figure 7: DszC PstI2 mutagenesis PCR with varying concentrations of template plasmid was performed. The gel shows the PCR products that were run on a gel. +C lanes show the PCR products of the control. The control was pWhitescript™ 4.5-kb control plasmid from Stratagene mutagenesis kit which was mutated with primers also from the kit. Bands indicate successful amplification of the plasmid.]]<br />
[[File:UCalgary 02.08.12 dszC psti digest mutagenesis.jpg|thumb|700px|center|Figure 8: The control and the mutated plasmids were digested with PstI restriction enzyme. The control is dszC biobrick. Other lanes show the plasmids purified from the cells transformed with PstI2 mutagenesis PCR products followed by digestion. The control shows three bands since it has three PstI cut sites. The other lanes show two bands which indicates one of the cut sites has been eliminated through mutagenesis. ]]<html><br />
<br />
</html>[[File:Ucalgary2012 2.8.2012 finding the right buffer for multisite mutagenesis.png|thumb|800px|center|Figure 9: The multisite mutagenesis using the Knight procedure was tried in different buffers to find out the buffer that Kappa works optimally at. All the PCRs were performed on the pWhitescript™ 4.5-kb control plasmid from Stratagene mutagenesis kit which contains the beta-galactosidase gene. Based on this gel, the optimal buffer is composed of 50% kappa buffer and 50% Taq ligase buffer.]]<html><br />
<br />
</html>[[File:Ucalgary 3.8.2012 optimal buffer for Taq ligase.png|thumb|800px|center|Figure 10: Using the control provided in the NEB Taq ligase buffer (BsteII digested lambda DNA), we tried to find if the Taq ligase enzyme can function in combinations of Kappa Hifi buffer and Taq ligase buffer. Lane two is the control which is only the digested lambda DNA. Lanes 1 and 3 show that some of the bands compared to control have been ligated together. Therefore, Taq ligase functions just as good in the buffer composed of 50% kappa hifi buffer and 50% Taq ligase as it would in its own buffer.]]<html><br />
<br />
<h2>Week 15 (August 6 - August 11)</h2><br />
<br />
<p>Sequencing results for <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A> <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> returned negative, so a 3-part ligation method was used to retry this construction. The following parts were ligated with the restriction enzymes indicated in brackets after each: <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>(EcoRI/SpeI) + <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> (XbaI/PstI) + <A HREF=" http://partsregistry.org/Part:PSB1K3">BBa_PSB1K3</A> (EcoRI/PstI). Also, the more conventional construction (only 1 insert) of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>(SpeI/PstI) + <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A> (XbaI/PstI) was reattempted. Furthermore, 3-way ligations were also attempted for <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
+<A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A>+<A HREF="http://partsregistry.org/Part:PSB1K3">BBa_PSB1K3</A>, and <a href="http://partsregistry.org/Part:BBa_R0011"> BBa_R0011</a>+<a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
+<A HREF="http://partsregistry.org/Part:PSB1C3"> PSB1C3</A>, as well as the two-way contruction of just <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> after the <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a>. After plating these transformations, colony PCRs were carried out and samples that gave an indication of being successful on the gels were used to prepare O/N cultures followed by miniprep. With regards to the site-directed mutagenesis side of the experimentation, <i>dszA</i>-PstI1 (the first PstI cut site in <i>dszA</i>) ,<i> dszB</i>-PstI and <i>dszC</i>(PstI2 mutated)-PstI1 mutagenesis were performed following the procedure explained in the previous week. The gel below shows the successful result of digest confirmation (Fig. 8). <br />
Multisite mutagenesis (Knight method) was repeated using the modified buffer (half Taq ligase buffer and half Kappa buffer). However it was not successful again. We also tried doing multisite mutagenesis using Pfu Turbo polymerase and following the Knight procedure without any buffer modifications. No successful results were observed. </p><br />
<br />
<br />
<br />
</html>[[File:Ucalgary2012 Digestion confirmation of mutagenesis in dszAPstI1, dszBPstI and dszCPstI1 (PstI2 site mutated)..jpg|thumb|800px|center|Figure 11: Confirmation of site-directed mutagensis to eliminate PstI cut sites in: <i>dszA</i>PstI1 cut site, <i>dszB</i>PstI cut site and <i>dszC</i>PstI1 (PstI2 site mutated). Note that in each case, the unmutated controls for each gene are to the left of each set of plasmids derived from mutagenic PCRs. Plasmids from cloned <i>dsz</i> genes are smaller than the bands to the right of them (which have undergone mutagenesis), indicating that in the plasmids on the right, the PstI cut site was lost. ]]<html><br />
<br />
<br />
<h2>Week 16 (August 12 - August 18)</h2><br />
<br />
<br />
<p>The progress in mutagenesis of <i>dsz</i> genes was continued from the previous week: <i>dszB</i>(PstI mutated)-Y63F and <i>dszA</i>(PstI1 mutated)-PstI3 mutagenesis. The gel below shows the digest confirmation.</p><br />
<br />
<br />
</html><br />
[[File:Ucalgary2012 15.08.2012 dszAPstI1&amp;3 dszB Psti y63f muta diges-1.jpg|thumb|800px|center|Figure 12: Digestion confirmation of biobricks of a) dszA (PstI1 mutated) PstI3 mutagenesis and b) dszB (PstI mutated) Y63F mutagenesis. Cutting dszA (PstI1 mutated) with PstI is expected to produce fragments of 2790, 333, 255, and 114bp. Cutting dszA (PstI1 and PstI3 mutated) is expected to produce fragments of 2730, 588, and 114bp. The primers for dszB-Y63F mutagenesis introduce an HpyAV cut site. pSB1C3 inteslf has two more HpyAV cut sites. Before the mutation bands of 2858 abd 310bp are expected, and after successful mutation bands of 1502, 1356, 310 are expected. Lane legend: 1- dszA (PstI1 mutated). 2-dszA (PstI1 mutated) digested with PstI. 3-dszA (PstI1 mutated) PstI3 mutagenesis c1 Digested with PstI. 4- dszA (PstI1 mutated) PstI3 mutagenesis c2 Digested with PstI. 5-dszA (PstI1 mutated) PstI3 mutagenesis c3 Digested with PstI. 6- dszA (PstI1 mutated) PstI3 mutagenesis c4 Digested with PstI. 7- Fermentas 1kb Plus Ladder. 8- Empty. 9- dszB (PstI mutated). 10-dszB (PstI mutated) digested with HpyAV. 11- dszB (PstI mutated) Y63F mutagenesis c1 digested with HpyAV. 12- dszB (PstI mutated) Y63F mutagenesis c2 digested with HpyAV. 13- dszB (PstI mutated) Y63F mutagenesis c3 digested with HpyAV. 14-dszB (PstI mutated) Y63F mutagenesis c4 digested with HpyAV.]]<html></p><br />
<br />
<p>We attempted a different approach to speed up the turnover time of the mutagenesis PCR. Briefly, after the PCR mutagenesis the PCR products were purified and then incubated with T4 polynucleotide kinase (PNK) and ligase. After heat inactivating the ligase and T4 PNK, the products were DpnI digested. Subsequently another round of DNA purification was performed. However, the results were unsatisfactory after the digest confirmation.</p><p><br />
Sequencing results came back. <i>dszA</i> (PstI1 and PstI3 mutated) and <i>dszB</i>(PstI and Y63F mutated) were good. However <i>dszC</i> (PstI1 and PstI2 mutated) had an insertion next to the PstI1 cut site. Mutagenesis was repeated on the <i>dszC</i>(PstI2 mutated).<br />
<i>dszB</i>(PstI and Y63F mutated)-NotI and <i>dszA</i>(PstI1 and PstI3 mutated)-PstI4 mutagenesis were also performed.</p><br />
<p> To investigate the desulfurisation capability of the <i>Rhodococcus</i> sp. from which we cloned the <i>dsz</i> operon, a desulfurization assay was prepared by inoculating different treatments of M9 media. We also prepared some solutions that will be needed for analysis in the following week: a conditioning agent composed of 100ml of 95% ethanol, 50ml glycerol, 30ml of 12M HCl (aq) and 70g of NaCl(s) was prepared. The assay relies on the turbidity of a sample containing sulphate ions which are precipitated (hence the turbidometric nature of the assay) upon adding BaCl2(s), therefore if the <i>dsz</i> pathway is active, we expect a more turbid solution to form than in control samples. </p><br />
<br />
<br />
<br />
<h2>Week 17 (August 19 - August 25)</h2><br />
<br />
<br />
<p>This week, progress was made in determining the desulfurization activity of our <i>Rhodococcus</i> strain as measured by the sulfate release using a turbidometric assay. We encountered several challenges in our prescribed protocol as the concentrations that we used to prepare the standard curve may have been too dilute, or the composition of out conditioning agent may have been flawed. Additionally, steps were taken to determine the decomposition of DBT to 2-HBP through Gas Chromatograph-Mass Spectroscopy (GC-MS) analysis, but due to a preparation error, the DBT was added to a growth solution of M9 media prematurely and the autoclaving process decomposed the DBT releasing a yellow colouration into the solution. These two approaches in determining the desulfurization capability of the <i>dsz</i> operon will be further investigated. </p><br />
<br />
<p>Since the <i>dszC</i> second mutagenesis had proven to be unsuccessful last week, the <i>dszC</i>(PstI2 mutated)PstI1 mutagenesis was repeated. Also <i>dszA</i>(PstI1,3,4 mutated) PstI2 mutagenesis was performed. <i>dszA</i> and <i>dszC</i> were sent for sequencing on Wednesday. <i>dszB</i> was sent for sequencing on Friday. Sequencing results of <i>dszA</i> and <i>dszC</i> were back by Friday. <i>dszC</i> was successful. However, <i>dszA</i> contained an insertion next to the binding site of PstI4 cut sit, so the last two mutations must be redone. <i>dszB</i>(PstI and Y63F mutated)-NotI-mutagenesis was also repeated in case the result of the sequencing was not successful. These constructions were repeated. <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>-<A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
-<i>dszC</i> constructions were attempted, however they were not successful as indicated by colony PCR. Constructions of <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>/<i>hpaC</i> were carried out and also came back negative in sequencing, however <a href="http://partsregistry.org/Part:BBa_K902059">BBa_B0034<br />
/<i>katG-LAA</i> (BBa_K902059)</a> was sequence confirmed.</p><br />
</html>[[File:Ucalgary2012 20.8.2012 dszC(psti2)psti1 mutagenesis.png|thumb|700px|center|Figure 13: Another attempt to mutagenize dszC(PstI2 mutated)at PstI1 site. The gel shows the confirmation digestion. C is the control which is dszC(PstI2 mutated). Since the control has two PstI cut sites, two bands are observed on the gel. The mutagenesis has been successful since only one band is observed on the other lanes indicating that one of the cut sites has been eliminated during mutagenesis.]]<html><br />
<br />
</html>[[File:Ucalgary2012 21.8.2012 dszA final mutagenesis digest.png|thumb|700px|center|Figure 14: DszA (PstI1, PstI2, PstI3 mutated)PstI4 mutagenesis digestion confirmation gel. All the lanes show the results of the plasmids being cut with PstI enzyme. C indicates the lane containing the control which is dszA(PstI1, PstI3 and PstI4 mutated). The other lanes are the digestions of the plasmids that are mutagenized, from different colonies. Control plasmid has two PstI cut sites and therefore the two bands observed on the gel were expected. The other lanes show only one band of about 3500bp which is about the right size for dszA in a psb1c3 vector. Therefore one of the cutsites has been eliminated during mutagenesis.]]<html><br />
<br />
<br />
<br />
<br />
<h2>Week 18 (August 26 - September 1)</h2><br />
<br />
<p> <A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i> (BBa_K902052)</i></A> sequencing results came back as successful. <i>dszA</i>(PstI1,3 mutated)-PstI2-mutagenesis was performed and sent for sequencing. Also <i>dszA</i>(PstI1,2,3 mutated)-PstI4-mutagenesis was performed, and this was also sent for sequencing. </p><br />
<p>Constructions of <A HREF="http://partsregistry.org/Part:BBa_K902053">P<sub>tetR</sub>-RBS/<i>dszB</i> (BBa_K902053)</a> and <a href="http://partsregistry.org/Part:BBa_K902056">BBa_B0034<br />
/<i>dszC</i> (BBa_K902056)</a> were attempted, verification digested, and sent for sequencing. Sequencing results for these constructs came back as positive, along with successful mutagenesis of <A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i> (BBa_K902050)</A>.</p><br />
<p>At this point, all of the <i>dsz</i> genes have been successfully made biobrick compatible, and <i>hpaC</i> has been biobricked. We have also successfully constructed <a href="http://partsregistry.org/Part:BBa_K902059">BBa_B0034 with <i>katG-LAA</i></a> to be used in the optimization circuit, as well as <A HREF="http://partsregistry.org/Part:BBa_K902053"><i>P<sub>tetR</sub>-RBS</i>/<i>dszB</i> (BBa_K902053) </a>and <a href="http://partsregistry.org/Part:BBa_K902056">BBa_B0034/<i>dszC</i> (BBa_K902056)</a>.<br />
<p> Constructions of <A HREF="http://partsregistry.org/Part:BBa_J04500">P<sub>lacI</sub>-RBS</A>with <A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> were performed. As well, attempts to construct <A HREF="http://partsregistry.org/Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></a>/<a href="http://partsregistry.org/Part:BBa_K902056">RBS-<i>dszC</i></a> as well as <A HREF="http://partsregistry.org/Part:BBa_J13002">P<sub>tetR</sub>-RBS</A>/<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
/<A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i></A>, and <A HREF="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</A>/<A HREF="http://partsregistry.org/Part:BBa_K137068"> <i>katG-LAA</i></A> were also carried out. These parts are intended as construction intermediates towards building the final systems, as well as providing a way of testing the genes functionality (namely, to test HpaC for oxidoreductase activity and to test if over-expression of KatG in the cell will increase its ability to survive H<sub>2</sub>O<sub>2</sub> stress). Transformations of all these constructions were carried out at the end of the week.</p><br />
<br />
<h2>Week 19 (September 2- September 8)</h2><br />
<p>Confirmation digests on colonies of the previous constructions that gave bands of the expected size with cPCR were performed. Positive results were found for colonies of <A HREF="http://partsregistry.org/Part:BBa_J04500"><i>P<sub>lacI</sub>-RBS</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902057"> <i>hpaC</i></A>, <A HREF="http://partsregistry.org/Part:BBa_J04500"><i>P<sub>lacI</sub>-RBS</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902052"> <i>dszB</i></A>, and <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a><br />
/<A HREF="http://partsregistry.org/Part:BBa_K902050"> <i>dszA</i></A>. Sequencing was sent, and results indicated that the constructions of <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS/hpaC</i> (BBa_K902058)</a> were successful, meaning that after many months of trying we FINALLY have a promoter in front of the <i>hpaC</i> gene and can proceed to test the parts functionality. Attempts to construct <i>hpaC</i> with the (<A HREF="http://partsregistry.org/Part:BBa_J13002">evil TetR promotor, BBa_J13002</A>) were abandoned, as it was believed that this construction was failing due to toxicity of over-expressing the protein, and it was determined that this part was not necessary after all. <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub>-RBS/dszB</i>, (BBa_K902054)</a> also worked, though this was less exciting. <a href="http://partsregistry.org/Part:BBa_K902051">BBa_B0034<br />
/<i>dszA</i></a> came back as a bad read despite looking very good on the confirmation digest gel, so this part will be resent for sequencing. Constructions of <A HREF="http://partsregistry.org/Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> were reattempted, and confirmation digests for this part looked good, and so samples were sent for sequencing.</p><br />
<br />
<br />
<h2>Week 20 (September 9- September 15)</h2><br />
<p>Construction attempts on <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> were performed. Colonies grew for the constructions, however further confirmation results were dissapointing (only 2 clones of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a> appeared to have been successful). These clones were sent for sequencing, and constructions were reattempted.</p><p> However, when sequencing came back, somehow reads indicated that these clones were in fact a gene from the Denitrogenation project (which is 990bp and a completely different band then what we saw on the gel). We believe, somewhere, something has gone very wrong- further investigation into this will be carried out. In the meantime, the above constructions were reattempted, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902051">BBa_B0034/<i>dszA</i></A> was re-prepped in case a contaminant in the plasmid stock was to blame for the bad reads found in this batch of sequencing as well as the last. In addition, plasmid switches of multiple sequence confirmed parts into a <a href=" http://partsregistry.org/Part:pSB1C3"> pSB1C3</a> backbone were carried out. </p><br />
<br />
<h2>Week 21 (September 16- September 22)</h2><br />
<p>Colonies for the transforms of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> have been few and far between, and cPCR results are always discouraging. Sequencing results for other sections of the project have once again come back very confusing, and further research continues into the source of this madness. </p><br />
<p> The sulfur compound degradation assay was set up to test the desulfurization rate of the original <i>Rhodococcus baikonurensis </i> (refer to the protocol page).</p><br />
<br />
<br />
<h2>Week 22 (September 23- September 29)</h2><br />
<p>Attempts to construct <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, , and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A> continue. In the meantime, <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> was tested for functionality. In order to do this, cultures of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"><i>P<sub>lacI</sub></i>-<i>KatG</i></A> were grown up overnight in LB media. A strain carrying <A HREF="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</A> only was used as a negative control. The following morning, 20 &micro;L of each culture was inoculated into 3 mL of LB with various concentrations of hydrogen peroxide; 0 mM, 1 mM, 5 mM, and 10 mM. These cultures were then allowed to grow overnight, and culture turbidity was observed. It was found that the negative control exhibited no growth after 12h at 1 mM peroxide, however cultures with induced expression of catalase were turbid after 12 h of growth at this concentration (Fig. 10). This demonstrated the ability of the catalase to protect the cells from excessive peroxide concentrations.</p><p><br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 15: Catalase Assay. Overnight cultures of J04500 and J04500-<i>KatGLAA</i> were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbitity was observed.]]<html></p><br />
</p><br />
<p>In addition to this, activity of <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-hpaC</i></a> was tested. In order to do this, cultures of <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-hpaC</i></a> and <a href="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub>-dszB</i></a> were grown up overnight in LB with appropriate antibiotics. Following this, protein expression was induced with IPTG, after which the assay was carried out as described in the following figure and on the protocols page.</p><br />
<p> </html> <br />
[[File:Ucalgary2012 DesulfurizationGroup HpaC assayTake1.png|center|600px|thumb|Figure 16: HpaC Assay. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 2 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, 1 mL of cell lysate was transferred to a cuvette, and a spectrophotometer was blanked at 340 nm (maximal absorbance of NADH) with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p>When the assay was run, it was found that NADH does not convert readily to NAD+ on its own. When cell lysate containing the naturally expressed amounts of oxidoreductase was added, a decrease in absorbance could quickly be observed as the NADH was converted to NAD+. When cultures over-expressing HpaC were tested, the absorbance levels were found to start much lower than the control. We believe that this is because with the amount of cell lysate tested, when the HpaC protein is overexpressed the NADH is consumed almost immediately and therefore the data reflecting the drop in absorbance is missed. Further tests will use differing amounts of cell lysate in order to try to capture data that shows the drop in absorbance for HpaC cultures.</p><br />
<br />
<h2>Week 23 (September 30-October 3): Wiki-Freeze PANIC!!</h2><br />
<br />
<p>The GCMS results of the sulfur assay were received. DBT desulfurization was successful (figures below). Some compounds were not detected by GCMS since they were too polar. However, 2 additional compounds showed degradation in addition to DBT, indicating that the pathway has a wider substrate specificity!</p><br />
<p></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 17: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol in the protocols section for details). Samples were taken out at different time points and were run through GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria, run through the GCMS after 6 days of being in the incubator to account for abiotic degradation. ]]<html></p<br />
<br />
<p></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 18: The peak in this mass spectra demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound (A: dibenzothiophene, B: tetrahydro-4h-thiopyran-4-one, and C: benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdownn. Degradation is seen for DBT (the model compound) as well as other sulfur containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<p>The HpaC assay was repeated using 100 &micro;L of cell lysate instead of 1 mL of 2x concentrated cell lysate, following the previously used protocol. With this smaller amount, a clear sharp decrease in the absorbance of NADH can be observed, indicating a very fast conversion by HpaC, and further confirming that the part was functional.</p><br />
<br />
<p></html>[[File:Ucalgary2012Desulfurization-Hpacasay2.PNG|center|550px|thumb|Figure 20: HpaC Assay. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, 100 &micro;L of cell lysate was transferred to a cuvette, and a spectrophotometer was blanked at 340 nm (maximal absorbance of NADH) with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<h2>Week 24 (October 4- October 11)</h2><br />
<p>This week, before the Americas West Regional Competition, attempts to construct the previously mentioned parts continued. Unfortunately, they were unsuccessful.<br />
<br />
<h2>Week 25 (October 16- October 20)</h2><br />
<p>Traditional attempts to build the sulfur operon and its sub-parts were repeated, however attempts failed once more. During this week, it was decided that due to time consstraints, alternative approaches to construct the operon would be undertaken. Due to this, splice-overlap-extention (SOE) PCR primers (containing scar-sites where appropriate) were designed according to <b>protocol</b> and ordered.</p><br />
<p><br><br />
<br />
<br />
Primer: 1a (BBK)-J13/DszB F - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGTCCCTATCAGTGATAGAGATTGACATCCC-3'<br />
<br><br><br />
Primer: 1b (BBK)-J04/DszB F - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 2 Jxx/B-(RBS/C) R - 5'-GGTGACAGTGTCATCTAGTATTTCTCCTCTTTCTAGTACTATCGGTGGCGATTGAGGCTGTTG-3'<br />
<br><br><br />
Primer: 3 (Jxx/B)-RBS/C F - 5'-CAACAGCCTCAATCGCCACCGATAGTACTAGAAAGAGGAGAAATACTAGATGACACTGTCACC-3'<br />
<br><br><br />
Primer: 4 RBC/C-(RBS/A) R - 5'-GCCAGATGCATTTGTCGTTGTTGAGTCATCTAGTATTTCTCCTCTTTCTAGTATCAGGAGGTGAAGCCGGGAATCG-3'<br />
<br><br><br />
Primer: 5 (RBS/C)-RBS/A F - 5'-CGATTCCCGGCTTCACCTCCTGATACTAGAAAGAGGAGAAATACTAGATGACTCAACAACGACAAATGCATCTGGC-3'<br />
<br><br><br />
Primer: 6 RBS/A-(J04/HpaC) R - 5'-GGGGAGAGGCGGTTTGCGTATTGCTAGTATCATGAAGGTTGTCCTTGCAGTTGTGG-3'<br />
<br><br><br />
Primer: 7 RBS/A-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATCATGAAGGTTGTCCTTGCAGTTGTGG-3'<br />
<br><br><br />
Primer: 8 (BBK)-J04/HpaC F - 5'-CGAATTCGCGGCCGCTTCTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 9 (RBS/A)-J04/HpaC F - 5'-CCACAACTGCAAGGACAACCTTCATGATACTAGCAATACGCAAACCGCCTCTCCCC-3'<br />
<br><br><br />
Primer: 10 J04/HpaC-(RBS/Kat) R - 5'-CTGACGTGCTCATCTAGTATTTCTCCTCTTTCTAGTATTAAATCGCAGCTTCCATTTCCAGCATCAC-3'<br />
<br><br><br />
Primer: 11 J04/HpaC-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTAAATCGCAGCTTCCATTTCCAGCATCAC-3'<br />
<br><br><br />
Primer: 12 (J04/HpaC)-RBS/Kat F - 5'-GTGATGCTGGAAATGGAAGCTGCGATTTAATACTAGAAAGAGGAGAAATACTAGATGAGCACGTCAG-3'<br />
<br><br><br />
Primer: 13 RBS/Kat-(BBK) R - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTATTAAGCAGCCAGAGCGTAGTTTTCGTC-3'<br />
</p><br><br />
<p><br />
Colony PCR was done on potential colonies of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A>/<A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a>, , and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058"><i>P<sub>lacI</sub></i>-<i>hpaC</i></a>/<A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902059">BBa_B0034-<i>katG-LAA</i></A>. Faint bands around the expected size were seen, and these were stabbed with a pipette tip and stirred into water in order to be used as template in a further round of PCR to get amplification of the part. The plan was that these amplicons could then be used as an insert in further constructions, however no amplification was seen. Further optimization of this procedure could possibly result in a short-cut to construction, however the time needed to do this is a luxury that we do not have.</p><p>Additional attempts to create inserts by doing PCR on ligation reactions in addition to gel extraction of colony PCR bands were attempted as well, however these attempts were unsuccessful as well. SOE PCR and possible Gibson Assembly appear to be the Sulfur Teams last hope at this point, and we are eagerly awaiting our primers.<br />
</p><br />
<br />
<br />
<h2>Week 26 (October 21- October 26)</h2><br />
<p><br />
Much to our delight, SOE PCR primers were recieved this week. Therefore, alongside traditional construction of the aforementioned parts, SOE PCR was carried out according to <b>protocol</b> and these parameters:</p><br />
<br><br />
<h4>Round 1:</h4><p><br />
<ul><br />
<li>Primers 1a and 2 with <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902053"><i>P<sub>tetR</sub></i>-<i>dszB</i></A> as a template = Product L1 (J13002/DszB with BBk prefix and B0034/DszC overlap)<br />
<br></li><li><br />
Primers 1b and 2 with <A HREF="http://partsregistry.org/Part:BBa_K902054"><i>P<sub>lacI</sub></i>-<i>dszB</i></A> as a template = Product L2 (J04500/<i>dszB</i> with BBk prefix and B0034/<i>dszC</i> overlap)<br />
<br></li><li><br />
Primers 3 and 4 with <A HREF="http://partsregistry.org/Part:BBa_K902056">BBa_B0034-<i>dszC</i></a> as a template = Product L3 (B0034/<i>dszC</i> with <i>dszB</i> and B0034/<i>dszA</i> overlap)<br />
<br></li><li><br />
Primers 5 and 6 with <A HREF="http://partsregistry.org/Part:BBa_K902050"><i>dszA</i></a> as a template = Product L4 (B0034/<i>dszA</i> with <i>dszC</i> and J04500/<i>hpaC</i> overlap)<br />
<br></li><li><br />
Primers 5 and 7 with <A HREF="http://partsregistry.org/Part:BBa_K902050"><i>dszA</i></a> as a template = Product L5 (B0034/<i>dszA</i> with <i>dszC</i> overlap and BBk suffix)<br />
<br></li><li><br />
Primers 8 and 10 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L6 (J04500/<i>hpaC</i> with BBk prefix and B0034/<i>katG-LAA</i> overlap)<br />
<br></li><li><br />
Primers 9 and 10 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L7 (J04500/<i>hpaC</i> with <i>dszA</i> and B0034/<i>katG-LAA</i> overlap)<br />
<br></li><li><br />
Primers 9 and 11 with <A HREF="http://partsregistry.org/Part:BBa_K902058"><i>Plac-hpaC</i></a> as a template = Product L8 (J04500/<i>hpaC</i> with <i>dszA</i> overlap and BBk suffix)<br />
<br></li><li><br />
Primers 12 and 13 with <A HREF="http://partsregistry.org/Part:BBa_K902059"><i>RBS-katG-LAA</i></a> as a template = Product L9 (B0034/<i>katG-LAA</i> with J04500/<i>hpaC</i> overlap and BBk suffix)<br />
</li></ul><br />
</p><br />
<p><br />
</html>[[File:Ucalgary2012 Spliceoverlapextentionpcr1.png|center|thumb|800px|Figure 21: Round 1 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with varying amounts of DNA template as indicated above the wells. Cycling conditions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 55%deg;C for 1 min, 72%deg;C for 2 min. 30 s.)x 31, Stage 3 (72%deg;C for 10 min.). Bands seen at ~1500 bp for L1 through L5, ~800 bp for L6-L8, and ~2200 bp for L9 indicate successful amplification of the desired product.]]<html><br />
</p><br />
<h4>Round 2</h4><br />
<p><br />
<ul><br />
<li>Primers 1a and 4 with templates L1 and L3 = Product X1 (J13002/<i>dszB</i>/B0034/<i>dszC</i> with BBk prefix and B0034/<i>dszA</i> overlap)</li><br />
<li>Primers 1b and 4 with templates L2 and L3 = Product X2 (J04500/<i>dszB</i>/B0034/<i>dszC</i> with BBk prefix and B0034/<i>dszA</i> overlap)</li><br />
<li>Primers 3 and 6 with templates L3 and L4 = Product X3 (B0034/<i>dszC</i>/B0034/<i>dszA</i> with <i>dszB</i> and J04500/<i>hpaC</i> overlap)</li><br />
<li>Primers 3 and 7 with templates L3 and L5 = Product X4 (B0034/<i>dszC</i>/B0034/<i>dszA</i> with <i>dszB</i> overlap and BBk suffix)</li><br />
<li>Primers 5 and 10 with templates L4 and L7 = Product X5 (B0034/<i>dszA</i>/J04500/<i>hpaC</i> with <i>dszC</i> and B0034/<i>katG-LAA</i> overlap)</li><br />
<li>Primers 5 and 11 with templates L4 and L8 = Product X6 (B0034/<i>dszA</i>/J04500/<i>hpaC</i> with <i>dszC</i> overlap and BBk suffix)</li><br />
<li>Primers 8 and 13 with templates L6 and L9 = Product X7 (J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with BBk prefix and suffix)</li><br />
<li>Primers 9 and 13 with templates L7 and L9 = Product X8 (J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with <i>dszA</i> overlap and BBk suffix)</li><br />
</ul></p><p><br />
</html>[[File:Ucalgary2012 Spliceoverlapextentionpcr2.png|center|thumb|800px|Figure 22: Round 2 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with varying amounts of gel-extracted DNA template from the previous PCR round as indicated above the wells. Bands were extracted using Qiagen QIAquick Gel Extraction Kit. Cycling conditions for the PCR reactions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 65%deg;C for 1 min, 72%deg;C for 3 min. 30 s.)x 31, Stage 3 (72%deg;C for 10 min.). Faint bands seen at ~3000bp indicate amplification of the desired product.]]<html> <br />
</p><br />
<h4>Round 3</h4><br />
<p><br />
<ul><br />
<li>Primers 1a and 10 with templates X1 and X5 = Product Z1 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with B0034/<i>katG-LAA</i> overlap and BBk prefix)</li><br />
<li>Primers 1a and 11 with templates X1 and X6 = Product Z2 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with BBk prefix and suffix)</li><br />
<li>Primers 1a and 7 with templates X1 and L5 = Product Z3 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 10 with templates X2 and X5 = Product Z4 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with B0034/<i>katG-LAA</i> overlap and BBk prefix)</li><br />
<li>Primers 1b and 7 with templates X2 and L5 = Product Z5 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 11 with templates X2 and X6 = Product Z6 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i>/J04500/<i>hpaC</i> with BBk prefix and suffix)</li><br />
<li>Primers 1b and 6 with templates L2 and X3 = Product Z7(J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with J04500/<i>hpaC</i> overlap and BBk prefix)</li><br />
<li>Primers 1b and 7 with templates L2 and X4 = Product Z8 (J04500/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 1a and 6 with templates L1 and X3 = Product Z9 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with J04500/<i>hpaC</i> overlap and BBk prefix)</li><br />
<li>Primers 1a and 7 with templates L1 and X4 = Product Z10 (J13002/<i>dszB</i>/B0034/<i>dszC</i>/B0034/<i>dszA</i> with BBk prefix and suffix)</li><br />
<li>Primers 5 and 13 with templates X5 and X8 = Product Z11 (B0034/<i>dszA</i>/J04500/<i>hpaC</i>/B0034/<i>katG-LAA</i> with <i>dszC</i> overlap and BBk suffix)</li><br />
</ul></p><p><br />
</html>[[File:Ucalgary2012 sulfurSpliceoverlapextentionpcr3.png|center|thumb|800px|Figure 23: Round 3 of Splice-Overlap-Extention PCR. Reactions were carried out as described above, with 50 ng of gel-extracted DNA template from the previous PCR round. Bands were extracted using Qiagen QIAquick Gel Extraction Kit. Cycling conditions for the PCR reactions were: Stage 1 (95%deg;C for 2 min.), Stage 2 (94%deg;C for 1 min., 65%deg;C for 1 min, 72%deg;C for 6 min.)x 31, Stage 3 (72%deg;C for 10 min.). A faint band (~6000bp) can be seen in lane 4, indicating possible amplification of the desired product. Other reactions appear to have failed, as this bands are not present elsewhere.]]<html> <br />
</p><br />
<p><br />
Though it appears that S.O.E. PCR consists of crushed dreams and lies (or that extensive optimization would have to be performed in order to amplify the correct product), extraction was carried out on the single band seen, in the hopes that a subsequent round of PCR with the terminal primers for this product would possibly amplify it to a concentration that can be used in construction in the next week to assemble and test the sulfur operon constructs before competition. Conveniently, the first round of PCR creates <b>Gibson Assembly</b> compatible products. Because a kit is available in the lab, this will be attempted in the following days as well. Meanwhile, a round of traditional construction appears to have finally created the parts <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902079"><i>P<sub>tetR</sub></i>-<i>dszB</i>-B0034-<i>dszC</i></A>, <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902080"><i>P<sub>lacI</sub></i>-<i>dszB</i>-B0034-<i>dszC</i></A>, and <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902081"><i>P<sub>lacI</sub></i>-<i>hpaC</i>-B0034-<i>katG</i></A>. Though sequence confirmation could not be obtained before Wiki-Freeze, we are confident that these parts are correct due to the clear size difference seen between these parts and their sub-parts. A plasmid switch was attempted the night before DNA submission had to be sent, however due to technical issues the incubator stayed at a low temperature overnight, and cultures did not grow enough to obtain the first two parts in a pSB1C3 backbone. Because of the difficulty assembling these parts, the deadline for DNA submission, and the fact that having them in an alternate backbone in the registry would allow for easier and quicker assembly of the sulfur operon by others (no plasmid switches of large parts would have to be performed, as we have found that plasmid switching the larger constructs is quite difficult.) we decided to submit these parts in an ampicillin backbone. Further attempts to switch the backbone for these parts will continue, and hopefully we will be able to submit them in the standard backbone at a later date before the competition. In addition, a reconstruction of <A HREF="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902051">B0034-<i>dszA</i></A> was obtained and finally, at long last, sequence confirmed. Because of the verification of this part and the excellent sequence read when compared to those obtained for the previously submitted version, we decided to also resubmit this part to the registry.</p><p>Construction attempts of the operon will continue into the following week, as only 1 additional construction is needed to assemble a testable version of the operon. Hopefully, this data will be obtained before the competition, and documented on the respective parts pages.</p><br />
<p><br><br>This is the Desulfurization Team, signing off.</p><br />
</h><br />
</html>}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/OSCAR/DesulfurizationTeam:Calgary/Project/OSCAR/Desulfurization2012-10-26T18:12:41Z<p>Lisa.O: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Desulfurization|<br />
<br />
CONTENT=<html><br />
<img src="https://static.igem.org/mediawiki/2012/5/5e/UCalgary2012_OSCAR_Desulfurization_Low-Res.png" style="float: right; padding: 10px;"></img><br />
<br />
<h2>Why Remove Sulfur?</h2><br />
<br />
<p><br />
Sulfur is the third most abundant element in crude oil (Ma, 2010), and when sulfur containing hydrocarbons are burned they release S0<sub>2</sub> and S0<sub>3</sub> gasses into the atmosphere. Not only does this reduce the efficiency and value of our product, but it also contributes to global warming, acid rain, and various health issues due to the pollution (Reichmuth <i>et al</i>., 2000). Strict regulation on sulfur in fuels are now in place and low-sulfur gasoline is mandated across all of Canada (Source: Environment Canada). To upgrade the quality of our fuel we need to remove the sulfur but keep the hydrocarbon backbone for combustion.</p><br />
<br />
<h2>Our Vision</h2><p><br />
Though a few pathways for biodesulfurization exist in the microbial world, most involve the destruction of part of the carbon skeleton (an example would be the Kodama pathway)(Soleimani <i>et al</i>., 2007). This would effectively reduce the quality of our product. With this in mind the pathway we have chosen is the 4S pathway found in <i>Rhodococcus spp</i>. It has been characterized and shown to remove sulfur from the model substrate dibenzothiophene (DBT) and convert it to 2-hydroxybiphenyl (2-HBP) in a non-destructive manner. DBT and its derivatives make up 70% of the organic sulfur compounds found in crude oil (Ma 2010), and are also some of the most difficult to remove through chemical means. By using the 4S pathway we will be able to upgrade our fuel and remove recalcitrant compounds at the same time. <br />
</p><br />
<br />
</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|750px|thumb|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD.]]<html></p><br />
<br />
<h2>4S pathway</h2><br />
<p><br />
Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO<sub>2</sub>) through the addition of 2 oxygen atoms to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the sulfur, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-HBP. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.</p><p> <br />
The first three steps of the 4S pathway require FMNH<sub>2</sub> and subsequently reduces the reductive power of the cell. WIn order to regain this power an oxidoreductase (DszD) uses NADH to recycle the FMNH<sub>2</sub>, allowing the reaction to proceed. Without DszD the desulfurization pathway would grind to a halt.</p><p align="justify"><br />
The <i>dszA</i>,<i>B</i>, and <i>C</i> genes form an operon on the pSOX plasmid of <i>R. erythropolis</i>, while <i>dszD</i> is found in the chromosome. Naturally this pathway is slow, however using synthetic biology approaches this process can be optimized.</p><br />
<br />
<h2>Our Approach</h2><br />
<a name="Degradation"></a><h3>1) Find the genes!</h3><br />
<p>We isolated the plasmid containing the <i>dsz</i> genes from a desulfurising environmental isolate of <i>Rhodococcus</i> using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">modified miniprep procedure</a>. As the native promoter has been shown to be repressed by various sulfur-containing compounds (Li <i>et al</i>., 1996), we designed primers for just the coding sequences of the <i>A, B, </i> and <i>C</i> genes. As these genes all have some illegal cutsites in them we constructed them into the PSB1C3 vector and started our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis protocol</a>.</p><br />
<p> We performed an experiment to measure the desulfurization rate of select organosulfur compounds by our <i>Rhodococcus</i> strain (Figures 4-6 below). These experiments monitored the degradation of the compounds by our strain over time. We discovered that the <i>dsz</i> operon is capable of desulfurizing a wider range of compounds than just the commonly studied DBT. This shows that this pathway could be a promising solution for degradation of a wide variety of sulfur containing toxins, including those that resemble naphthenic acids. </p> <br />
<br />
<p></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 2: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through the GC/MS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GC/MS after 6 days of incubation. ]]<html></p<br />
<br />
<p></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 3: The peak in this mass spectrum demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound (A: dibenzothiophene, B: tetrahydro-4h-thiopyran-4-one, and C: benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdownn. Degradation is seen for DBT (the model compound) as well as other sulfur containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
<br />
<h3>2) Mutagenesis: Biobrick Compatability and Increasing DszB Activity </h3><br />
<p>In total the <i>dszABC</i> genes had 7 PstI sites and 1 NotI site that needed to be mutated for the biobrick standard. The primers were designed such that the site was removed without the amino acid being changed. In addition, a point mutation of Y63F in DszB increased the activity of the protein (Oshiro <i>et al</i>., 2007), and was included in the mass mutagenesis we undertook. Mutagenesis was performed as described in <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">this protocol.</a></p><br />
<br />
<a name="catalase"></a><h3>3) Replacing DszD with HpaC & Introducing Catalase </h3><br />
<p><br />
As FMNH<sub>2</sub> is consumed in the first three steps of the pathway it needs to be regenerated or the process will grind to a halt. This usually falls to the <i>dszD</i> gene, however it has been shown that the <i>hpaC</i> gene from <i>E. coli</i> performs the same function more efficiently (Gala´n <i>et al</i>., 2000). One problem arises from this though, as high levels of FMNH<sub>2</sub> cause the production of toxic hydrogen peroxide inside the cell (Gala´n <i>et al</i>. 2000). To address this issue we have included a catalase gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"> <i>P<sub>lacI</sub>-katG-LAA</i></a>) that will remove the peroxide that would be toxic to the cell.</p><br />
<br />
<p></html>[[File:Ucalgary_sulfur_constructs_KatandHpaC.PNG|center|250px|thumb|Figure 7: Diagrammatic representation of the full "optimization circuit", consisting of the oxidoreductase HpaC and a catalase (KatG).]]<html></p><br />
<br />
<br />
<h3>Results</h3><br />
<p>To show that catalase activity increased <i>E. coli</i> survivability in peroxide we cultured the inducible catalase against a catalase-free control with varying levels of peroxide. After growing overnight the negative didn't grow in any culture except in the absence of peroxide, while the catalase cultures could tolerate peroxide. This is shown below.</p><p><br />
<br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 8: Catalase Assay. Overnight cultures of P<sub>lacI</sub> and P<sub>lacI</sub>-KatGLAA were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbidity was observed. It was found that at 1 mM of peroxide, cultures with just the lacI promotor perished, however when KatG-LAA was expressed, the cells survived.]]<html></p><br />
<br />
<br />
<p>To test the action of HpaC to use NADH to recycle FMN into FMNH<sub>2</sub> cell lysates were exposed to NADH and it's absorbance at 340nm (Kamali <i>et al</i>., 2010) was measured over time. Both native HpaC expression and an induced <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> system were tested as well as a negative control. The results are shown below.</p><br />
<br />
<p> </html> <br />
[[File:Ucalgary2012 HpaC assaycumulativeforthedatapage.png|center|850px|thumb|Figure 9: HpaC Assay with '''A)''' 2 mL cell lysate and '''B)''' 100 &micro;L cell lysate. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, various amounts of cell lysate were transferred to a cuvette, and a spectrophotometer was blanked at 340 nm with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p>The assay showed that NADH does not abiotically convert into NAD+, however the native expression of HpaC did show a steady decrease in the levels of NADH. The induced overexpression of HpaC caused extremely rapid conversion into NAD+ as reflected by a sharp drop in the absorbance of NADH (see figure B). This drop was much sharper than what was seen when native levels of oxidoreductases were tested, showing that the <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> was functional and that it would effectively recycle FMN.</p><br />
<br />
<br />
<a name="UBC"></a><h3>4) Optimizing Gene Order</h3><br />
<br />
<p>Further optimization of the system was achieved through reorganization of the reconstructed operon. Natively the genes are arranged ABC, however the catalytic efficiency of the protein products are 25:1:5 for A:B:C respectively (Li <i>et al</i>., 2008). By rearranging the genes into BCA there is stronger transcription of the weaker proteins, giving a more balanced system overall. These would all be constructed with the same strong ribosomal binding site, <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>.</p><br />
<br />
</html>[[File:DszOperonOptimize.png|center|400px|thumb|Figure 10: Method of optimizing gene order. The top circuit represents that found natively in the organism, with the bottom circuit representing our modified version.]]<html><br />
</p><br />
<br />
<a name="sulfur"></a><h2>Final Constructs</h2><br />
<p>After all of the above considerations are met, four final constructs for our system will be made to allow us to test desulfurization under different conditions.</p><p><br />
<br />
</html>[[File:WikiConstructs_ucalgary_sulfur_2012_final_systems.png|center|700px|thumb|Figure 11: First set of final constructs for the desulfurization operon, with constitutive Dsz expression and inducible expression of the optimization proteins; either HpaC on its own or coexpressed with KatG]]<html></p><br />
<br />
<p><br />
The first two constructs have the modified <i>dsz</i> operon (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902052"><i>dszB</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><i>dszC</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902050"><i>dszA</i></a>) under the control of a constitutive TetR promotor (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>) This is to allow for the testing of the optimization circuit, which is under the control of a lacI promotor inducible by IPTG (<a href="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</a>). The set-up of these two constructs will therefore allow for the expression of the <i>dsz</i> genes with the ability to test and compare their desulfurization rates <br> A) On their own <br> B) With the addition of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> <br> C) With the addition of both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K137068"><i>katG-LAA</i></a></p><br />
<br />
<p>This will allow us to determine what the optimal construct and expression levels of the additional genes must be in order to have the most effective sulfur removal system.</p><br />
<br />
</html>[[File:WikiConstructs2 sulfur ucalgary induciblesytems.PNG|center|700px||thumb|Figure 12: Second set of final constructs for the desulfurization operon, with all genes under an IPTG inducible promotor.]]<html><br />
<br />
<p><br />
Due to the large number of proteins being expressed in this system, the possibility of forming inclusion bodies is present. As such, a backup system was built where both the optimization circuit and the <i>dsz</i> operon were under the control of the inducible lacI promoter. This system would allow us to tune the expression of the genes, and determine which expression level is optimal for desulfurization in our bioreactor.</p> <br />
<br />
<p>Currently the final steps of construction of these constructs is underway, following which functionality tests will begin.</p><br />
<br />
<br />
</html><br />
}}</div>Lisa.Ohttp://2012.igem.org/Team:Calgary/Project/OSCAR/DesulfurizationTeam:Calgary/Project/OSCAR/Desulfurization2012-10-26T18:11:26Z<p>Lisa.O: </p>
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<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Desulfurization|<br />
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<img src="https://static.igem.org/mediawiki/2012/5/5e/UCalgary2012_OSCAR_Desulfurization_Low-Res.png" style="float: right; padding: 10px;"></img><br />
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<h2>Why Remove Sulfur?</h2><br />
<br />
<p><br />
Sulfur is the third most abundant element in crude oil (Ma, 2010), and when sulfur containing hydrocarbons are burned they release S0<sub>2</sub> and S0<sub>3</sub> gasses into the atmosphere. Not only does this reduce the efficiency and value of our product, but it also contributes to global warming, acid rain, and various health issues due to the pollution (Reichmuth <i>et al</i>., 2000). Strict regulation on sulfur in fuels are now in place and low-sulfur gasoline is mandated across all of Canada (Source: Environment Canada). To upgrade the quality of our fuel we need to remove the sulfur but keep the hydrocarbon backbone for combustion.</p><br />
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<h2>Our Vision</h2><p><br />
Though a few pathways for biodesulfurization exist in the microbial world, most involve the destruction of part of the carbon skeleton (an example would be the Kodama pathway)(Soleimani <i>et al</i>., 2007). This would effectively reduce the quality of our product. With this in mind the pathway we have chosen is the 4S pathway found in <i>Rhodococcus spp</i>. It has been characterized and shown to remove sulfur from the model substrate dibenzothiophene (DBT) and convert it to 2-hydroxybiphenyl (2-HBP) in a non-destructive manner. DBT and its derivatives make up 70% of the organic sulfur compounds found in crude oil (Ma 2010), and are also some of the most difficult to remove through chemical means. By using the 4S pathway we will be able to upgrade our fuel and remove recalcitrant compounds at the same time. <br />
</p><br />
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</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|750px|thumb|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD.]]<html></p><br />
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<h2>4S pathway</h2><br />
<p><br />
Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO<sub>2</sub>) through the addition of 2 oxygen atoms to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the sulfur, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-HBP. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.</p><p> <br />
The first three steps of the 4S pathway require FMNH<sub>2</sub> and subsequently reduces the reductive power of the cell. WIn order to regain this power an oxidoreductase (DszD) uses NADH to recycle the FMNH<sub>2</sub>, allowing the reaction to proceed. Without DszD the desulfurization pathway would grind to a halt.</p><p align="justify"><br />
The <i>dszA</i>,<i>B</i>, and <i>C</i> genes form an operon on the pSOX plasmid of <i>R. erythropolis</i>, while <i>dszD</i> is found in the chromosome. Naturally this pathway is slow, however using synthetic biology approaches this process can be optimized.</p><br />
<br />
<h2>Our Approach</h2><br />
<a name="Degradation"></a><h3>1) Find the genes!</h3><br />
<p>We isolated the plasmid containing the <i>dsz</i> genes from a desulfurising environmental isolate of <i>Rhodococcus</i> using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">modified miniprep procedure</a>. As the native promoter has been shown to be repressed by various sulfur-containing compounds (Li <i>et al</i>., 1996), we designed primers for just the coding sequences of the <i>A, B, </i> and <i>C</i> genes. As these genes all have some illegal cutsites in them we constructed them into the PSB1C3 vector and started our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis protocol</a>.</p><br />
<p> We performed an experiment to measure the desulfurization rate of select organosulfur compounds by our <i>Rhodococcus</i> strain (Figures 4-6 below). These experiments monitored the degradation of the compounds by our strain over time. We discovered that the <i>dsz</i> operon is capable of desulfurizing a wider range of compounds than just the commonly studied DBT. This shows that this pathway could be a promising solution for degradation of a wide variety of sulfur containing toxins, including those that resemble naphthenic acids. </p> <br />
<br />
<p></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 2: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through the GC/MS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GC/MS after 6 days of incubation. ]]<html></p<br />
<br />
<p></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 3: The peak in this mass spectrum demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
<br />
</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound (A: dibenzothiophene, B: tetrahydro-4h-thiopyran-4-one, and C: benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdownn. Degradation is seen for DBT (the model compound) as well as other sulfur containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html><br />
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<h3>2) Mutagenesis: Biobrick Compatability and Increasing DszB Activity </h3><br />
<p>In total the <i>dszABC</i> genes had 7 PstI sites and 1 NotI site that needed to be mutated for the biobrick standard. The primers were designed such that the site was removed without the amino acid being changed. In addition, a point mutation of Y63F in DszB increased the activity of the protein (Oshiro <i>et al</i>., 2007), and was included in the mass mutagenesis we undertook. Mutagenesis was performed as described in <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">this protocol.</a></p><br />
<br />
<a name="catalase"></a><h3>3) Replacing DszD with HpaC & Introducing Catalase </h3><br />
<p><br />
As FMNH<sub>2</sub> is consumed in the first three steps of the pathway it needs to be regenerated or the process will grind to a halt. This usually falls to the <i>dszD</i> gene, however it has been shown that the <i>hpaC</i> gene from <i>E. coli</i> performs the same function more efficiently (Gala´n <i>et al</i>., 2000). One problem arises from this though, as high levels of FMNH<sub>2</sub> cause the production of toxic hydrogen peroxide inside the cell (Gala´n <i>et al</i>. 2000). To address this issue we have included a catalase gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"> <i>P<sub>lacI</sub>-katG-LAA</i></a>) that will remove the peroxide that would be toxic to the cell.</p><br />
<br />
<p></html>[[File:Ucalgary_sulfur_constructs_KatandHpaC.PNG|center|250px|thumb|Figure 7: Diagrammatic representation of the full "optimization circuit", consisting of the oxidoreductase HpaC and a catalase (KatG).]]<html></p><br />
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<br />
<h3>Results</h3><br />
<p>To show that catalase activity increased <i>E. coli</i> survivability in peroxide we cultured the inducible catalase against a catalase-free control with varying levels of peroxide. After growing overnight the negative didn't grow in any culture except in the absence of peroxide, while the catalase cultures could tolerate peroxide. This is shown below.</p><p><br />
<br />
<br />
<br />
</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 8: Catalase Assay. Overnight cultures of P<sub>lacI</sub> and P<sub>lacI</sub>-KatGLAA were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbidity was observed. It was found that at 1 mM of peroxide, cultures with just the lacI promotor perished, however when KatG-LAA was expressed, the cells survived.]]<html></p><br />
<br />
<br />
<p>To test the action of HpaC to use NADH to recycle FMN into FMNH<sub>2</sub> cell lysates were exposed to NADH and it's absorbance at 340nm (Kamali <i>et al</i>., 2010) was measured over time. Both native HpaC expression and an induced <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> system were tested as well as a negative control. The results are shown below.</p><br />
<br />
<p> </html> <br />
[[File:Ucalgary2012 HpaC assaycumulativeforthedatapage.png|center|850px|thumb|Figure 9: HpaC Assay with '''A)''' 2 mL cell lysate and '''B)''' 100 &micro;L cell lysate. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, various amounts of cell lysate were transferred to a cuvette, and a spectrophotometer was blanked at 340 nm with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
<br />
<p>The assay showed that NADH does not abiotically convert into NAD+, however the native expression of HpaC did show a steady decrease in the levels of NADH. The induced overexpression of HpaC caused extremely rapid conversion into NAD+ as reflected by a sharp drop in the absorbance of NADH (see figure B). This drop was much sharper than what was seen when native levels of oxidoreductases were tested, showing that the <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> was functional and that it would effectively recycle FMN.</p><br />
<br />
<br />
<a name="UBC"></a><h3>4) Optimizing Gene Order</h3><br />
<br />
<p>Further optimization of the system was achieved through reorganization of the reconstructed operon. Natively the genes are arranged ABC, however the catalytic efficiency of the protein products are 25:1:5 for A:B:C respectively (Li <i>et al</i>., 2008). By rearranging the genes into BCA there is stronger transcription of the weaker proteins, giving a more balanced system overall. These would all be constructed with the same strong ribosomal binding site, <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>.</p><br />
<br />
</html>[[File:DszOperonOptimize.png|center|400px|thumb|Figure 10: Method of optimizing gene order. The top circuit represents that found natively in the organism, with the bottom circuit representing our modified version.]]<html><br />
</p><br />
<br />
<a name="sulfur"></a><h2>Final Constructs</h2><br />
<p>After all of the above considerations are met, four final constructs for our system will be made to allow us to test desulfurization under different conditions.</p><p><br />
<br />
</html>[[File:WikiConstructs_ucalgary_sulfur_2012_final_systems.png|center|700px|thumb|Figure 11: First set of final constructs for the desulfurization operon, with constitutive Dsz expression and inducible expression of the optimization proteins; either HpaC on its own or coexpressed with KatG]]<html></p><br />
<br />
<p><br />
The first two constructs have the modified <i>dsz</i> operon (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902052"><i>dszB</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><i>dszC</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902050"><i>dszA</i></a>) under the control of a constitutive TetR promotor (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>) This is to allow for the testing of the optimization circuit, which is under the control of a lacI promotor inducible by IPTG (<a href="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</a>). The set-up of these two constructs will therefore allow for the expression of the <i>dsz</i> genes with the ability to test and compare their desulfurization rates <br> A) On their own <br> B) With the addition of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> <br> C) With the addition of both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K137068"><i>katG-LAA</i></a></p><br />
<br />
<p>This will allow us to determine what the optimal construct and expression levels of the additional genes must be in order to have the most effective sulfur removal system.</p><br />
<br />
</html>[[File:WikiConstructs2 sulfur ucalgary induciblesytems.PNG|center|700px||thumb|Figure 12: Second set of final constructs for the desulfurization operon, with all genes under an IPTG inducible promotor.]]<html><br />
<br />
<p><br />
Due to the large number of proteins being expressed in this system, the possibility of forming inclusion bodies is present. As such, a backup system was built where both the optimization circuit and the <i>dsz</i> operon were under the control of the inducible lacI promoter. This system would allow us to tune the expression of the genes, and determine which expression level is optimal for desulfurization in our bioreactor.</p> <br />
<br />
<p>Currently, assembly of these final constructs is underway, with only a couple more construction steps before functionality tests can begin.</p><br />
<br />
<br />
</html><br />
}}</div>Lisa.O