http://2012.igem.org/wiki/index.php?title=Special:Contributions/Stephanie0101&feed=atom&limit=50&target=Stephanie0101&year=&month=2012.igem.org - User contributions [en]2024-03-29T08:28:31ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Calgary/Project/FRED/DetectingTeam:Calgary/Project/FRED/Detecting2012-10-04T03:35:35Z<p>Stephanie0101: </p>
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This year, our team wanted to identify a novel responsive element capable of detecting and quantifying 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 />
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<h2>Transposons: What, How, Why?</h2><br />
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The transposable element (TE), Tn5, is a conservative transposon that is able to insert a segment of genes bordered by specific 19bp insertion sequences (IS) from one part of the genome (e.g. plasmid vector) randomly to another location, such as the 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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<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 />
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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 />
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<h2>Toxin-Degrading Organism Used</h2><br />
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<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 />
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<h2>Method Design</h2><br />
<h3>Mutant Library Generation</h3><br />
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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 />
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</html>[[File:Transposonproject Tn5OT182constructucalgary.png|thumb|750px|center|Figure 1: 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 />
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<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 2Transposons: 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 3: 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 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. 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 5: 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 6: 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 />
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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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<h2>The Risks of FRED and OSCAR</h2><br />
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<p>Our project utilizes two types of engineered bacteria to detoxify tailings water. To quantify the amount of the toxic compounds present in the tailings water we are relying on <a href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a>, a biosensor bacterium, that will work inside a closed environment to detect the toxins. Within our bioreactor system, we intend to introduce <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a>, a bacterium capable of detoxifying toxic compounds, to process large volumes of tailings water. </p><br />
<p>Whenever engineered bacteria are used, there is an inherent risk that the bacteria might escape from their containment vessels into the surrounding environment. There is no direct evidence to suggest the genetic systems we have implemented would have a negative impact on the environment. However, the implications of horizontal gene transfer to native microorganisms in tailings ponds and the surrounding environment must be addressed. This issue has been raised by numerous leaders in the oil industry as well as individuals living near tailing ponds. Our approach to biosafety was inspired by a comment published in Nature (Dana <i>et al</i>., 2012) which suggested multiple ways to prevent “Synthetic Biology Disaster”. We strongly believe that we must tackle four major safety concerns with our project. First, the synthetically engineered bacteria may be harmful to natural flora in the environment. Second, the bacteria may not only survive in the tailing pond environment but thrive in it, allowing it to outcompete naturally occurring bacteria. Third, genes may be transferred from our synthetic bacteria to native organisms. Fourth, if any of our genetically modified bacteria were to be able to grow in the tailings pond, evolution may allow for mutations which prevent our safety measures from working.</p><br />
<p> To address these four major safety concerns, we have engineered mechanical and biological safety measures that function to contain genetic elements of our synthetic bacteria. The first two concerns have been addressed through mechanical engineered controls by physically separating our organisms from the native environment. The third concern has been addressed through the development of a novel <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">kill switch system</a> to prevent our engineered organisms' DNA from spreading to other organisms. The fourth concern will be addressed by producing redundancy in our kill switch system which can be applied in the scale-up process of our project. By integrating these controls, we have taken a proactive approach to the biosafety of FRED and OSCAR.</p><br />
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<h2>Mechanical Engineered Controls</h2><br />
<h3>FRED</h3><br />
<p>Our team has established a series of controls which we hope to implement in our biosensor during field testing and optimization of the final product. The final product will contain FRED within one-time use closed containers, with one-way valves. The operator will insert water samples through the one way valve, isolating FRED from the operator and the environment. Once testing is completed, the operator will be instructed to simply twist the cap of the tube to release a pre-measured aliquot of bleach to destroy FRED prior to proper disposal of the container. </p><br />
<h3>OSCAR</h3><br />
<p>The goal of OSCAR is to be able to detoxify tailings ponds compounds through the removal of nitrogen, sulfur, and carboxylic acid functional groups. OSCAR has been designed to function in a closed system to which tailings pond water is added, as opposed to adding OSCAR directly into the environment. Hydrocarbon products generated in the bioreactor after microbial remediation are collected from the culture with a continuous belt skimming device. There is potential for OSCAR in the bioreactor vessel to escape by adhering to the belt. To counter this risk, the belt is treated with UV irradiation as it exits the bioreactor solution. This process will destroy OSCAR on the belt while simultaneously maintaining integrity of the generated hydrocarbons. The extracted solution is then sent through fractional distillation, a process which heats the hydrocarbon solution to over 400&deg;C, killing any OSCARs that may have survived UV exposure.</p><br />
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<h2>Biological Engineered Controls</h2><br />
<p>Kill switches that have previously been entered into the registry often rely on methods that induce cell lysis. In these systems, genetic material is left intact, allowing for the remaining DNA to be taken up by bacteria and introducing the possibility that synthetic genes escape into the environment. We feel that lysis based kill switches are insufficient for use in FRED and OSCAR, necessitating design of novel kill switches. </p><br />
<p>To ensure that synthetic genetic elements cannot escape the closed systems in which FRED and OSCAR will be used, we engineered novel biological kill switches which we named "Ribo-Kill-Switches." These Ribo-Kill-Switches initiate cell death through degradation of genomic and plasmid DNA. Through a unique cell culture condition within the closed bioreactor and biosensor systems the kill switch genes can be suppressed. Should bacteria escape, the lack of the unique suppression conditions enables the kill switch system to become active. </p><br />
<p>Activation of the kill switch system causes the engineered cell to produce micrococcal nuclease and CviAII restriction enzyme. Our kill switch mechanisms are superior to previous nuclease-based kill switches because we have improved the completeness of DNA degradation. CviAII and micrococcal nuclease work in tandem: the endonuclease CviAII creates DNA double strand breaks at multiple sites while the micrococcal exonuclease activity degrades remaining strands into single nucleotides. The degradative enzymes chosen for our system were specifically selected for their ability to function at low temperatures, in variable pH conditions, and to work quickly to degrade as much of the genetic material as possible. These engineered biological controls ensure that synthetic genetic elements are completely destroyed in the event that FRED or OSCAR escape from the closed bioreactor or biosensor systems.</p><br />
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<h2>Laboratory Personal Safety</h2><br />
<p>All of the students working with the iGEM 2012 Calgary Team received appropriate safety training as described by the University of Calgary’s safety policies. This included a Biosafety course which introduced the students to proper handling of biological materials. In addition, all iGEM students were required to attend proper Workplace Hazardous Materials Information System (WHMIS) training sessions. All safety procedures and guidelines of “Level 2 Biohazard Labs” were followed. Students were also supervised at all times by at least one authorized senior member, lab coordinator, teaching assistant, or professor. </p><br />
<p>The bacterial strains (<i>Nocardia</i>, <i>Rhodoccocus</i>, <i>Pseudomonas</i>, and <i>Escherichia</i>) used in the research are lab strains rated as Biosafety Level 1 and do not pose a health risk to laboratory workers, the general public, or the environment. The team also practiced appropriate procedures for working with and the disposal of tailings water samples. Appropriate handling measures were also applied for genetically modified bacteria and materials contaminated with bacteria. All measures outlined in the Material Safety Data Sheets (MSDS) and the biosafety regulations present at the University of Calgary were followed. </p> <br />
<p>Through these procedures, none of the genetically modified bacteria could have a chance of being introduced into the environment. The constructs that we have built to test our systems in the laboratory all used a safe, non-pathogenic bacterial strain of <i>E. coli</i> commonly used in labs worldwide. Other bacteria which were used for characterization of their genes, as listed above, are also non-pathogenic.</p><br />
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<h2>Background</h2><br />
<p>When designing our actual kill genes, we needed to consider again the challenges of our environment. Many of the restriction enzymes found in the registry such as BamHI and BglII are active only at temperatures around 37&deg; C. Although our bioreactor may be at this temperature, the surrounding environment would be much cooler. Since the kill genes <i>should</i> be active in the surrounding environment, we needed to pick enzymes that would be active at lower temperatures. In addition to that we also wanted enzymes that would cut very frequently in the <i>E. coli</i> genome to limit the chance of horizontal gene transfer. Finally, as we chose to use an inducible system, which can easily be mutated, we wanted to introduce some redundancy by using two different kill genes. </p><br />
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<p> We ended up choosing genes for two novel kill enzymes: S7 micrococcal nuclease (<a href="http://partsregistry.org/Part:BBa_K902019">BBa_K902019</a>) and CviAII (<a href="http://partsregistry.org/Part:BBa_K902022">BBa_K902022</a>). Both of these enzymes are active at much lower temperatures than restriction enzymes. Through sequence analysis of the <i>E. coli</i> genome, we also determined that they cut extremely frequently in the genome, much more so than BglII or BamHI even combined (figure 1).<br />
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<h2>Kill Gene: S7 micrococcal nuclease</h2><br />
<p>S7 nuclease is native to <i>Staphylococcus aureus. S. aureus </i> uses this enzyme to destroy extracellular DNA when it infects humans to evade the immune system. S7 has both endo and exonuclease activity, and has a preference for -AT rich regions as opposed to -GC rich regions. Due to the non-specificity and high activity of this enzyme, it digests the DNA into <200 bp fragments. Ideally this enzyme will be present both intracellularly and extracellularly. The intracellular fraction would degrade the <i>E. coli</i> genome and the extracellular fraction would degrade any free floating DNA thereby reducing the chances of horizontal gene transfer. We synthesized this enzyme from IDT, however it came with a mutation which altered a lysine residue to an isoleucine making the enzyme dysfunctional. In order to overcome this issue, constructs with S7 were subjected to site-directed mutagenesis to restore the activity of the enzyme(Dingwall <i>et al</i>. 1985). </p><br />
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<h2>Kill Gene: CviAII Restriction Enzyme</h2><br />
<p>CviAII is a restriction endonuclease that was sourced from the <i>Chlorella</i> virus PBCV-1 (Zhang <i>et al</i>., 1992). Our team selected this enzyme for three reasons. Firstly, this enzyme recognizes small, four-base pair restriction sites as opposed to other restriction enzymes such as the six-base cutter BamHI from the 2007 Berkely team (<a href="http://partsregistry.org/Part:BBa_I716462">BBa_I716462</a>). Because of this, the CviAII restriction site is 16 times more prevalent in the E. coli genome and causes more thorough degradation of genetic material. Secondly, CviAII is able to cut Dam and Dcm methylated sites in the <i>E. coli</i> genome, and this decreased selectivity increases prevalence of cut sites. Finally, the temperature optimum for the enzyme is 23&deg;C (Zhang <i>et al</i>., 1992). This optimum is closer to temperature conditions in the tailings ponds, and thus, CviAII will exhibit better enzyme activity as opposed to other enzymes in the registry with higher optimal temperatures.</p><br />
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<h2>Nuclease Assay</h2><br />
</html>[[File:UCalgary2012 RE-S7&amp;CviaII.png|thumb|300px|right|Figure 2: This assay compares the enzymes present in the regitry i.e, BglII and BamHI to the enzymes added by us, S7 and CviAII. This shows that S7 and CviAII degrade the DNA much quicker than BglII and BamHI combined.]]<html> <p>In order to compare the S7 and CviAII to other nucleases in the 2011 registry (BglII and BamHI), we used combinations of commercial enzymes from New England Biolabs to digest <i>E. coli</i> genomic preps. Please view the <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/nucleaseassay">nuclease assay protocol</a> for more details on how this was done. As shown in figure 2, S7 activity is extremely rapid and shows degradation at the zero time point. Following 45 minutes of incubation time S7 and CviAII have digested the <i>E. coli</i> genome into small fragments, whereas BamHI and BglII treated fragments are significantly larger. After 90 minutes, S7 and CviAII have sheared the genome into very small fragments (less than 200 bp in size) while there are no difference in the lanes with BglII and BamHI which are similar to the 45 minutes time point.<br />
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}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/RegulationTeam:Calgary/Project/HumanPractices/Killswitch/Regulation2012-10-04T02:32:27Z<p>Stephanie0101: </p>
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<h2> Tight Regulation </h2><br />
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<p>Inducible kill systems are not new to iGEM. Looking through the registry, there are several constructs such as the inducible BamHI system contributed by Berkley in 2007 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_I716462">BBa_I716462</a>) and <a href="http://partsregistry.org/Image:UoflBamHIdatasheet.png">tested by Lethbridge in 2011</a>. This uses a <i>BamHI</i> gene downsteam of an arabinose-inducible promoter. Another example is an IPTG inducible Colicin construct (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K117009">BBa_K117009</a>) submitted by NTU-Singapore in 2008. One major problem with these systems however is a lack of tight control. As was demonstrated by the Lethbridge team, this part has leaky expression when inducer compound is not present. The frequently used lacI promoter has similar problems when not used in conjunction with strong plasmid-mediated expression of lacI. This can be seen in our electrochemical characterization of the uidA hydrolase enzyme (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902002">BBa_K902002</a>) shown here. Tight control is not only a problem for kill switch application, but for any application requiring strict regulation. As such, we decided that expanding the registry repertoire of control elements would be useful for our system as well as a variety of other applications. </p><br />
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<h2> Introducing the Riboswitch </h2><br />
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<p>Riboswitches are small pieces of mRNA which bind small molecules to modify translation of downstream genes. These sites are engineered into circuits by replacing traditional ribosome binding sites with riboswitches. The riboswitch is able to bind its respective ligand to inhibit or promote binding of translational machinery(Vitreschak <i>et al</i>, 2004). Riboswitches can be used in tandem with an appropriate promoter to enable tighter control of gene expression. Given this opportunity for control, and that ligands for riboswitches are often inexpensive small ions, these methods might be a feasible solution for controlling the kill switch in our industrial bioreactor.</p><br />
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<p>We explored 3 different riboswitches, each responsive to a different metabolite (magnesium, manganese or molybdate) that would be cheap to implement into a bioreactor environment. Additionally, we also investigated a repressible and inducible promoter responsive to glucose and rhamnose respectively.</p><br />
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<h2>Magnesium riboswitch</h2><br />
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<p>The magnesium riboswitch that we looked at is repressed in the presence of magnesium ions. This system has two control components – a promoter and a riboswitch. Normally the magnesium (mgtA) promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and the magnesium (mgtA) riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902009</a>) are activated if there is a deficiency of magnesium in the cell (Winnie and Groisman, 2010). The sequence of the <i>mgtA</i> promoter and riboswitch was obtained from Winnie and Groisman. The lack of magnesium activates other genes in <i>E. coli </i>to allow influx of magnesium into the cell. The two proteins in the cascade that activate the system are <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>) and <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>). <i>PhoQ</i> is the trans-membrane protein which gets activated in the absence of magnesium and phosphorylates <i>PhoP</i>. <i>PhoP</i> in turn binds to the mgtA promoter and transcribes genes downstream(Winnie and Groisman, 2010).</p><br />
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<h2>Manganese riboswitch</h2><br />
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<p> Manganese is an essential micronutrient. It is an important co-factor for enzymes and it also reduces oxidative stress in the cell (Waters <i>et al</i>. 2011). Despite being an important micronutrient, it is toxic to cells at high levels. MntR protein detects the level of manganese in the cell and acts as a transcription factor to control the expression of manganese transporter such as mntH, mntP and mntABCDE. In order to regulate these genes <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) binds to the mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>). The manganese homeostasis is also controlled by the manganese riboswitch <i>mntPrb</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K90274</a>). The sequences of the <i>mntP</i> promoter and the <i>mntP</i> riboswitch was obtained from the Waters et al, 2011.</p><br />
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[[File:Ucalgary2012 KillswitchstuffsystemsAandB.png|thumb|800px|left|Figure 1: '''A)''' MgtA pathway in <i>E. coli</i>. <i>PhoQ</i> is the transmembrane receptor which, upon detecting low magnesium concentrations, phosphorylates <i>PhoP</i> which acts as a transcription factor, transcribing genes downstream of the MgtA promoter necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript, thus inhibiting translation. '''B)''' In the presence of manganese, the <i>MntR</i> protein represses the <i>mntH</i> transporter, preventing the movement of manganese and also upregulating the putative efflux pump. Genes downstream of the mntP promoter are thus transcribed in the presence of manganese. The addition of the <i>MntR</i> protein in this system allows for tighter regulation of the system.]]<html><br />
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<h2> The Moco Riboswitch </h2><br />
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<p>The molybdenum cofactor (moco) riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>) is an RNA element which responds to the presence of the metabolite molybdenum cofactor(MOCO) (Regulski et al, 2008). This RNA element is located in the <i>E.coli</i> genome just upstream of the <i>moaABCDE</i> operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>), which contain the important moco synthesis genes. Moco is an important co-factor in many different enzymes. The moco riboswitch has 2 regions: an aptamer domain and the expression platform. When moco is present in the cell it will bind to the aptamer region in the riboswitch causing an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site which prevents translation.</p><br />
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[[File:Moco_riboswitchCalgary2012.jpg|thumb|750px|center|Figure 2: This picture depicts the Moco RNA motif which is upstream of the <i>moaABCDE</i> operon. ]]<html> <br />
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<h2> Building the Systems </h2><br />
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<p> Using these riboswitches, we wanted to design a system where we would place our kill genes downstream, and then supplement our bioreactor with the appropriate ions to keep the systems turned off. We biobricked and submitted DNA for the the <i>mgtaP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>) as well as their respective riboswitches (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902008</a>) (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K902074</a>) and the moco riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>). In addition, we also biobricked some of the regulatory proteins: <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>), <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>), <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) and the Moa Operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>) . Our final system would inovolve constitutive expression of these necessary regulatory elements upstream of our riboswitches and kill genes. An example of the manganese system is shown in figure 3. </p><br />
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</html>[[File:U.Calgary.2012_10.02.2012_Final_Construct_1.png|thumb|600px|center|Figure 3: Final construct for the manganese system. The circuit includes a TetR promoter, RBS, mntR, double terminator, mntP promoter, mntP riboswitch, <i>S7</i>, mntP riboswitch and <i>CViAII</i>.]]<html><br />
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<h2> Characterizing the riboswitches </h2><br />
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<h3> GFP testing</h3><br />
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</html>[[File:MgtA circuits Ucalgary1.png|thumb|150px|right|Figure 4: In these set of circuits, <i>TetR</i>-RBS-K082003 serves as a positive control and the <i>mgtAp-mgtArb</i> serves as a negative control.]]<html><br />
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<p> In order to test the control of these promoters and riboswitches, we constructed them independently and together upstream of GFP (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K082003">BBa_K082003</a>) with an LVA tag. Figure 4 shows these circuits for the mgtA system. Identical circuits were designed for all three systems, however only the top two were needed for the mocoriboswitch system.</p><br />
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<p>We then tested the aforementioned circuits by growing cells containing our circuits with varying concentrations of their respective ions. Our detailed protocols can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">here</a>. We then measured fluorescent output, normalizing to a negative control not expressing GFP.</p><br />
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<h3> Results </h3><br />
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<p>So far, we have been able to obtain results for our magnesium system, as can be seen in figure 5. </html><br />
[[File:Magmesium graph ucalgary2.png|thumb|500px|left|Figure 5: This graph represents the relative fluorescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html></p><br />
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<p>As the graph shows, there is a much larger decrease in the GFP output when the mgtA promoter and riboswitch are working together as compared to the <i>mgtA</i> riboswitch alone under the control of TetR promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_J13002">BBa_J13002</a>). This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that the magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.</p><br />
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<p> It is important to consider however that the control elements of the system, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010"><i>PhoP</i> </a> and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011"> <i>PhoQ</i></a>, that were described above were not present in the circuits tested and therefore there is GFP expression in at the inhibitory concentration (10mM MgCl<sub>2</sub>). We believe that having the regulatory elements would give us better control and limit the leakiness.</p><br />
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<p>Although the magnesium system is highly regulated, it is not a suitable system for the purposes of our bioreactor. The tailings are composed of very high concentration of magnesium, as high as 120mM (Kim <i>et al</i>. 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria were to escape into the tailings, the kill genes would not be activated and the bacteria would be able to survive. However, we feel that this could still be an incredibly useful system for other teams for both killswtitch and non-killswitch-related applications, making it still a valuable contribution to the registry. </p><br />
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<h3> Kill Gene Testing </h3><br />
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<p> While building our systems with GFP in order to test their control, we also constructed them with our kill genes. This was delayed substantially however due to problems in their synthesis. Specifically, the micrococcal nuclease that arrived from IDT had a 1bp point mutation which changed a isoleucine residue into a lysine. Initially, our systems resulted in no killing of cells. Therefore we had to mutate this residue using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis"> site-directed mutagenesis</a>. Once completed, we were able to begin testing. With our GFP data collected, we moved on to characterizing the mgtA control system upstream of our <i>S7</i> kill gene (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902019">BBa_K902019</a>). To test the circuits, we incubated cells expressing our construct with varying concentrations of magnesium. We then measured both Colony Forming Units (CFU) and OD 600. For a deatiled protocol, see <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">here</a>.</p><br />
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<h3> Results </h3><br />
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</html>[[File:24 hour assay with mgtap-rb-S7 Ucalgary.png|thumb|750px|center| Figure 6: This shows the OD600 values of mgtA circuits with S7 both mutated and unmutated. The negative control consists of <i>mgtAp-mgtArb</i>.]]<html><br />
<p> Figure 6 shows that the mgtAp-mgtArb-S7 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902018">BBa_K902018</a>) starts acting approximately 4 hours after induction. However, it also shows that 10mM MgCl<sub>2</sub> is not enough salt to inhibit the entire system because there is no difference in OD600 measurement at 4hr time point between 10mM and the 0mM concentrations. This test needs to be repeated with higher concentrations of Mg<sup>2+</sup> however this data suggests that the mutagenesis was successful and <i>S7</i> is active and killing the cells at approximately 4hr which does not necessarily reflect solely upon the activity of <i>S7</i> but also on the response time of the mgtA system.</p><br />
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<h2>An alternative: a glucose repressible system</h2><br />
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<p>Based on the problem with the magnesium system in relation to tailings pond conditions, we wanted to find an alternative (other than the manganese and moco systems, which require further testing). We found a promoter in the literature that was induced by rhamnose and repressed by glucose. This seemed to be a very suitable candidate for controlling the kill switch in the bioreactor since the promoter was shown to be tightly repressed by glucose and rhamnose is fairly inexpensive and not found in high concentrations in tailings ponds. We could supplement the bioreactor with glucose to inhibit expression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds would cause expression of the kill genes since glucose levels in the surrounding environment would be insufficient for deactivate the promoter.<br />
</p> <br />
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<p>This promoter, known as <i>pRha</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>), is responsible for regulating six genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see figure 7). <i>RhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) and <i>RhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) serve to regulate expression of the rhamnose metabolism genes <i>rhaB</i>, <i>rhaA</i>, and <i>rhaD</i> on the opposite side of the promoter. The <i>RhaR</i> transcription factor is activated by L-rhamnose to up-regulate expression <i>rhaSR</i> operon. In turn, the resulting <i>RhaS</i> activates the <i>rhaBAD</i> operon to generate the rhamnose metabolism genes (Egan & Schleif, 1993).</p><br />
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</html>[[File:NativeRhamnosePromoter_Calgary2012.jpg|thumb|750px|center|Figure 7: The rhamnose metabolism genes as they exist in Top Ten <i>E. coli</i>]]<br />
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<p>As a kill switch regulator, our team has harnessed global catabolite repression of the rhamnose promoter. Expression of the <i>rhaBAD</i> operon with <i>RhaS</i> requires the binding of catabolite receptor protein (CRP) cAMP complex to the promoter. When glucose is present, cAMP levels are low, such that CRP is unable to activate the promoter (Egan & Schleif, 1993). With this mechanism, our team set out to control our kill genes with glucose via the following rhamnose promoter construct:</p><br />
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<p>Our kill system is different from the native rhamnose system with the <i>rhaR</i> and <i>rhaS</i> control genes. We have constituitively expressed <i>RhaS</i> to overcome dependency on rhamnose to cause activation of the kill switch. While <i>RhaS</i> is continuously present, global catabolite repression prevents activation of the kill genes in the bioreactor, however in the outside environment glucose levels are lower such that <i>RhaS</i> is able to activate the kill genes.</p><br />
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<h3>Building the system</h3><br />
<p>Our team had <i>pRha</i> promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>) commercially synthesized as per the sequence given by Jeske and Altenbuchner (2010). The <i>rhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) and <i>rhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) genes were amplified via PCR from Top Ten <i>E. coli</i> using Kapa HiFi polymerase. </p><br />
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<p>Given the control gene modifications which we have engineered into our system to optimize it for the tailings ponds, we are working to determine whether glucose repression of our modified system can match patterns shown by Giacalone <i>et al</i>. (2006) and Jeske and Altenbuchner (2010). To this end, we have constructed the rhamnose promoter with GFP (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902066">BBa_K902066</a>) and are finalizing construction with constituitively expressed <i>RhaS</i>, so that we can characterize this promoter and test it in combination with our kill genes.</p><br />
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}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-04T02:25:08Z<p>Stephanie0101: </p>
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<h2>Toxins In Our Environment</h2><br />
<p>During petroleum extraction and refinement, many toxic compounds are produced. These have become a huge problem in our society resulting in land, water, and air contamination. The toxins consist of a variety different types of compounds. Air contaminants consist of NO<sub>x</sub> (nitrogen containing compounds) and SO<sub>x</sub> (sulfur containing compounds) which contribute to a variety of environmental issues including green house gas accumulation and acid rain (Schneider, 2006; Environmental protection agency, 1999). Similarly water contaminants often consist of complex mixtures of compounds including highly toxic phenols and aromatic compounds, corrosive and additionally toxic carboxylic acid containing compounds, sulfur, and nitrogen containing compounds. These often have complex structures and cause acute toxicity to wild life. Classical examples of water contamination include tailing ponds produced from the oil extraction process. Finally, land areas can become contaminated as a result of these toxic compounds leaching into ground water sources, spills or accidental release of waste products into the environment, and other ways. </p><br />
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</html>[[Image:Calgary_EnviroToxins.jpg|thumb|600px|center|Figure 1: Environmental toxins contaminate air, water, and land masses. These can consist of various compounds which could be divided into sulfur, nitrogen, carboxylic acid, and phenolic based compounds. What can we do to solve this problem?]]<html><br />
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<h2>Synthetic Biology As A Platform For Remediation</h2><br />
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<p>Presently, there are a variety of solutions to remove these compounds from the environment. This has been facilitated by stricter regulations from government bodies as well as better chemical and physical techniques for reducing the release of these compounds. Toxins which are released into the environment can be removed using various chemical agents or by physically removing contaminated soil or water areas and storing these products in contained areas(Scott <i>et al</i>. 2005). However there is still no efficient, environmentally friendly mechanism for this to occur. <b>What needs to occur in order to better remediate these toxin's from the environment?</b> We require a method to be able to easily detect where toxins are and also have a system for remediating them. Microorganisms in the environment have evolved to be able to do both of these functions, responding to compounds in their environment and transform them into other products. Therefore we can harness these abilities to apply synthetic biology to these problems.</p><br />
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<p><b>What if we could detect toxins in our environment using a synthetically engineered organism? What if we could use a second organism to take these compounds and not only <u>degrade</u> them but convert them into <u>useful</u> compounds like hydrocarbons!</b></p><br />
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<h2>Introducing...</h2><br />
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</html>[[File:Calgary FredandOscarDef.jpg|thumb|600px|center|Figure 2: Introducing our dynamic duo FRED and OSCAR! This biosensor/bioreactor team is ready to detect and remediate toxins in the environment. Not only can OSCAR break down toxic carboxylic acid containing compounds such as naphthenic acids, but we also demonstrated that he can turn them into functional hydrocarbons!]]<html><br />
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<p><br />
We would like to introduce FRED and OSCAR! Our dynamic biosensor/bioreactor duo designed to be able to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and also be able to convert these toxic components into useable hydrocarbons. FRED or the Functional Robust Electrochemical Detector, is capable of detecting various toxic components through an electrochemical response at the same time. We illustrated how this sensor could work by showing that it has the potential to detect toxins in contaminated water. Additionally, we developed a minaturized circuit for a prototype, validated that this device worked in the wetlab, and designed our own software available to everyone to be used with a home made potentiostat. <br />
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<p><br />
OSCAR or the Optimized System for Carboxylic Acid Remediation is designed specifically to target naphthenic acids (NAs) the carboxylic acid containing compounds in the tailing ponds for their degradation. Using the PetroBrick (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025</a>) we were able to convert various different naphthenic acid based compounds into their hydrocarbon analogs. Additionally, we wanted to be able to degrade other toxic components of tailings so we used the <i>xylE</i> gene in order to cleave catechol, an abundant intermediate in many toxic areas. Not only did we set out to break down catechol but we attempted to see if we could further reduce the toxicity of the catechol breakdown product through use of the petrobrick. <b>FILL THIS IN DEPENDENT ON CATECHOL ASSAY DATA TONIGHT!</b><br />
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<h2>Taking A Step Back - Human Practices Inspired Our Project!</h2><br />
<img src="https://static.igem.org/mediawiki/2012/1/17/UCalgary2012_FRED_and_OSCAR_HP.png" style="float: right; width: 280px; padding: 10px;"></img><br />
<p>Before starting our project, the Calgary iGEM team felt it would be important to answer a few questions about how FRED and OSCAR could be applied into the oil and gas sector.</p> <br />
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<p><b>Would oilsands industry be interested in a biosensor and bioreactor for remediation purposes?</b> Yes! In fact, our meeting with the Oilsands Leadership Initiative (OSLI) has led us to believe that industry is interested in potentially using synthetic biology for remediation of toxins.</p> <br />
<p><b>What would people think about using synthetic biology<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> in the oilsands? Do they have any concerns about its implementation?</b> We went to talk to two professionals related to biotechnology and ecological development in Alberta. Both of them made it clear that while the concept sounds great its important that we keep in mind the safety and ethics of our project.</p> <br />
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<p><b>How can OSCAR and FRED be designed with safety in mind?</b> From our various conversations our team looked towards both physical <img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_FRED_and_OSCAR_Design.png" style="float: right; padding: 10px; width: 250px;"></img>and genetic design considerations to ensure that both FRED and OSCAR were designed form the beginning in a safe and functional way. This involved developing biosensor and bioreactor containment devices as well as kill switch.</p> <br />
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<p><b>How can we teach people more about FRED, OSCAR, and Synthetic Biology?</b> From our interviews it was clear that not many people knew much about synthetic biology or its applications in the oil and gas sector. For this we partnered with the Telus Spark Centre, the local Science Centre in Calgary to help communicate synthetic biology to them. We also developed a video game that we took to the centre and better educated adults and kids on synthetic biology! </p><br />
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}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Project/OSCAR/DesulfurizationTeam:Calgary/Project/OSCAR/Desulfurization2012-10-04T02:17:09Z<p>Stephanie0101: </p>
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<h2>Why Remove Sulfur?</h2><br />
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<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 />
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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 />
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<h2>Our Approach</h2><br />
<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 pocedure</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 eight compounds by our <i>Rhodococcus</i> strain (figures 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 GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GCMS after 6 days of being in the incubator. ]]<html></p<br />
<br />
<p></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 3: 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 />
<p></html>[[File:Ucalgary2012 DBT GCMS bargraph.PNG|center|650px|thumb|Figure 4: <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 GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GCMS after 6 days of being in the incubator. Comparing the control with the sample incubated for 6 days shows that the presence of bacteria increases the degradation rate by about ten times.]]<html></p><br />
<br />
<br />
<p></html>[[File:Ucalgary2012DesulfurizationTetrahydrdegradation.png|center|600px|thumb|Figure 5: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM compound with no other sulfur containing compounds (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 DBT. The control only contained modified M9 but no bacteria and it was run through the GCMS at the last time point. Degradation is seen, indicating that the pathway has wider substrate specificity than previously thought.]]<html></p><br />
<br />
<p></html>[[File:UcalgaryBenzothiopenecarbozyaldehydedegradation.png|center|600px|thumb|Figure 6: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM compound with no other sulfur containing compounds (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 DBT. The control only contained modified M9 but no bacteria and it was run through the GCMS at the last time point. Degradation is seen, indicating that the pathway has wider substrate specificity than previously thought.]]<html></p><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. It was also shown that a point mutation changing <i>dszB</i>'s 63rd amino acid from Y to F increases the activity of the protein (Oshiro <i>et al</i>., 2007). This mutation was also included in the mass mutagenesis we undertook. Mutagenesis was performed as per <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">this protocol.</a></p><br />
<br />
<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>pLacI-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 pLacI and pLacI-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>pLacI-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 pLacI-hpaC and pLacI-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. pLacI-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the pLacI-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>pLacI-RBS-hpaC</i></a> was functional and that it would effectively recycle FMN.</p><br />
<br />
<br />
<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 />
<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>, <i>dszC</i>, <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>Stephanie0101http://2012.igem.org/Team:Calgary/Project/OSCAR/DesulfurizationTeam:Calgary/Project/OSCAR/Desulfurization2012-10-04T02:11:48Z<p>Stephanie0101: </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 />
<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 pocedure</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 eight compounds by our <i>Rhodococcus</i> strain (figures 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 GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GCMS after 6 days of being in the incubator. ]]<html></p<br />
<br />
<p></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 3: 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 />
<p></html>[[File:Ucalgary2012 DBT GCMS bargraph.PNG|center|650px|thumb|Figure 4: <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 GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GCMS after 6 days of being in the incubator. Comparing the control with the sample incubated for 6 days shows that the presence of bacteria increases the degradation rate by about ten times.]]<html></p><br />
<br />
<br />
<p></html>[[File:Ucalgary2012DesulfurizationTetrahydrdegradation.png|center|600px|thumb|Figure 5: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM compound with no other sulfur containing compounds (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 DBT. The control only contained modified M9 but no bacteria and it was run through the GCMS at the last time point. Degradation is seen, indicating that the pathway has wider substrate specificity than previously thought.]]<html></p><br />
<br />
<p></html>[[File:UcalgaryBenzothiopenecarbozyaldehydedegradation.png|center|600px|thumb|Figure 6: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM compound with no other sulfur containing compounds (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 DBT. The control only contained modified M9 but no bacteria and it was run through the GCMS at the last time point. Degradation is seen, indicating that the pathway has wider substrate specificity than previously thought.]]<html></p><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. It was also shown that a point mutation changing <i>dszB</i>'s 63rd amino acid from Y to F increases the activity of the protein (Oshiro et al., 2007). This mutation was also included in the mass mutagenesis we undertook. Mutagenesis was performed as per <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">this protocol.</a></p><br />
<br />
<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>pLacI-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 pLacI and pLacI-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 et al., 2010) was measured over time. Both native HpaC expression and an induced <a href="http://partsregistry.org/Part:BBa_K902058"><i>pLacI-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 pLacI-hpaC and pLacI-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. pLacI-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the pLacI-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>pLacI-RBS-hpaC</i></a> was functional and that it would effectively recycle FMN.</p><br />
<br />
<br />
<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 et al., 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 />
<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>, <i>dszC</i>, <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 />
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}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Project/OSCAR/DecarboxylationTeam:Calgary/Project/OSCAR/Decarboxylation2012-10-04T02:05:50Z<p>Stephanie0101: </p>
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<h2>Why Decarboxylation?</h2><br />
<br />
<p>Among the toxins found in the tailing ponds, naphthenic acids (NAs) are among the most harmful and the most common. Though there is great diversity within the NAs class of compounds, all share the common chemical feature of a carboxylic acid group. The carboxyl group is the primary cause for their toxicity, allowing these chemicals to traverse cell membranes <br />
and react with cellular materials (Frank <i>et al</i>. 2009). NAs are recalcitranct (not easily degraded), potentially harmful to the surrounding ecosystem (Clemente & Fedorak, 2005) and corrosive to extraction and transport equipment of petroleum materials (Slavcheva <i>et al</i>. 1999). Corrosion of pipelines leads to higher maintenance costs as well as the grim possibility of these and other toxins leaking into the environment. <br />
There is a need for methods to degrade NAs that are not prohibitively expensive or that would result in production of other hazardous chemicals.</p> <br />
<br />
<p>The main goal of OSCAR is to turn toxins like these into useable hydrocarbons by removing the carboxylic acid group(s) (Behar & Albrecht, 1984). <br />
<br />
Since NAs from petroleum deposits are a variable mixture, an enzymatic process with broad<br />
specificity is necessary. With the removal of the carboxylic acid moiety, we aim to produce alkanes suitable for use as fuel. The goal of this subproject was to find one or more suitable pathways to accomplish the decarboxylation of compounds such as NAs with the broadest specificity.</p><br />
<br />
<br />
<h2>The PetroBrick</h2><br />
<br />
<p>The 2011 Washington iGEM team developed the PetroBrick (<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025.</a>), a BioBrick consisting of two primary genes. These include acyl-ACP reductase (<i>AAR</i>), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (<i>ADC</i>), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain (Sukovich, 2010). Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that the fatty acids that the PetroBrick targets, have a very similar structure to NAs.</p><br />
<br />
</html>[[File:UCalgary-Fatty-Acids-vs-NAs.jpg|550px|centre|thumb|Figure 1. A comparison of the structure of fatty acids and naphthenic acid]]<html><br />
<br />
<br />
<br />
<p>This lead us to believe that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the Petrobrick, we needed to verify that the Petrobrick would work in our hands as it did for the 2011 Washington team. <br />
<br />
Figures 2 and 3 demonstrate the function of the Petrobrick.</p><br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 2: Gas Chromatograph demonstrating the differences in peak composition between an <i>E.coli</i> control and the Petrobrick. There was a large increase in a peak with a retention time of 12.25 min. suggesting that the Petrobrick was producing a new compound.|650px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center|thumb|Figure 3: Mass Spectra of the gas chromatograph peak at 12.25 min. The spectra suggests that the Petrobrick is selectively producing a C15 alkane. This is what was expected as determined by the Washington 2011 iGEM team.|750px]]<html><br />
<br />
<p>With the Petrobrick shown to be able to successfully produce alkanes, it was time to test it out on NAs, to see if <br />
they could be selectively converted into alkanes! This experiment used commercially available NAs fractions including a large number of different complex NAs compounds. </p><br />
<br />
<h2>Successful conversion of NA's into Hydrocarbons!</h2><br />
<br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 4: The relative intensity of alkane production over a retention time in both <i>E.coli</i> that contain the PetroBrick, and in <i>E.coli</i> that are lacking the PetroBrick, as measured with GC-MS. NAs were used as a substrate. A NA standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 5: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from <i>E.coli</i> containing the PetroBrick as in Figure 2, using NAs as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
<br />
<p> The above graphs indicate that hydrocarbons were successfully produced from <i>E.coli</i> that contained the PetroBrick plasmid, as analysed with GC-MS. In Figure 2, <i>E.coli</i> containing the PetroBrick had significantly higher hydrocarbon peaks than in a control of <i>E.coli</i> that did not contain the PetroBrick plasmid. Not only was the PetroBrick able to degrade NAs into alkanes, but it was also able to produce alkenes as shown by Figure 3, indicating that the PetroBrick worked how we had expected it to! </p><br />
<br />
<br />
<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)- Can we do better?</h2><br />
<br />
<p>Although we were successful using the PetroBrick to remove carboxyl groups from NAs, we wanted to improve on our results to see if we could get a higher yield or possibly target other compounds. One of our original fears in using the PetroBrick to decarboyxlate NAs was that the first enzyme AAR was reported to be highly specific for fatty acids bound to <i>ACP</i>. We had concerns about its compatibility with NAs and therefore sought another enzyme in the literature called carboxylic acid reductase (CAR) that was documented to perform a similar task as AAR, converting fatty acids to aldehydes, but with much lower specificity (He <i>et al</i>. 2004). This enzyme, from <i>N. iowensis</i> does not require covalent attachment to ACP so would likely be much broader in substrate specificity. It requires a second gene from <i>N. iowensis</i>, called Nocardia phosphopantetheinyl transferase (NPT) necessary to append a 4’- phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian et al, 2006).</p><br />
<br />
</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|450px|thumb|Figure 6. Mechanism of action of CAR]]<html><br />
<br />
<p>Another enzyme with the potential to remove carboxyl groups from NAs is olefin-forming fatty acid decarboxylase (<i>OleT</i>) from <i>Jeotgalicoccus</i> sp. ATCC 8456. This is a decarboxylase of the cytochrome P450 family that acts on fatty acids, but has also been documented to have low substrate specificity (Rude <i>et al</i>. 2011). What was attractive with this was that it was one single enzyme that go do the job of the PetroBrick! Now that we knew that our decarboxylation approach was valid, it was time to start testing and comparing this gene to the PetroBrick.</p><br />
<br />
<h2> Progress so far </h2><br />
<br />
<p>Genes <i>car</i> and <i>npt</i> were cloned from the host organism <i>N. iowensis</i> (NRRL 5646). <i>car</i> was ligated into the pET vector and verified by a restriction digest while <i>npt</i> was cloned into pSB1C3(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902061">BBa_K902061</a>) and similarly verified.</p><br />
<br />
<p><i>CAR</i> was cloned into pET47b+ plasmid due to six illegal cut sites(one XbaI site, two EcoRI sites, and three NotI sites) which made it unsuitable for the BioBrick construction vectors. We first attempted to use a multi-site mutagenesis derived from the QuikChange® Multi Site Directed Mutagenesis Kit, but this showed little success. Instead, a more time-consuming but effective series of conventional single-site <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis procedure</a> was favoured, using the KAPA Hi-Fi polymerase. The XbaI and EcoRI sites were eliminated <br />
<br />
first so that <i>car</i> can be moved from the pET Vector and ligated into the PSB1C3 vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902062">BBa_K902062</a>). The <i>oleT</i> was successfully amplified from the <i>Jeotgalicoccus</i> sp. ATCC 8456.<p> <br />
</p><br />
<p>Like <i>car</i>, <i>oleT</i> was inserted in a pET47b+ (Novagen) vector before placing it into a BioBrick vector, as two illegal cut sites adjacent to one another needed to be mutagenized. This part is now being ligated into pSB1C3. We are currently in the process of constructing all three parts under control of a <i>tetR</i> promoter and ribosomal binding site (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>), and then constructing these composite parts together as outlined below.</p><br />
<br />
<h2>Final testing constructs</h2><br />
<br />
<p>Final testing constructs are almost complete. These are illustrated in figure 7 and will allow us to compare the three different approaches. Unfortunately, as Washington only sent us the PetroBrick and not the two individual components, we will have to compare a combination of the PetroBrick and <i>CAR/NPT</i> to the PetroBrick alone and to <i>OleT</i>. </p><br />
<br />
<p></html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|centre|600px]]<html></html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|centre|400px|thumb|Figure 7. Final constructs required for validating and comparing different decarboxylation approaches]]<html></p><br />
<br />
<h2> Testing OleT </h2><br />
<br />
<p>One major stumbling block in testing out <i>oleT</i> has been significant difficulty in trying to ligate it into a vector, which has prevented us from submitting it as a BioBrick. As such, we chose to try some assays on the host organism: <i>Jeotgalicoccus</i> sp. ATCC 8456. This way we could at least validate that this gene was functional before we had our BioBricks. We started by trying to verify what was found by Rude <i>et al</i>., 2011, namely that this enzyme could convert fatty acids into alkenes. To do so, we grew up cultures according to this <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT_in_Validation_Assay">protocol</a> and used and GC-MS in order to analyze any alkene production. Our results are shown below. </p><br />
<br />
<h2> Formation of alkanes by <i>Jeotgalicoccus</i> sp. ATCC 8456</h2><br />
<br />
</html>[[File:UofC_OleT_Assay_1.png|centre|650px|thumb|Figure 8. Gas chromatograph demonstrating the production of olefins (alkenes) from fatty acids as shown from the increase in the peak with a retention time of 14.7 min. The dramatic change in peak intensity at this point suggests that we are producing hydrocarbons.]]<br />
[[File:UofC_OleT_2nd_Assay.png|centre|650px|thumb|Figure 9. Mass spectra of the peak in Figure 8 at retention time 14.7 min. Demonstrating that this peak is an olefin which was previously suggested to be produced using this species of bacteria. This provides a proof of concept that the <i>Micrococcus</i> species we have can degrade fatty acids into olefins. ]]<html><br />
<br />
<p>Based on the additional peak we saw in the gas chromatograph, we were able to show that our organism was indeed able to produce alkenes, as expected. This is possible improvement over the PetroBrick as it uses only one enzyme instead of two, however future testing is still needed. Now that we have validated the functionality of this enzyme in producing alkenes, the next step is to test it out on complex naphthenic acids in order to compare it to the PetroBrick. This testing is still underway.</p><br />
<br />
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}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Project/OSCAR/UpgradingTeam:Calgary/Project/OSCAR/Upgrading2012-10-04T01:59:55Z<p>Stephanie0101: </p>
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<p><br />
While we have been able to show that we can convert toxins such as naphthenic acids into useful hydrocarbons there is still a lot of compounds in the tailings that will decrease the value of our product as a fuel. These compounds include sulfur and nitrogen containing heterocyclic atoms such as dibenzothiophene (DBT) and carbazole. Normally these compounds are removed through chemical means, but this requires expensive machinery, extreme pressurs and temperatures, and the addition of organic solvents. By using synthetic biology to do this in a simple bioreactor we can save the energy that is required to produce these conditions and reduce the cost of the expensive processes.</p><br />
<br />
<h2>Why Use Synthetic Biology? Why Not Chemical Methods?</h2><br />
<br />
<p>The most widely used chemical method for removing nitrogen from fuel sources is called hydrodenitrogenation (HDN). This process is not very efficient, as only 77% of nitrogen containing compounds are actually removed (Zeuthen <i>et al</i>. 2001). It also requires harsh conditions, for example temperatures upwards of 350&deg; C and pressures up to 30 Bar. This is mostly because the nitrogen atoms in the ring must be hydrogenated before the carbon-nitrogen bond can be cleaved because this (Katzer & Sivasubramanian, 1979). The input of molybdenum based chemical catalysts required for this reaction is also very costly and can produce toxic by-products of its own (Zhu <i>et al</i>. 2008). Desulfurization has similar problems. Hydrodesulfurization for example, requires high temperature and pressure and also releases the sulfur in the form of hydrogen sulfide gas, a toxic compound which is then converted to elemental sulfur or sulfuric acid.</p><br />
<br />
<p></html>[[File:Ucalgary2012 upgradingOscarinapot.png|centre]]<html><br />
<br />
<br />
<br />
<p>In contrast to this, microbes have been found capable of harvesting both sulfur and nitrogen out of hydrocarbons under physiological conditions, making them a much more environmentally sound and economic approach to carry out on an industrial scale. Synthetic biology can potentially offer a new avenue to address these problems, with much more potential for innovation and new ideas. </p><br />
<br />
<p>We took two main approaches for upgrading our hydrocarbons. The first method focused on reducing the sulfur content of the product through the action of <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">desulfurization</a>. This would prevent the production of toxic sulfur gasses when the hydrocarbons are combusted in an engine. Our second approach uses <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">denitrogenation</a> to remove nitrogen containing compounds from the product. This will reduce the emissions of nitrous oxide gasses that are heavily involved in global warming.</p><br />
<br />
<h2>Click on OSCAR to learn more about what he can do!</h2><br />
<br />
<a style="margin-left: 20px;" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation"><img src="https://static.igem.org/mediawiki/2012/7/71/Calgary2012_Upgrading_Nitrogen.png"></img></a> <br />
<a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization"><img src="https://static.igem.org/mediawiki/2012/3/37/Calgary2012_Upgrading_Sulfur.png"></img></a><br />
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}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradationTeam:Calgary/Project/OSCAR/CatecholDegradation2012-10-04T01:55:11Z<p>Stephanie0101: </p>
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<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_OSCAR_Catechol_Low-Res.png" style="float: right; padding: 10px;"></img><br />
<br />
<br />
<p>Catechol is a toxic compound found in tailings ponds that is a by-product of polyaromatic hydrocarbon metabolism (Vaillancourt <i>et al.</i>, 2006, Schweigert <i>et al.</i>, 2001)). The chemical properties of catechol allow it to react with biomolecules, causing cellular damage including DNA damage, enzyme inactivation and membrane uncoupling (Schweigert <i>et al.</i>, 2001). </p><br />
<p><br />
Catechol is characterized as having a benzene ring with two hydroxyl groups at the 2,3 position. It can be converted to 2-hydroxymuconic acid by the enzyme catechol 2,3-dioxygenase, encoded by the <i>xylE</i> gene on the Tol plasmid of <i>Pseudomonas putida</i> (Nakai <i>et al.</i>, 1983).</p><br />
<br />
<p><br />
Currently the registry has two BioBricks available of <i>xylE</i>. One contained <i>xylE</i> with its native ribosome-binding site (<a href=http://partsregistry.org/Part:BBa_J33204>BBa_J33204</a>), while the other part contained <i>xylE</i> under the glucose-repressible promoter <i>cstA </i>(<a href=http://partsregistry.org/Part:BBa_K118021>BBa_K118021</a>). Given that <i>E. coli</i> is grown in the presence of glucose, we designed a new construct to keep <i>xylE</i> repressed by using the <i>tetR</i> promoter (<a href= http://partsregistry.org/Part:BBa_R0040>BBa_R0040</a>).</p> <br />
<br />
</html>[[File:UCalgary2010_R0040-XylE.png|400px|thumb|Figure 1: BioBrick genetic circuit for catechol degradation showing <i>xylE</i> under the ''tetR'' promoter|center]]<html><br />
<h3></h3><br />
<br />
<p>Catechol 2,3-dioxygenase is an extradiol dioxygenase which cleaves catechol adjacent to the two hydroxyl groups. When this occurs 2-hydroxymuconate semialdehyde is produced, which is yellow in colour. This change in colour allows for visual assay to assess the activity of XylE.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_to_2-HMS.PNG|400px|thumb|Figure 2: Catechol 2,3-dioxygenase (XylE) converts catechol to 2-Hydroxymuconate semialdehyde in the presence of oxygen. Adapted from Shu <i>et al</i>., 1995.|center]]<html><br />
<br />
<p>The visual assays were performed with <i>E. coli</i> cells transformed with (<a href=http://partsregistry.org/Part:BBa_K118021>BBa_K118021</a>) as well as with <i>E. coli</i> cells transformed with the newly constructed part (<a href=http://partsregistry.org/Part:BBa_K902048 >BBa_K902048</a>) by bringing the supernatant of an overnight culture to a concentration of 0.1 M of catechol. When the part (<a href=http://partsregistry.org/Part:BBa_K118021>BBa_K118021</a>) was used, the pellet was first washed in M9-MM and centrifuged before catechol was added to the supernatant. This was necessary to avoid the glucose in the LB from repressing the cstA promoter (<a href=http://partsregistry.org/Part:BBa_K118011>BBa_K118011</a>). Catechol was added to the supernatant because the reaction takes place outside of the cell. Within minutes of the addition of catechol to the supernatant, the solution turned from the pale yellow of LB to a bright yellow. This was indicative that catechol was breaking down into 2-Hydroxymuconate semialdehyde, which was exactly what we expected! This assay was completed by following the protocol written by the 2008 Edinburgh iGEM team.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Figure 3: Results of the catechol visual assay using ''xylE'' [http://partsregistry.org/Part:BBa_K118021 BBa_K118021]. Cultures were grown overnight in LB and the pellets were washed with M9-MM at various times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). Cells were then spun down and catechol was added to the supernatant to 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E. coli</i> cells without the <i>xylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added. |center]]<html><br />
<br />
<h2> Converting Catechol into hydrocarbons? </h2><br />
<p>After verifying that we could in fact degrade catechol into 2-hydroxymuconate semialdehyde using our <i>xylE</i> construct (<a href=http://partsregistry.org/Part:BBa_J33204>BBa_J33204</a>), we wondered if we could take this any further. What if we could convert this by-product page into hydrocarbons too? As catechol is the breakdown product of a number of different degradation pathways, this could be particularly useful.</p><br />
<br />
<p>As 2-hydroxymuconate semialdehyde can be further metabolized to pyruvate and acetaldehyde (Harayama S et al., 1987), it seemed possible that these products could be routed into the fatty acid biosynthesis pathway and converted to alkanes using the PetroBrick or the <i>OleT</i> enzyme. Given that the Catechol 2,3-dioxygenase reaction is extracellular, it creates a possible scenario in which cells with the <i>xylE</i> construct could be co-cultured with Petrobrick-containing cells to cooperatively metabolise catechol into hydrocarbons. </p><br />
<br />
<p> In order to test this, we followed this <a href=https://2012.igem.org/Team:Calgary/Notebook/Protocols/decatecholization>protocol</a>, where we co-cultured cells expressing our <i>xylE</i> construct with either <i>E. coli</i> cells expressing the PetroBrick, or <i>Jeotgalicoccus sp. ATCC 8456</i> cells expressing <i>OleT</i>. in the presence of catechol.<br />
<br />
<br />
</html>[[File:Calgary PetrobrickCatechol.jpg|600px|thumb|centre|Figure 4: Gas chromatograph of catechol degradation assay using the PetroBrick. While there is limited differences between <i>xylE</i> incubated with and without the Petrobrick, there was one peak with a retention time of 10.5 min which was dramatically increased in the co-culture.]]<html><br />
<br />
</html>[[File:Calgary MSCatecholPetroPeak.jpg|600px|thumb|centre|Figure 5: Mass spectra of the Petrobrick/<i>xylE</i> co-culture retention peak at 10.5 min from Figure 3. While the identity of this compound is unknown to us presently, it is clear that there is changes occuring to some of the catechol breakdown products.]]<html><br />
<br />
</html>[[File:Calgary CatechololeTGC.jpg|600px|thumb|centre|Figure 6: Gas chromatograph of catechol degradation assay using <i>Micrococcus cadicans</i> a species of bacteria shown to be able to convert fatty acids into alkenes. This identified a similar peak change is in the Petrobrick with a retention time of 10.5 min. This provides additional support for the idea that the Petrobrick and this organism can further degrade catechol breakdown products.]]<html><br />
<br />
</html>[[File:Calgary CatecholMSoleT.jpg|600px|thumb|centre|Figure 7: Mass spectra of <i>Micrococcus</i>/<i>xylE</i> co-culture retention peak at 10.5 min from Figure 5. The similarity of this peak to the one found from the PetroBrick co-culture suggests that there may be similar breakdown products for both of these enzymes suggesting that we are capable of modifying catechol further than with <i>xylE</i> alone. We have yet to identify what this compound is.]]<html><br />
<br />
<br />
<br />
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</html>}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Project/OSCAR/DecarboxylationTeam:Calgary/Project/OSCAR/Decarboxylation2012-10-04T01:53:18Z<p>Stephanie0101: </p>
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<div>{{Team:Calgary/TemplateProjectBlue|<br />
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CONTENT=<html><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_OSCAR_Decarboxylation_Low-Res.png" style="float: right; padding: 10px; width: 300px;"></img><br />
<h2>Why Decarboxylation?</h2><br />
<br />
<p>Among the toxins found in the tailing ponds, naphthenic acids (NAs) are among the most harmful and the most common. Though there is great diversity within the NAs class of compounds, all share the common chemical feature of a carboxylic acid group. The carboxyl group is the primary cause for their toxicity, allowing these chemicals to traverse cell membranes <br />
and react with cellular materials (Frank <i>et al</i>. 2009). NAs are recalcitranct (not easily degraded), potentially harmful to the surrounding ecosystem (Clemente & Fedorak, 2005) and corrosive to extraction and transport equipment of petroleum materials (Slavcheva <i>et al</i>. 1999). Corrosion of pipelines leads to higher maintenance costs as well as the grim possibility of these and other toxins leaking into the environment. <br />
There is a need for methods to degrade NAs that are not prohibitively expensive or that would result in production of other hazardous chemicals.</p> <br />
<br />
<p>The main goal of OSCAR is to turn toxins like these into useable hydrocarbons by removing the carboxylic acid group(s) (Behar & Albrecht, 1984). <br />
<br />
Since NAs from petroleum deposits are a variable mixture, an enzymatic process with broad<br />
specificity is necessary. With the removal of the carboxylic acid moiety, we aim to produce alkanes suitable for use as fuel. The goal of this subproject was to find one or more suitable pathways to accomplish the decarboxylation of compounds such as NAs with the broadest specificity.</p><br />
<br />
<br />
<h2>The PetroBrick</h2><br />
<br />
<p>The 2011 Washington iGEM team developed the PetroBrick (<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025.</a>), a BioBrick consisting of two primary genes. These include acyl-ACP reductase (<i>AAR</i>), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (<i>ADC</i>), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain (Sukovich, 2010). Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that the fatty acids that the PetroBrick targets, have a very similar structure to NAs.</p><br />
<br />
</html>[[File:UCalgary-Fatty-Acids-vs-NAs.jpg|550px|centre|thumb|Figure 1. A comparison of the structure of fatty acids and naphthenic acid]]<html><br />
<br />
<br />
<br />
<p>This lead us to believe that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the Petrobrick, we needed to verify that the Petrobrick would work in our hands as it did for the 2011 Washington team. <br />
<br />
Figures 2 and 3 demonstrate the function of the Petrobrick.</p><br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 2: Gas Chromatograph demonstrating the differences in peak composition between an <i>E.coli</i> control and the Petrobrick. There was a large increase in a peak with a retention time of 12.25 min. suggesting that the Petrobrick was producing a new compound.|650px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center|thumb|Figure 3: Mass Spectra of the gas chromatograph peak at 12.25 min. The spectra suggests that the Petrobrick is selectively producing a C15 alkane. This is what was expected as determined by the Washington 2011 iGEM team.|750px]]<html><br />
<br />
<p>With the Petrobrick shown to be able to successfully produce alkanes, it was time to test it out on NAs, to see if <br />
they could be selectively converted into alkanes! This experiment used commercially available NAs fractions including a large number of different complex NAs compounds. </p><br />
<br />
<h2>Successful conversion of NA's into Hydrocarbons!</h2><br />
<br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 4: The relative intensity of alkane production over a retention time in both <i>E.coli</i> that contain the PetroBrick, and in <i>E.coli</i> that are lacking the PetroBrick, as measured with GC-MS. NAs were used as a substrate. A NA standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 5: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from <i>E.coli</i> containing the PetroBrick as in Figure 2, using NAs as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
<br />
<p> The above graphs indicate that hydrocarbons were successfully produced from <i>E.coli</i> that contained the PetroBrick plasmid, as analysed with GC-MS. In Figure 2, <i>E.coli</i> containing the PetroBrick had significantly higher hydrocarbon peaks than in a control of <i>E.coli</i> that did not contain the PetroBrick plasmid. Not only was the PetroBrick able to degrade NAs into alkanes, but it was also able to produce alkenes as shown by Figure 3, indicating that the PetroBrick worked how we had expected it to! </p><br />
<br />
<br />
<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)- Can we do better?</h2><br />
<br />
<p>Although we were successful using the PetroBrick to remove carboxyl groups from NAs, we wanted to improve on our results to see if we could get a higher yield or possibly target other compounds. One of our original fears in using the PetroBrick to <br />
decarboyxlate NAs was that the first enzyme <i>AAR</i> was reported to be highly specific for fatty acids bound to <i>ACP</i>. We had concerns about its compatibility with NAs and therefore sought another enzyme in the literature called carboxylic acid reductase (<i>CAR</i>) that was documented to perform a similar task as <i>AAR</i>, converting fatty acids to aldehydes, but with much lower specificity (He <i>et al</i>. 2004). This enzyme, from <i>N. iowensis</i> does not require covalent attachment to <i>ACP</i> so would <br />
likely be much broader in substrate specificity. It requires a second gene from <i>N. iowensis</i>, called Nocardia phosphopantetheinyl transferase (<i>NPT</i>) necessary to append a 4’- phosphopantetheine prosthetic group to <i>CAR</i> required for its full function (Venkitasubramanian et al, 2006).</p><br />
<br />
</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|450px|thumb|Figure 6. Mechanism of action of CAR]]<html><br />
<br />
<p>Another enzyme with the potential to remove carboxyl groups from NAs is olefin-forming fatty acid decarboxylase (<i>OleT</i>) from <i>Jeotgalicoccus</i> sp. ATCC 8456. This is a decarboxylase of the cytochrome P450 family that acts on fatty acids, but has also been documented to have low substrate specificity (Rude <i>et al</i>. 2011). What was attractive with this was that it was one single enzyme that go do the job of the PetroBrick! Now that we knew that our decarboxylation approach was valid, it was time to start testing and comparing this gene to the PetroBrick.</p><br />
<br />
<h2> Progress so far </h2><br />
<br />
<p>Genes <i>car</i> and <i>npt</i> were cloned from the host organism <i>N. iowensis</i> (NRRL 5646). <i>car</i> was ligated into the pET vector and verified by a restriction digest while <i>npt</i> was cloned into pSB1C3(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902061">BBa_K902061</a>) and similarly verified.</p><br />
<br />
<p><i>CAR</i> was cloned into pET47b+ plasmid due to six illegal cut sites(one XbaI site, two EcoRI sites, and three NotI sites) which made it unsuitable for the BioBrick construction vectors. We first attempted to use a multi-site mutagenesis derived from the QuikChange® Multi Site Directed Mutagenesis Kit, but this showed little success. Instead, a more time-consuming but effective series of conventional single-site <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis procedure</a> was favoured, using the KAPA Hi-Fi polymerase. The XbaI and EcoRI sites were eliminated <br />
<br />
first so that <i>car</i> can be moved from the pET Vector and ligated into the PSB1C3 vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902062">BBa_K902062</a>). The <i>oleT</i> was successfully amplified from the <i>Jeotgalicoccus</i> sp. ATCC 8456.<p> <br />
</p><br />
<p>Like <i>car</i>, <i>oleT</i> was inserted in a pET47b+ (Novagen) vector before placing it into a BioBrick vector, as two illegal cut sites adjacent to one another needed to be mutagenized. This part is now being ligated into pSB1C3. We are currently in the process of constructing all three parts under control of a <i>tetR</i> promoter and ribosomal binding site (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>), and then constructing these composite parts together as outlined below.</p><br />
<br />
<h2>Final testing constructs</h2><br />
<br />
<p>Final testing constructs are almost complete. These are illustrated in figure 7 and will allow us to compare the three different approaches. Unfortunately, as Washington only sent us the PetroBrick and not the two individual components, we will have to compare a combination of the PetroBrick and <i>CAR/NPT</i> to the PetroBrick alone and to <i>OleT</i>. </p><br />
<br />
<p></html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|centre|600px]]<html></html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|centre|400px|thumb|Figure 7. Final constructs required for validating and comparing different decarboxylation approaches]]<html></p><br />
<br />
<h2> Testing <i>OleT</i> </h2><br />
<br />
<p>One major stumbling block in testing out <i>oleT</i> has been significant difficulty in trying to ligate it into a vector, which has prevented us from submitting it as a BioBrick. As such, we chose to try some assays on the host organism: <i>Jeotgalicoccus</i> sp. ATCC 8456. This way we could at least validate that this gene was functional before we had our BioBricks. We started by trying to verify what was found by Rude <i>et al</i>., 2011, namely that this enzyme could convert fatty acids into alkenes. To do so, we grew up cultures according to this <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT_in_Validation_Assay">protocol</a> and used and GC-MS in order to analyze any alkene production. Our results are shown below. </p><br />
<br />
<h2> Formation of alkanes by <i>Jeotgalicoccus</i> sp. ATCC 8456</h2><br />
<br />
</html>[[File:UofC_OleT_Assay_1.png|centre|650px|thumb|Figure 8. Gas chromatograph demonstrating the production of olefins (alkenes) from fatty acids as shown from the increase in the peak with a retention time of 14.7 min. The dramatic change in peak intensity at this point suggests that we are producing hydrocarbons.]]<br />
[[File:UofC_OleT_2nd_Assay.png|centre|650px|thumb|Figure 9. Mass spectra of the peak in Figure 8 at retention time 14.7 min. Demonstrating that this peak is an olefin which was previously suggested to be produced using this species of bacteria. This provides a proof of concept that the <i>Micrococcus</i> species we have can degrade fatty acids into olefins. ]]<html><br />
<br />
<p>Based on the additional peak we saw in the gas chromatograph, we were able to show that our organism was indeed able to produce alkenes, as expected. This is possible improvement over the PetroBrick as it uses only one enzyme instead of two, however future testing is still needed. Now that we have validated the functionality of this enzyme in producing alkenes, the next step is to test it out on complex naphthenic acids in order to compare it to the PetroBrick. This testing is still underway.</p><br />
<br />
</html><br />
<br />
}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Project/OSCAR/DecarboxylationTeam:Calgary/Project/OSCAR/Decarboxylation2012-10-04T01:47:58Z<p>Stephanie0101: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Decarboxylation|<br />
<br />
CONTENT=<html><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_OSCAR_Decarboxylation_Low-Res.png" style="float: right; padding: 10px; width: 300px;"></img><br />
<h2>Why Decarboxylation?</h2><br />
<br />
<p>Among the toxins found in the tailing ponds, naphthenic acids (NAs) are among the most harmful and the most common. Though there is great diversity within the NAs class of compounds, all share the common chemical feature of a carboxylic acid group. The carboxyl group is the primary cause for their toxicity, allowing these chemicals to traverse cell membranes <br />
and react with cellular materials (Frank <i>et al</i>. 2009). NAs are recalcitranct (not easily degraded), potentially harmful to the surrounding ecosystem (Clemente & Fedorak, 2005) and corrosive to extraction and transport equipment of petroleum materials (Slavcheva <i>et al</i>. 1999). Corrosion of pipelines leads to higher maintenance costs as well as the grim possibility of these and other toxins leaking into the environment. <br />
There is a need for methods to degrade NAs that are not prohibitively expensive or that would result in production of other hazardous chemicals.</p> <br />
<br />
<p>The main goal of OSCAR is to turn toxins like these into useable hydrocarbons by removing the carboxylic acid group(s) (Behar & Albrecht, 1984). <br />
<br />
Since NAs from petroleum deposits are a variable mixture, an enzymatic process with broad<br />
specificity is necessary. With the removal of the carboxylic acid moiety, we aim to produce alkanes suitable for use as fuel. The goal of this subproject was to find one or more suitable pathways to accomplish the decarboxylation of compounds such as NAs with the broadest specificity.</p><br />
<br />
<br />
<h2>The PetroBrick</h2><br />
<br />
<p>The 2011 Washington iGEM team developed the PetroBrick (<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025.</a>), a BioBrick consisting of two primary genes. These include acyl-ACP reductase (<i>AAR</i>), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (<i>ADC</i>), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain (Sukovich, 2010). Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that the fatty acids that the PetroBrick targets, have a very similar structure to NAs.</p><br />
<br />
</html>[[File:UCalgary-Fatty-Acids-vs-NAs.jpg|550px|centre|thumb|Figure 1. A comparison of the structure of fatty acids and naphthenic acid]]<html><br />
<br />
<br />
<br />
<p>This lead us to believe that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the Petrobrick, we needed to verify that the Petrobrick would work in our hands as it did for the 2011 Washington team. <br />
<br />
Figures 2 and 3 demonstrate the function of the Petrobrick.</p><br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 2: Gas Chromatograph demonstrating the differences in peak composition between an <i>E.coli</i> control and the Petrobrick. There was a large increase in a peak with a retention time of 12.25 min. suggesting that the Petrobrick was producing a new compound.|650px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center|thumb|Figure 3: Mass Spectra of the gas chromatograph peak at 12.25 min. The spectra suggests that the Petrobrick is selectively producing a C15 alkane. This is what was expected as determined by the Washington 2011 iGEM team.|750px]]<html><br />
<br />
<p>With the Petrobrick shown to be able to successfully produce alkanes, it was time to test it out on NAs, to see if <br />
they could be selectively converted into alkanes! This experiment used commercially available NAs fractions including a large number of different complex NAs compounds. </p><br />
<br />
<h2>Successful conversion of NA's into Hydrocarbons!</h2><br />
<br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 4: The relative intensity of alkane production over a retention time in both <i>E.coli</i> that contain the PetroBrick, and in <i>E.coli</i> that are lacking the PetroBrick, as measured with GC-MS. NAs were used as a substrate. A NA standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 5: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from <i>E.coli</i> containing the PetroBrick as in Figure 2, using NAs as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
<br />
<p> The above graphs indicate that hydrocarbons were successfully produced from <i>E.coli</i> that contained the PetroBrick plasmid, as analysed with GC-MS. In Figure 2, <i>E.coli</i> containing the PetroBrick had significantly higher hydrocarbon peaks than in a control of <i>E.coli</i> that did not contain the PetroBrick plasmid. Not only was the PetroBrick able to degrade NAs into alkanes, but it was also able to produce alkenes as shown by Figure 3, indicating that the PetroBrick worked how we had expected it to! </p><br />
<br />
<br />
<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)- Can we do better?</h2><br />
<br />
<p>Although we were successful using the PetroBrick to remove carboxyl groups from NAs, we wanted to improve on our results to see if we could get a higher yield or possibly target other compounds. One of our original fears in using the PetroBrick to <br />
decarboyxlate NAs was that the first enzyme AAR was reported to be highly specific for fatty acids bound to ACP. We had concerns about its compatibility with NAs and therefore sought another enzyme in the literature called carboxylic acid reductase (CAR) that was documented to perform a similar task as AAR, converting fatty acids to aldehydes, but with much lower specificity (He <i>et al</i>. 2004). This enzyme, from <i>N. iowensis</i> does not require covalent attachment to ACP so would <br />
likely be much broader in substrate specificity. It requires a second gene from <i>N. iowensis</i>, called Nocardia phosphopantetheinyl transferase (NPT) necessary to append a 4’- phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian et al, 2006).</p><br />
<br />
</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|450px|thumb|Figure 6. Mechanism of action of CAR]]<html><br />
<br />
<p>Another enzyme with the potential to remove carboxyl groups from NAs is olefin-forming fatty acid decarboxylase (<i>OleT</i>) from <i>Jeotgalicoccus</i> sp. ATCC 8456. This is a decarboxylase of the cytochrome P450 family that acts on fatty acids, but has also been documented to have low substrate specificity (Rude <i>et al</i>. 2011). What was attractive with this was that it was one single enzyme that go do the job of the PetroBrick! Now that we knew that our decarboxylation approach was valid, it was time to start testing and comparing this gene to the PetroBrick.</p><br />
<br />
<h2> Progress so far </h2><br />
<br />
<p>Genes <i>car</i> and <i>npt</i> were cloned from the host organism <i>N. iowensis</i> (NRRL 5646). <i>car</i> was ligated into the pET vector and verified by a restriction digest while <i>npt</i> was cloned into pSB1C3(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902061">BBa_K902061</a>) and similarly verified.</p><br />
<br />
<p><i>CAR</i> was cloned into pET47b+ plasmid due to six illegal cut sites(one XbaI site, two EcoRI sites, and three NotI sites) which made it unsuitable for the BioBrick construction vectors. We first attempted to use a multi-site mutagenesis derived from the QuikChange® Multi Site Directed Mutagenesis Kit, but this showed little success. Instead, a more time-consuming but effective series of conventional single-site <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis procedure</a> was favoured, using the KAPA Hi-Fi polymerase. The XbaI and EcoRI sites were eliminated <br />
<br />
first so that <i>car</i> can be moved from the pET Vector and ligated into the PSB1C3 vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902062">BBa_K902062</a>). The <i>oleT</i> was successfully amplified from the <i>Jeotgalicoccus</i> sp. ATCC 8456.<p> <br />
</p><br />
<p>Like <i>car</i>, <i>oleT</i> was inserted in a pET47b+ (Novagen) vector before placing it into a BioBrick vector, as two illegal cut sites adjacent to one another needed to be mutagenized. This part is now being ligated into pSB1C3. We are currently in the process of constructing all three parts under control of a <i>tetR</i> promoter and ribosomal binding site (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>), and then constructing these composite parts together as outlined below.</p><br />
<br />
<h2>Final testing constructs</h2><br />
<br />
<p>Final testing constructs are almost complete. These are illustrated in figure 7 and will allow us to compare the three different approaches. Unfortunately, as Washington only sent us the PetroBrick and not the two individual components, we will have to compare a combination of the PetroBrick and <i>CAR/NPT</i> to the PetroBrick alone and to <i>OleT</i>. </p><br />
<br />
<p></html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|centre|600px]]<html></html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|centre|400px|thumb|Figure 7. Final constructs required for validating and comparing different decarboxylation approaches]]<html></p><br />
<br />
<h2> Testing <i>OleT</i> </h2><br />
<br />
<p>One major stumbling block in testing out <i>oleT</i> has been significant difficulty in trying to ligate it into a vector, which has prevented us from submitting it as a BioBrick. As such, we chose to try some assays on the host organism: <i>Jeotgalicoccus</i> sp. ATCC 8456. This way we could at least validate that this gene was functional before we had our BioBricks. We started by trying to verify what was found by Rude <i>et al</i>., 2011, namely that this enzyme could convert fatty acids into alkenes. To do so, we grew up cultures according to this <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT_in_Validation_Assay">protocol</a> and used and GC-MS in order to analyze any alkene production. Our results are shown below. </p><br />
<br />
<h2> Formation of alkanes by <i>Jeotgalicoccus</i> sp. ATCC 8456</h2><br />
<br />
</html>[[File:UofC_OleT_Assay_1.png|centre|650px|thumb|Figure 8. Gas chromatograph demonstrating the production of olefins (alkenes) from fatty acids as shown from the increase in the peak with a retention time of 14.7 min. The dramatic change in peak intensity at this point suggests that we are producing hydrocarbons.]]<br />
[[File:UofC_OleT_2nd_Assay.png|centre|650px|thumb|Figure 9. Mass spectra of the peak in Figure 8 at retention time 14.7 min. Demonstrating that this peak is an olefin which was previously suggested to be produced using this species of bacteria. This provides a proof of concept that the <i>Micrococcus</i> species we have can degrade fatty acids into olefins. ]]<html><br />
<br />
<p>Based on the additional peak we saw in the gas chromatograph, we were able to show that our organism was indeed able to produce alkenes, as expected. This is possible improvement over the PetroBrick as it uses only one enzyme instead of two, however future testing is still needed. Now that we have validated the functionality of this enzyme in producing alkenes, the next step is to test it out on complex naphthenic acids in order to compare it to the PetroBrick. This testing is still underway.</p><br />
<br />
</html><br />
<br />
}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Project/OSCAR/DecarboxylationTeam:Calgary/Project/OSCAR/Decarboxylation2012-10-04T01:43:12Z<p>Stephanie0101: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Decarboxylation|<br />
<br />
CONTENT=<html><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_OSCAR_Decarboxylation_Low-Res.png" style="float: right; padding: 10px; width: 300px;"></img><br />
<h2>Why Decarboxylation?</h2><br />
<br />
<p>Among the toxins found in the tailing ponds, naphthenic acids (NAs) are among the most harmful and the most common. Though there is great diversity within the NAs class of compounds, all share the common chemical feature of a carboxylic acid group. The carboxyl group is the primary cause for their toxicity, allowing these chemicals to traverse cell membranes <br />
and react with cellular materials (Frank <i>et al</i>. 2009). NAs are recalcitranct (not easily degraded), potentially harmful to the surrounding ecosystem (Clemente & Fedorak, 2005) and corrosive to extraction and transport equipment of petroleum materials (Slavcheva <i>et al</i>. 1999). Corrosion of pipelines leads to higher maintenance costs as well as the grim possibility of these and other toxins leaking into the environment. <br />
There is a need for methods to degrade NAs that are not prohibitively expensive or that would result in production of other hazardous chemicals.</p> <br />
<br />
<p>The main goal of OSCAR is to turn toxins like these into useable hydrocarbons by removing the carboxylic acid group(s) (Behar & Albrecht, 1984). <br />
<br />
Since NAs from petroleum deposits are a variable mixture, an enzymatic process with broad<br />
specificity is necessary. With the removal of the carboxylic acid moiety, we aim to produce alkanes suitable for use as fuel. The goal of this subproject was to find one or more suitable pathways to accomplish the decarboxylation of compounds such as NAs with the broadest specificity.</p><br />
<br />
<br />
<h2>The PetroBrick</h2><br />
<br />
<p>The 2011 Washington iGEM team developed the PetroBrick (<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025.</a>), a BioBrick consisting of two primary genes. These include acyl-ACP reductase (<i>AAR</i>), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (<i>ADC</i>), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain (Sukovich, 2010). Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that the fatty acids that the PetroBrick targets, have a very similar structure to NAs.</p><br />
<br />
</html>[[File:UCalgary-Fatty-Acids-vs-NAs.jpg|550px|centre|thumb|Figure 1. A comparison of the structure of fatty acids and naphthenic acid]]<html><br />
<br />
<br />
<br />
<p>This lead us to believe that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the Petrobrick, we needed to verify that the Petrobrick would work in our hands as it did for the 2011 Washington team. <br />
<br />
Figures 2 and 3 demonstrate the function of the Petrobrick.</p><br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 2: Gas Chromatograph demonstrating the differences in peak composition between an <i>E.coli</i> control and the Petrobrick. There was a large increase in a peak with a retention time of 12.25 min. suggesting that the Petrobrick was producing a new compound.|650px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center|thumb|Figure 3: Mass Spectra of the gas chromatograph peak at 12.25 min. The spectra suggests that the Petrobrick is selectively producing a C15 alkane. This is what was expected as determined by the Washington 2011 iGEM team.|750px]]<html><br />
<br />
<p>With the Petrobrick shown to be able to successfully produce alkanes, it was time to test it out on NAs, to see if <br />
they could be selectively converted into alkanes! This experiment used commercially available NAs fractions including a large number of different complex NAs compounds. </p><br />
<br />
<h2>Successful conversion of NA's into Hydrocarbons!</h2><br />
<br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 4: The relative intensity of alkane production over a retention time in both <i>E.coli</i> that contain the PetroBrick, and in <i>E.coli</i> that are lacking the PetroBrick, as measured with GC-MS. NAs were used as a substrate. A NA standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 5: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from <i>E.coli</i> containing the PetroBrick as in Figure 2, using NAs as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
<br />
<p> The above graphs indicate that hydrocarbons were successfully produced from <i>E.coli</i> that contained the PetroBrick plasmid, as analysed with GC-MS. In Figure 2, <i>E.coli</i> containing the PetroBrick had significantly higher hydrocarbon peaks than in a control of <i>E.coli</i> that did not contain the PetroBrick plasmid. Not only was the PetroBrick able to degrade NAs into alkanes, but it was also able to produce alkenes as shown by Figure 3, indicating that the PetroBrick worked how we had expected it to! </p><br />
<br />
<br />
<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)- Can we do better?</h2><br />
<br />
<p>Although we were successful using the PetroBrick to remove carboxyl groups from NAs, we wanted to improve on our results to see if we could get a higher yield or possibly target other compounds. One of our original fears in using the PetroBrick to <br />
decarboyxlate NAs was that the first enzyme AAR was reported to be highly specific for fatty acids bound to ACP. We had concerns about its compatibility with NAs and therefore sought another enzyme in the literature called carboxylic acid reductase (CAR) that was documented to perform a similar task as AAR, converting fatty acids to aldehydes, but with much lower specificity (He et al, 2004). This enzyme, from <i>N. iowensis</i> does not require covalent attachment to ACP so would <br />
likely be much broader in substrate specificity. It requires a second gene from <i>N. iowensis</i>, called Nocardia phosphopantetheinyl transferase (NPT) necessary to append a 4’- phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian et al, 2006).</p><br />
<br />
</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|450px|thumb|Figure 6. Mechanism of action of CAR]]<html><br />
<br />
<p>Another enzyme with the potential to remove carboxyl groups from NAs is olefin-forming fatty acid decarboxylase (OleT) from <i>Jeotgalicoccus</i> sp. ATCC 8456. This is a decarboxylase of the cytochrome P450 family that acts on fatty acids, but has also been documented to have low substrate specificity (Rude et al, 2011). What was attractive with this was that it was one single enzyme that go do the job of the PetroBrick! Now that we knew that our decarboxylation approach was valid, it was time to start testing and comparing this gene to the PetroBrick.</p><br />
<br />
<h2> Progress so far </h2><br />
<br />
<p>Genes <i>car</i> and <i>npt</i> were cloned from the host organism <i>N. iowensis</i> (NRRL 5646). <i>car</i> was ligated into the pET vector and verified by a restriction digest while <i>npt</i> was cloned into pSB1C3(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902061">BBa_K902061</a>) and similarly verified.</p><br />
<br />
<p><i>CAR</i> was cloned into pET47b+ plasmid due to six illegal cut sites(one XbaI site, two EcoRI sites, and three NotI sites) which made it unsuitable for the BioBrick construction vectors. We first attempted to use a multi-site mutagenesis derived from the QuikChange® Multi Site Directed Mutagenesis Kit, but this showed little success. Instead, a more time-consuming but effective series of conventional single-site <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis procedure</a> was favoured, using the KAPA Hi-Fi polymerase. The XbaI and EcoRI sites were eliminated <br />
<br />
first so that <i>car</i> can be moved from the pET Vector and ligated into the PSB1C3 vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902062">BBa_K902062</a>). The <i>oleT</i> was successfully amplified from the <i>Jeotgalicoccus</i> sp. ATCC 8456.<p> <br />
</p><br />
<p>Like <i>car</i>, <i>oleT</i> was inserted in a pET47b+ (Novagen) vector before placing it into a BioBrick vector, as two illegal cut sites adjacent to one another needed to be mutagenized. This part is now being ligated into pSB1C3. We are currently in the process of constructing all three parts under control of a <i>tetR</i> promoter and ribosomal binding site (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>), and then constructing these composite parts together as outlined below.</p><br />
<br />
<h2>Final testing constructs</h2><br />
<br />
<p>Final testing constructs are almost complete. These are illustrated in figure 7 and will allow us to compare the three different approaches. Unfortunately, as Washington only sent us the PetroBrick and not the two individual components, we will have to compare a combination of the PetroBrick and CAR/NPT to the PetroBrick alone and to OleT. </p><br />
<br />
<p></html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|centre|600px]]<html></html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|centre|400px|thumb|Figure 7. Final constructs required for validating and comparing different decarboxylation approaches]]<html></p><br />
<br />
<h2> Testing OleT </h2><br />
<br />
<p>One major stumbling block in testing out oleT has been significant difficulty in trying to ligate it into a vector, which has prevented us from submitting it as a BioBrick. As such, we chose to try some assays on the host organism: <i>Jeotgalicoccus</i> sp. ATCC 8456. This way we could at least validate that this gene was functional before we had our BioBricks. We started by trying to verify what was found by Rude et al., 2011, namely that this enzyme could convert fatty acids into alkenes. To do so, we grew up cultures according to this <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT_in_Validation_Assay">protocol</a> and used and GC-MS in order to analyze any alkene production. Our results are shown below. </p><br />
<br />
<h2> Formation of alkanes by <i>Jeotgalicoccus</i> sp. ATCC 8456</h2><br />
<br />
</html>[[File:UofC_OleT_Assay_1.png|centre|650px|thumb|Figure 8. Gas chromatograph demonstrating the production of olefins (alkenes) from fatty acids as shown from the increase in the peak with a retention time of 14.7 min. The dramatic change in peak intensity at this point suggests that we are producing hydrocarbons.]]<br />
[[File:UofC_OleT_2nd_Assay.png|centre|650px|thumb|Figure 9. Mass spectra of the peak in Figure 8 at retention time 14.7 min. Demonstrating that this peak is an olefin which was previously suggested to be produced using this species of bacteria. This provides a proof of concept that the <i>Micrococcus</i> species we have can degrade fatty acids into olefins. ]]<html><br />
<br />
<p>Based on the additional peak we saw in the gas chromatograph, we were able to show that our organism was indeed able to produce alkenes, as expected. This is possible improvement over the PetroBrick as it uses only one enzyme instead of two, however future testing is still needed. Now that we have validated the functionality of this enzyme in producing alkenes, the next step is to test it out on complex naphthenic acids in order to compare it to the PetroBrick. This testing is still underway.</p><br />
<br />
</html><br />
<br />
}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Notebook/CatecholDegradationTeam:Calgary/Notebook/CatecholDegradation2012-10-04T01:35:46Z<p>Stephanie0101: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decatecholization Journal|<br />
<br />
CONTENT=<html><br />
<br />
<h2>Week 1 and 2 (May 1-4 and May 7-11)</h2><br />
<br />
<p>The Catechol degradation part of the project started off as general ring cleavage. The goal was to cleave both the aromatic and aliphatic rings contained in many toxic compounds in the tailings ponds. The first two weeks involved getting familiar with lab procedures and researching aromatic ring cleaving using intra- and extradiol dioxygenases from species of <i>Pseudomonas</i> and <i>Bacillus</i>. We also began literature searches on aliphatic ring cleavage performed by monooxygenases. The project was focused on ring cleavage from week 1-12 and catechol degradation the following weeks.</p><br />
<br />
<h2>Week 3 (May 14 - May 18)</h2><br />
<br />
<p>In the second week we continued to do more research on the degradation of alicyclic compounds and found two strains of bacteria that contained genes needed for this process. The first strain, <i>Thauera butanivorans</i>, contains the genes required to activate the ring by adding a hydroxyl group. The enzyme is called butane monooxygenase and is composed of three subunits, a hydroxylase, a reductase, and a regulatory component. The second strain, <i> Acinetobacter sp. </i> SE19, produces the enzyme needed to oxidize the alcohol, formed by butane monooxygenase, and to cleave the ring. There is a cluster of nine genes that perform this oxidation and cleavage but only six are involved directly.</p><br />
<br />
<h2>Week 4 (May 22 - May 25)</h2><br />
<br />
<p>We started looking into more organisms that can carry out cyclohexane degradation and found one called <i>Brachymonas petroleovorans</i>. This organism is capable of both hydroxylating cyclohexane and carrying out cyclohexanol oxidation. We were also hoping to find evidence that <i>Pseudomonas fluorescens</i>, Pf-5 produces enzymes that can cleave aliphatic rings, however, first we have to determine if Pf-5 can grow in the presence of butylcyclohexane. To do so we carried out a growth assays in LB and the compound butylcyclohexane, testing if it was toxic to the strain Pf-5. After 24 hours of incubation we used a spectrophotometer to take OD600 readings indicating cells density. The results suggested that the presence of butylcyclohexane did not have a detrimental effect on cell growth. We also looked up <i>Pseudomonas fluorescens</i> Pf-5 in the Pseudomonas Genome Database and found genes that code for proteins with similar functions to what we have found for aromatic and aliphatic ring cleavage. </p><br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<br />
<p>This week we performed the growth/toxin survival assay with Pf-5 and butylcyclohexane using glass vials instead of plastic 15mL tubes. These vials allowed for more surface and better containment of the volatile compound. We carried out this assay using both LB and M9-MM (M9 minimal media). After 24 hours we took spectrophotometer readings at OD600 and obtained similar results as last week. The growth was considerably decreased in the M9-MM, however we are not sure whether the bacteria were able to use the compound as a carbon source because the MM contained glucose. The fact that the growth decreased showed that they might depend on the glucose in the media to grow. </p><br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<br />
<p> This week the experiment of testing survival and growth was repeated with the intention of determining if butylcyclohexane could be metabolized by the cells. To conclude this, the cells needed to be cultured without glucose. We subcultured from Pf-5 in M9-MM into M9-MM without glucose to avoid carry-over and ensure that the bacteria use butylcyclohexane as their only carbon source. From these cultures we also obtained GC-MS (Gas Chromatography Mass Spectroscopy) readings and compared experimental values of the compound to standards made with water and butylcyclohexane. The Pf-5 samples demonstrated a decrease in amount of butylcyclohexane in the headspace (area above the liquid culture) compared to standards, indicating that the compound was being degraded by Pf-5. Furthermore, we made subcultures from original Pf-5 in M9-MM without glucose into M9-MM and toluene to test for pathways that degrade aromatic structures. </p><br />
<p>We also wanted to identify which genes of Pf-5 that were part of the pathway to degrade toluene and butylcycohexane. We started running blasts on the Pf-5 genome looking for genes that are homologous to ones that we have been looking at for the two ring cleavage pathways (aromatic and aliphatic). In addition we started a verification of <i>XylE</i> (aromatic ring cleavage enzyme) by transforming part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) from the parts registry. We performed two colony PCRs. Unfortunately none of them showed successful results and only the positive control showed an appropriate length DNA. </p><br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<br />
<p> Continuing with the growth assays of Pf-5 in butylcyclohexane and toluene, we obtained more GC-MS readings. At this point the Pf-5 had been incubated with butylcyclohexane for 6 days and with toluene for 5 days. The amount of both butylcyclohexane and toluene decreased by a large amount in the experimental cultures but the amount of each in the abiotic standards also largely decreased by about the same amount. This lead us to conclude that the reason for decrease was not due to the bacteria metabolizing the compound, but due to unknown abiotic factors. To measure relative amounts of bacterial growth in these cultures we used a spectrophotometer at OD600 to read absorbance for both cultures. Optical density for both cultures was very low indicating very little bacterial growth. </p><br />
<p>The verification of <i>XylE</i> from the parts registry also continued. Three overnight cultures were miniprepped from part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. Unfortunately, the DNA concentrations were very high, likely due to genomic contamination of the miniprep. Also, a second part (<a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) containing <i>XylE</i> and an rbs site was transformed and a colony PCR was performed on part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. The gel did not indicate successful results, as again, only the positive control PCR. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<br />
<p>This week we began a new growth assay of Pf-5, but instead of incubating it with butylcyclohexane, we incubated with naphthalene. Since naphthalene is not volatile, we no longer needed to sample the culture headspace but could directly sample remaining naphthalene in the culture itself. Like the previous experiment, we needed to establish rate of growth in M9-MM without glucose and with naphthalene as the sole carbon source. A spectrophotometer was used to measure bacterial growth. The original cultures were grown in LB from a Pf-5 culture in LB and subcultured into M9-MM without glucose. After 24 hours of growth these were subcultured into fresh M9-MM without glucose, again to prevent carryover of glucose from the LB. Positive controls were also made that consisted of M9-MM with glucose and Pf-5. Spectrophotometer readings of the subcultures were taken over a period of 8 days.</p><br />
<p>GC readings were taken of the Pf-5 incubated with butylcyclohexane after 11 days and with toluene after 10 days. The trend remained the same as the measurements taken after 5 and 6 days in the previous week. The amount of compound decreased in the experimental cultures but the amount of compound also decreased in the abiotic standards almost in equal proportions. With the results after 10 and 11 days we could see that the decrease was not due to the bacteria and we decided that the strain we were using did not contain the genes necessary for butylcyclohexane or toluene degradation.</p> <br />
<br />
<p>Throughout the week we continued the verification of <i>XylE</i> by performing a second colony PCR from the transformation plate using part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. Again, the gel did not contain DNA of the right lengths and only the positive control worked. </p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<br />
<p> After the 8 day incubation the readings of the naphthalene assay were graphed and there was a definite increase in the cell growth of the cultures containing naphthalene as a carbon source which indicated that the bacteria were most likely metabolizing the compound. A verification of another part, Alkane hydroxylase (<i>alkB2,rubB,rubA3,rubA4</i>) system (<a href=http://partsregistry.org/Part:BBa_K398014>K398014</a>), from the registry was started. The system consists of 4 genes that have been shown to hydroxylate C5-C8 cycloalkanes. We chose this part to assist in the degradation of cyclohexane and cyclopentane. The transformation was successful and the gel of the colony PCR showed one positive colony but since there was contamination in the gel we were not sure if we could conclude that the PCR had worked. Final GC readings on the butylcyclohexane and toluene cultures were taken and results were graphed. <br />
</p><br />
<br />
</html>[[File:UCalgary2012ButCH_graph_(1).PNG|frame|center]]<br />
[[File:UCalgary2012Toluene_graph_(1).PNG|frame|center]] <br />
[[File:UCalgary2012Week9gelAHS.PNG|500px|thumb|Fig.3 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies 1-5 that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top band in lane 6 indicated successful amplification of the alkane hydroxylase system (2932 bp).|center]]<br />
[[File:UCalgary2012Naphthalene_graph_(1).PNG|frame|center]]<html><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<br />
<p> This week we performed another colony PCR of 5 colonies from the transformation plate of part <a href=http://partsregistry.org/Part:BBa_K398014>K398014</a> (alkane hydrolase construct: <i>alkB2,rubB,rubA3,rubA4</i>) and ran the products on a gel. We obtained 4 positive colonies and made overnight cultures. We decided to stop using this part for the project and therefore did not require a further miniprep on these overnight cultures. However we made an overnight culture of the positive colony from the alkane hydroxylase system gel (colony 5) and miniprepped the overnight culture. This resulted in a good concentration of 345.6 ng/μL as we verification of<i> XylE</i> by doing two more colony PCRs of each part (<a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) but only the positive control worked when we ran the gel. </p><br />
<br />
</html>[[File:UCalgaryWeek10gelAHS.PNG|500px|thumb|Fig.5 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies A-E that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top bands in lanes 2-4 indicated successful amplification of the alkane hydroxylase system (2932 bp). There was also a band in the negative control indicating contamination so we cannot be positive that the cPCR worked.|center]]<html><br />
<br />
<h2>Week 11 (July 9 - July 13)</h2><br />
<br />
<p> Since we could not obtain any positive colonies from last weeks colony PCRs we began the transformation of each part again. The transformations were successful and when the colony PCR products were run on a gel there were two positive colonies for each part. Overnight cultures of the positive colonies were made. Minipreps were done on the overnight cultures and a restriction digest was performed on the products of the minipreps. The restriction digest products were run on a gel and there was one that looked like it had been successful but due to a poor quality ladder on this gel we could not make any final conclusions on the success of the restriction digest.</p><br />
<br />
</html>[[File:UCalgaryWeek11gelXylE.PNG|500px|thumb|Fig.6 XylE Restriction Digest Gel. Both XylE parts from the registry were transformed into E.coli. One part contained the XylE gene and a native RBS site (J33204) and the other contained glucose repressible promoter (Pcst) and the XylE gene and a native RBS site (K118021). After running a colony PCR on the transformation plates two colonies from each plate looked like they contained the XylE gene. Overnight cultures of these colonies were made and restriction digests were ran on the mini-prepped cells. Lanes 2 and 3 contain the restriction digest product from colonies 2 and 4 on the J33204 plate and lanes 4 and 5 contain the product from colonies 3 and 5 from the k118021 plate. Because of the poor 1 Kb plus ladder it was difficult to tell what size the amplified DNA was so the gel was run again. |center]]<html><br />
<br />
<h2>Week 12 (July 16 - July 20)</h2><br />
<br />
<p> We began the week by rerunning the restriction digest products on a gel with a good ladder and obtained one positive result with two bands. One band was around 1000 bp for the part and the other was at about 2000 bp for the plasmid backbone. We made a streak plate of the colony that had worked for the restriction digest and sent the miniprep product for sequencing. The colony that worked was from the transformation plate of part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. We also repeated a colony PCR of the transformants from part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> using 10 colonies and all of them looked like positive colonies. Overnight culutres were made and a miniprep and restriction digest was completed.</p><br />
<br />
</html>[[File:UCalgary2012Week12agelXylE.PNG|500px|thumb|Fig.7 XylE Restriction Digest Gel. Both XylE parts from the registry were transformed into E.coli. One part contained the XylE gene and a native RBS site (J33204) and the other contained glucose repressible promoter (Pcst) and the XylE gene and a native RBS site (K118021). After running a colony PCR on the transformation plates two colonies from each plate looked like they contained the XylE gene. Overnight cultures of these colonies were made and restriction digests were ran on the mini-prepped cells. Lanes 2 and 3 contain the restriction digest product from colonies 2 and 4 on the J33204 plate and lanes 4 and 5 contain the product from colonies 3 and 5 from the k118021 plate. The two bands in 3rd lane after the 1 Kb plus ladder indicate that the restriction digest for that plasmid was successful. The top band around 2000 base pairs represented the pSB1A3 vector (2155 bp) and the bottom band around 1000 base pairs represented the XylE gene (1097 bp).|center]]<br />
[[File:UCalgary2012Week12bgelXylE.PNG|500px|thumb|Fig.8 Colony PCR gel of XylE with a native rbs site (J33204). Lanes 2-11 represent colonies A to J from the transformation plate. All of these lanes contain a band at the expected size of 958 bp. Lane 12 and 13 represent the positive and negative controls.|center]] <br />
[[File:UCalgary2012Week12cgelXylE.PNG|500px|thumb|Fig.9 Restriction digest gel of XylE gene with rbs site (J33204). Overnight cultures were made from colonies A,B,D,F and J from the Fig. 8 gel. These were mini-prepped and the restriction digest product was run on a gel. The digest of the plasmids from colonies B and D was successful which is shown by the bands at the expected size of 958 bp.|center]]<html><br />
<br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<br />
<p>The colony PCR of part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i> with a native rbs site) was done again and all five colonies looked like they contained the plasmid after running the products on a gel. After miniprepping and running a restriction digest on these five colonies two of them were sent to sequencing. An assay using the compound catechol was also started this week. E.coli cells (top 10) transformed with the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) were used in this assay. Six overnight cultures were made, 3 using M9-MM and 3 using LB. These were spun down and resuspended in fresh M9-MM and LB and brought to a concentration of 0.1 M catechol by using a 1M stock solution. There seemed to be an initial colour change in the reactions from clear to yellow which was only visible in the cultures using M9-MM. All of the cultures were incubated overnight and they turned a black green colour. For this assay we should have used the supernatant because the reaction takes place outside of the cell so this is what we did next week.</p><br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<br />
<p>The sequencing results for the part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> matched the part so a construction was started. The construction was a 3-way ligation putting the tetR promoter (<a href= http://partsregistry.org/Part:BBa_R0040>R0040</a>) and <i>XylE</i> (<a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) into a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. The ligation product was transformed into competent cells and a colony PCR was done on the transformants using both bio brick and <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> primers. The primers for <i>XylT</i>, which is a ferredoxin that allows for <i>XylE</i> to be activated after a reaction with catechol, arrived and a colony PCR on four different strains of Pseudomonas putida was done. None of the strains seemed to contain the TOL plasmid, which is where <i>XylT</i> is found in certain strains of P. putida, because no bands showed up in the gel. The catechol assay was continued this week. Six overnight cultures were made, 3 using M9-MM and 3 using LB. They were spun down and the supernatant was brought to a catechol concentration of 0.1M, 0.2 M and 0.5 M using a 1M stock solution. The solutions turned light yellow initially, but after a few hours they turned brown. The assay was repeated with the same procedure as above but a control, a colony that did not contain the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>, was added. After the catechol was added to the supernatants there was a colour change from clear to yellow in all of the tubes, even the control. For a negative control catechol was added to M9-MM and this solution became pink. The tubes were incubated on the bench and checked regularly. The colour changed from yellow to pink to brown when they were left on the bench overnight.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>The Catechol assay was repeated with a new procedure. Overnight cultures were made in LB and washed the pellet in M9-MM without glucose for varying amounts of time. These cultures were spun down and the supernatant was brought to a concentration of 0.1 M. All of the supernatants became bright yellow except for the control which did not contain the <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. The colour change indicated that catechol was being converted to 2-HMS (2-Hydroxymuconate semialdehyde). The three way ligation didn’t work so a two-way ligation with <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> (tetR) in a <a href=http://partsregistry.org/Part:pSB1A3>pSB1A3</a> vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i>) in a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. One reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the insert, and another reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the insert and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the vector. Both ways worked and the new procedure for the catechol assay was done with the transformed cells and all of the supernatants turned bright yellow.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Fig.10 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added.|center]]<html><br />
<br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>The sequencing results of the ligation were positive so assays were started with the cells transformed with the construct tetR-XylE. The assays involved the use of the petrobrick and a species of micrococcus. The purpose of these experiments was to see if any of the oxygen groups on the product of catechol degradation, which is 2-HMS, were removed.</p><br />
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</html>}}</div>Stephanie0101http://2012.igem.org/File:UCalgary2012Naphthalene_graph_(1).PNGFile:UCalgary2012Naphthalene graph (1).PNG2012-10-04T01:33:55Z<p>Stephanie0101: </p>
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<div></div>Stephanie0101http://2012.igem.org/File:UCalgary2012Toluene_graph_(1).PNGFile:UCalgary2012Toluene graph (1).PNG2012-10-04T01:30:18Z<p>Stephanie0101: </p>
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<div></div>Stephanie0101http://2012.igem.org/File:UCalgary2012ButCH_graph_(1).PNGFile:UCalgary2012ButCH graph (1).PNG2012-10-04T01:29:29Z<p>Stephanie0101: </p>
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<div></div>Stephanie0101http://2012.igem.org/File:UCalgary2012Catechol_assay_(2).jpgFile:UCalgary2012Catechol assay (2).jpg2012-10-04T01:14:30Z<p>Stephanie0101: </p>
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<div></div>Stephanie0101http://2012.igem.org/File:UCalgary2012Catechol_assay2.jpgFile:UCalgary2012Catechol assay2.jpg2012-10-04T01:13:56Z<p>Stephanie0101: </p>
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<div></div>Stephanie0101http://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradationTeam:Calgary/Project/OSCAR/CatecholDegradation2012-10-03T09:00:29Z<p>Stephanie0101: </p>
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<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_OSCAR_Catechol_Low-Res.png" style="float: right; padding: 10px;"></img><br />
<p><b>****This section needs work. Why are we degrading catechol? What part did we use? What is the number?</b></p><br />
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<p>Catechol is a toxic compound found in tailings ponds that is a by-product of polyaromatic hydrocarbon metabolism (Vaillancourt <i>et al.</i>, 2006). Catechol is toxic to a wide range of organisms from microorganisms to mammals (Schweigert <i>et al.</i>, 2001). The chemical properties of catechol allow it to react with biomolecules like DNA, proteins and membranes (Schweigert <i>et al.</i>, 2001). These interactions can cause serious damage including DNA breakage, enzyme inactivation and membrane uncoupling (Schweigert <i>et al.</i>, 2001). Catechol can be degraded by the enzyme catechol 2,3-dioxygenase encoded by the <i>xylE</i> gene on the Tol plasmid of <i>Pseudomonas putida</i> (Nakai <i>et al.</i>, 1983). The current iGEM Part repository has two BioBricks available of <i>xylE</i>. One contained <i>XylE</i> with its native ribosome-binding site (part: <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>), while the other part contained <i>XylE</i> under the glucose-repressible promoter cstA (Part: <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>). Given that <i>E. coli</i> is grown in the presence of glucose, we designed a new construct to keep <i>XylE</i> repressed by using the TetR promoter (Part:<a href= http://partsregistry.org/Part:BBa_R0040>R0040</a>).</p> <br />
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<h3></h3><br />
</html>[[File:UCalgary2010_R0040-XylE.png|400px|thumb|Fig.1 Genetic circuit for catechol degradation showing <i>XylE</i> biobricked under the TetR promoter|center]]<html><br />
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<p>Catechol 2,3-dioxygenase is an extradiol dioxygenase which cleaves catechol adjacent to the two hydroxyl groups. When this occurs 2-hydroxymuconate semialdehyde is produced, which is yellow in colour. This change in colour allows for visual assay to assess the activity of <i>XlyE</i>.</p><br />
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</html>[[File:UCalgary2012_Catechol_to_2-HMS.PNG|400px|thumb|Fig.2 Catechol 2,3-dioxygenase (<i>XlyE</i>) converts catechol to 2-Hydroxymuconate semialdehyde in the presence of oxygen. Adapted from Shu ''et al''., 1995.|center]]<html><br />
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<p>The visual assays were performed with <i>E.coli</i> cells transformed with <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> as well as with <i>E.coli</i> cells transformed with the newly constructed part (<a href=http://partsregistry.org/Part:BBa_K902048 >K902048</a>) by bringing the supernatant of an overnight culture to a concentration of 0.1 M of catechol. When the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> was used the pellet was first washed in M9-MM and spun down before catechol was added to the supernatant. This was done to avoid the glucose in the LB from repressing the cstA promoter (<a href=http://partsregistry.org/Part:BBa_K118011 >K118011</a>). The catechol was added to the supernatant because the reaction takes place outside of the cell. Within minutes of the addition of catechol to the supernatant, the solution turned from the pale yellow of LB to a bright yellow. This assay was completed by following the previous assay done by the 2008 Edinburgh iGEM team.</p><br />
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</html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Fig.3 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added. |center]]<html><br />
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</html>}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Project/ReferencesTeam:Calgary/Project/References2012-10-03T08:59:06Z<p>Stephanie0101: </p>
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<ul><br />
<li>Asenjo J.A. (1949).Bioreactor system design. New York (NY): Marcel Dekker Inc. <br />
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<li>Dana G, Kuiken T, Rejeski D & Snow A (2012) Synthetic biology: Four steps to avoid a synthetic-biology disaster. Nature 483:29.</li><br><br />
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<li>United Nations Environment Programme: Sulfur Air Pollution, http://www.unep.org/transport/pcfv/pdf/Ethiopia-AirPollutionsulphur.pdf (Retrieved: 09/18/2012)</li><br><br />
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<li>United States Environmental Protection Agency: Sulfur Dioxide, http://www.epa.gov/air/sulfurdioxide/ (Retrieved: 09/18/2012)</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>2009 iGEM Calgary, 2009.igem.org/Team:Calgary/Notebook </li><br><br />
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<li> He A, Li T, Daniels L, Fotheringham I, Rosazza J.P.N. Nocardia sp. Carboxylic Acid Reductase: Cloning, Expression, and Characterization of a New Aldehyde Oxidoreductase Family. Applied and Environmental Microbiology 2004 Mar;70(3):1874–1881.</li><br><br />
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<li> Venkitasubramanian P, Daniels L, Rosazza J.P.N. 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> Rude M.A, Baron T.S, Brubaker S, Alibhai M, Del Cardayre S.B, and Schirmer A. Terminal olefin (1-alkene) biosynthesis by a novel p450 fatty acid decarboxylase from Jeotgalicoccus species. Applied and Environmental Microbiology 2011 Mar;77(5):1718–1727.</li><br><br />
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<li> Clemente J.S, Fedorak P.M. A review of the occurrences, analyses, toxicity, and biodegradation of naphthenic acids. Chemosphere 2005 Feb 6;60(5):585-600.</li><br><br />
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<li> Frank R.A, Fischer K, Kavanagh R, Burnison B.K, Aresenault G, Headley J, Peru K.M, VanDerKraak G, Solomon K. Effect of Carboxylic Acid Content on the Acute Toxicity of Oil Sands Naphthenic Acids. EnvironSciTechnol 2009 Dec 11;43(2):266–271.</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> Behar F.H, Albrecht P. Correlations between carboxylic acids and hydrocarbons in several crude oils alteration by biodegradation. Organic Geochemistry 1984;6:597-604. </li><br><br />
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<li>German Collection of Microorganisms and Cell Cultures (DSMZ): Nocardia iowensis. https://www.dsmz.de/catalogues/details/culture/DSM-45197.html (retrieved 8/28/2012)</li><br><br />
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<li>UW iGEM: Diesel Production Background. https://2011.igem.org/Team:Washington/Alkanes/Background (retrieved 8/28/2012)</li><br><br />
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<li>Parts Registry: The PetroBrick – Strong Constitutive Expression of ADC and AAR in pSB1C3. http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 (retrieved 8/28/2012)</li><br><br />
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<li>Asenjo J.A. (1949).Bioreactor system design. New York (NY): Marcel Dekker Inc.</li></br><br />
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<li>Reit K., & Tramper J. (1991). Basic bioreactor design. New York (NY):Marcel Dekker Inc.</li></br><br />
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<li>Becker SA, Feist AM, Mo ML, Hannum G, Palsson BØ, Herrgard MJ. Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox. Nature Protocols 2 2007 Mar; 727-723.</li></br><br />
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<li>Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BØ. A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information. Molecular Systems Biology 3 2007 Jun; 121.</li></br><br />
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<li>Gopinath AV, Russell D. An Inexpensive Field-Portable Programmable Potentiostat. Chem.Educator.2006 July;11:23-28.</li></br><br />
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<li>Waters LS, Sandoval M, and Storz G. <i>The Escherichia coli MntR miniregulon includes genes encoding a small protein and an efflux pump required for manganese homeostasis.</i> J Bacteriol 2011 Nov; 193(21) 5887-97.</li></br><br />
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<li>Ramesh A, Wakeman CA, and Winkler WC. <i>Insights into metalloregulation by M-box riboswitch RNAs via structural analysis of manganese-bound complexes.</i> J Mol Biol 2011 Apr 8; 407(4) 556-70. </li></br><br />
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<li>Helmann J. <i>Measuring metals with RNA.</i> J Mol Cell 2007 Sept 21; 27(6) 859-860. </li></br><br />
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<li>Daus B, Mattusch J, Paschke A, Wennrich R, and Weiss H. <i>Kinetics of the arsenite oxidation in seepage water from a tin mill tailings pond.</i> Talanta 2000 May 5; 51(6) 1087-95. </li></br><br />
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<div>{{Team:Calgary/TemplateOutreach|<br />
TITLE=Telus Spark Science Centre|<br />
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CONTENT=<html><br />
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</html>[[File:IGEMCalgary-Telus-Spark-Header.jpg]]<html><br />
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<h3> </h3><br />
<h2>Sharing Synthetic Biology</h2><br />
<br />
<p>Telus Spark has given us the amazing opportunity to share and engage with the general public to demonstrate through hands on activities what biology is and how we can use synthetic biology to solve problems. The Science Centre brings together a huge range of people, from young children and their parents, to adults of all ages at their monthly Adults Only Nights. We have shown off our work and an exhibit meant to demonstrate the cool factor of synthetic biology. The first event we ran was Bacterial Art, and the second was an public beta test of our video game.<br />
<br />
<h2>Bacterial Art</h2><br />
<br />
<p>We have been able to let hundreds of people try their hand at making magnificent art by drawing bacteria containing different fluorescent biobricks onto agar plates. Stocks of bacteria with various colors of fluorescent protein plasmids were prepared. Visitors were got to use their imaginations and 'draw' the bacteria onto agar plates to make pieces of art. These agar plates contained a antibiotic to ensure the plasmid containing the fluorescent marker was not shed by the bacteria. These plates were then incubated overnight, before being photographed under a UV light and being posted to Telus Spark's <a href=https://www.facebook.com/media/set/?set=a.10152086146370538.896423.24253660537>facebook page</a> for the public to tag as theirs.<br />
<br />
<p>This event let us go hands on with the public with bacteria that we use everyday in our lab. We got to explain what the bacteria were, how we changed them, and a bit of the biology on how biobricks coding for fluorescent proteins work. Not only that we got to challenge some of the negative press that bacteria have by showing people that there are bacteria out their that do not cause disease and that can do amazing things.<br />
<br />
<p><br />
<br />
<h2>LAB ESCAPE Video Game Premiere</h2><br />
<br />
<p>Our second major event was the first public beta test of the <a href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game</a> we have been developing throughout the summer. We spent two days on a weekend (September 29-30, 2012) letting the general public try out our video game and learn a thing or two about synthetic biology. Players of the game gave us valuable feedback on places where we could improve the game, make it more clear, and most importantly how to make it more fun!<br />
<br />
<h2>Synthetic Biology Survey</h2><br />
<br />
<p>At one of the Telus Spark Adult Only Nights our team conducted a synthetic biology survey. The purpose of this survey was to evaluate the general public’s knowledge and thoughts on synthetic biology. Our results showed that a large amount of the general public feels like they have at least some understanding of synthetic biology and that they feel genetic engineering will be useful in the future. This is important because, for new advances in biology to be accepted in the general public, there needs to be understanding and the knowledge that the advancement can be useful. With this survey we also found that support of genetic engineering companies would be dependent on the ethical integrity of the company. This shows the importance of genetic engineering companies keeping a good standing with the general public. Below is some graphical representations of the answers to our survey.<br />
<br />
</html>[[File:UCalgary2012_SurveyQuestion_1_(new).PNG|500px|thumb||left]]<html><br />
</html>[[File:UCalgary2012_SurveyQuestion_2_(graph_1)_(new).PNG|500px|thumb||right]]<html><br />
</html>[[File:UCalgary2012_SurveyQuestion_2(graph_2)_(new).PNG|500px|thumb||left]]<html><br />
</html>[[File:UCalgary2012_SurveyQuestion_3(new1).PNG|500px|thumb||right]]<html><br />
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}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Outreach/SparkTeam:Calgary/Outreach/Spark2012-10-03T08:41:25Z<p>Stephanie0101: </p>
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<div>{{Team:Calgary/TemplateOutreach|<br />
TITLE=Telus Spark Science Centre|<br />
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CONTENT=<html><br />
<br />
</html>[[File:IGEMCalgary-Telus-Spark-Header.jpg]]<html><br />
<br />
<h3> </h3><br />
<h2>Sharing Synthetic Biology</h2><br />
<br />
<p>Telus Spark has given us the amazing opportunity to share and engage with the general public to demonstrate through hands on activities what biology is and how we can use synthetic biology to solve problems. The Science Centre brings together a huge range of people, from young children and their parents, to adults of all ages at their monthly Adults Only Nights. We have shown off our work and an exhibit meant to demonstrate the cool factor of synthetic biology. The first event we ran was Bacterial Art, and the second was an public beta test of our video game.<br />
<br />
<h2>Bacterial Art</h2><br />
<br />
<p>We have been able to let hundreds of people try their hand at making magnificent art by drawing bacteria containing different fluorescent biobricks onto agar plates. Stocks of bacteria with various colors of fluorescent protein plasmids were prepared. Visitors were got to use their imaginations and 'draw' the bacteria onto agar plates to make pieces of art. These agar plates contained a antibiotic to ensure the plasmid containing the fluorescent marker was not shed by the bacteria. These plates were then incubated overnight, before being photographed under a UV light and being posted to Telus Spark's <a href=https://www.facebook.com/media/set/?set=a.10152086146370538.896423.24253660537>facebook page</a> for the public to tag as theirs.<br />
<br />
<p>This event let us go hands on with the public with bacteria that we use everyday in our lab. We got to explain what the bacteria were, how we changed them, and a bit of the biology on how biobricks coding for fluorescent proteins work. Not only that we got to challenge some of the negative press that bacteria have by showing people that there are bacteria out their that do not cause disease and that can do amazing things.<br />
<br />
<p><br />
<br />
<h2>LAB ESCAPE Video Game Premiere</h2><br />
<br />
<p>Our second major event was the first public beta test of the <a href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game</a> we have been developing throughout the summer. We spent two days on a weekend (September 29-30, 2012) letting the general public try out our video game and learn a thing or two about synthetic biology. Players of the game gave us valuable feedback on places where we could improve the game, make it more clear, and most importantly how to make it more fun!<br />
<br />
<h2>Synthetic Biology Survey</h2><br />
<br />
<p>At one of the Telus Spark Adult Only Nights our team conducted a synthetic biology survey. The purpose of this survey was to evaluate the general public’s knowledge and thoughts on synthetic biology. Our results showed that a large amount of the general public feels like they have at least some understanding of synthetic biology and that they feel genetic engineering will be useful in the future. This is important because, for new advances in biology to be accepted in the general public, there needs to be understanding and the knowledge that the advancement can be useful. With this survey we also found that support of genetic engineering companies would be dependent on the ethical integrity of the company. This shows the importance of genetic engineering companies keeping a good standing with the general public. Below is some graphical representations of the answers to our survey.<br />
<br />
</html>[[File:UCalgary2012_SurveyQuestion_1_(new).PNG|500px|thumb||left]]<html><br />
</html>[[File:UCalgary2012_SurveyQuestion_2_(graph_1)_(new).PNG|500px|thumb|Caption|right]]<html><br />
</html>[[File:UCalgary2012_SurveyQuestion_2(graph_2)_(new).PNG|500px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_SurveyQuestion_3(new1).PNG|500px|thumb|Caption|right]]<html><br />
<br />
</html><br />
}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Outreach/SparkTeam:Calgary/Outreach/Spark2012-10-03T08:35:54Z<p>Stephanie0101: </p>
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<div>{{Team:Calgary/TemplateOutreach|<br />
TITLE=Telus Spark Science Centre|<br />
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CONTENT=<html><br />
<br />
</html>[[File:IGEMCalgary-Telus-Spark-Header.jpg]]<html><br />
<br />
<h3> </h3><br />
<h2>Sharing Synthetic Biology</h2><br />
<br />
<p>Telus Spark has given us the amazing opportunity to share and engage with the general public to demonstrate through hands on activities what biology is and how we can use synthetic biology to solve problems. The Science Centre brings together a huge range of people, from young children and their parents, to adults of all ages at their monthly Adults Only Nights. We have shown off our work and an exhibit meant to demonstrate the cool factor of synthetic biology. The first event we ran was Bacterial Art, and the second was an public beta test of our video game.<br />
<br />
<h2>Bacterial Art</h2><br />
<br />
<p>We have been able to let hundreds of people try their hand at making magnificent art by drawing bacteria containing different fluorescent biobricks onto agar plates. Stocks of bacteria with various colors of fluorescent protein plasmids were prepared. Visitors were got to use their imaginations and 'draw' the bacteria onto agar plates to make pieces of art. These agar plates contained a antibiotic to ensure the plasmid containing the fluorescent marker was not shed by the bacteria. These plates were then incubated overnight, before being photographed under a UV light and being posted to Telus Spark's <a href=https://www.facebook.com/media/set/?set=a.10152086146370538.896423.24253660537>facebook page</a> for the public to tag as theirs.<br />
<br />
<p>This event let us go hands on with the public with bacteria that we use everyday in our lab. We got to explain what the bacteria were, how we changed them, and a bit of the biology on how biobricks coding for fluorescent proteins work. Not only that we got to challenge some of the negative press that bacteria have by showing people that there are bacteria out their that do not cause disease and that can do amazing things.<br />
<br />
<p><br />
<br />
<h2>LAB ESCAPE Video Game Premiere</h2><br />
<br />
<p>Our second major event was the first public beta test of the <a href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game</a> we have been developing throughout the summer. We spent two days on a weekend (September 29-30, 2012) letting the general public try out our video game and learn a thing or two about synthetic biology. Players of the game gave us valuable feedback on places where we could improve the game, make it more clear, and most importantly how to make it more fun!<br />
<br />
<h2>Synthetic Biology Survey</h2><br />
<br />
<p>At one of the Telus Spark Adult Only Nights our team conducted a synthetic biology survey. The purpose of this survey was to evaluate the general public’s knowledge and thoughts on synthetic biology. Our results showed that a large amount of the general public feels like they have at least some understanding of synthetic biology and that they feel genetic engineering will be useful in the future. This is important because, for new advances in biology to be accepted in the general public, there needs to be understanding and the knowledge that the advancement can be useful. With this survey we also found that support of genetic engineering companies would be dependent on the ethical integrity of the company. This shows the importance of genetic engineering companies keeping a good standing with the general public. Below is some graphical representations of the answers to our survey.<br />
<br />
</html>[[File:UCalgary2012_SurveyQuestion_1_(new).PNG|500px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_SurveyQuestion_2_(graph_1)_(new).PNG|500px|thumb|Caption|right]]<html><br />
</html>[[File:UCalgary2012_SurveyQuestion_2(graph_2)_(new).PNG|500px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_SurveyQuestion_3(new1).PNG|500px|thumb|Caption|right]]<html><br />
<br />
</html><br />
}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Outreach/SparkTeam:Calgary/Outreach/Spark2012-10-03T08:06:50Z<p>Stephanie0101: </p>
<hr />
<div>{{Team:Calgary/TemplateOutreach|<br />
TITLE=Telus Spark Science Centre|<br />
<br />
CONTENT=<html><br />
<br />
</html>[[File:IGEMCalgary-Telus-Spark-Header.jpg]]<html><br />
<br />
<h3> </h3><br />
<h2>Sharing Synthetic Biology</h2><br />
<br />
<p>Telus Spark has given us the amazing opportunity to share and engage with the general public to demonstrate through hands on activities what biology is and how we can use synthetic biology to solve problems. The Science Centre brings together a huge range of people, from young children and their parents, to adults of all ages at their monthly Adults Only Nights. We have shown off our work and an exhibit meant to demonstrate the cool factor of synthetic biology. The first event we ran was Bacterial Art, and the second was an public beta test of our video game.<br />
<br />
<h2>Bacterial Art</h2><br />
<br />
<p>We have been able to let hundreds of people try their hand at making magnificent art by drawing bacteria containing different fluorescent biobricks onto agar plates. Stocks of bacteria with various colors of fluorescent protein plasmids were prepared. Visitors were got to use their imaginations and 'draw' the bacteria onto agar plates to make pieces of art. These agar plates contained a antibiotic to ensure the plasmid containing the fluorescent marker was not shed by the bacteria. These plates were then incubated overnight, before being photographed under a UV light and being posted to Telus Spark's <a href=https://www.facebook.com/media/set/?set=a.10152086146370538.896423.24253660537>facebook page</a> for the public to tag as theirs.<br />
<br />
<p>This event let us go hands on with the public with bacteria that we use everyday in our lab. We got to explain what the bacteria were, how we changed them, and a bit of the biology on how biobricks coding for fluorescent proteins work. Not only that we got to challenge some of the negative press that bacteria have by showing people that there are bacteria out their that do not cause disease and that can do amazing things.<br />
<br />
<p><br />
<br />
<h2>LAB ESCAPE Video Game Premiere</h2><br />
<br />
<p>Our second major event was the first public beta test of the <a href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game</a> we have been developing throughout the summer. We spent two days on a weekend (September 29-30, 2012) letting the general public try out our video game and learn a thing or two about synthetic biology. Players of the game gave us valuable feedback on places where we could improve the game, make it more clear, and most importantly how to make it more fun!<br />
<br />
<h2>Synthetic Biology Survey</h2><br />
<br />
<p><br />
<br />
</html>[[File:UCalgary2012_SurveyQuestion_1_(new).PNG|500px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_SurveyQuestion_2_(graph_1)_(new).PNG|500px|thumb|Caption|right]]<html><br />
</html>[[File:UCalgary2012_SurveyQuestion_2(graph_2)_(new).PNG|500px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_SurveyQuestion_3(new1).PNG|500px|thumb|Caption|right]]<html><br />
<br />
</html><br />
}}</div>Stephanie0101http://2012.igem.org/File:UCalgary2012_SurveyQuestion_3(new1).PNGFile:UCalgary2012 SurveyQuestion 3(new1).PNG2012-10-03T08:04:48Z<p>Stephanie0101: </p>
<hr />
<div></div>Stephanie0101http://2012.igem.org/File:UCalgary2012_SurveyQuestion_2(graph_2)_(new).PNGFile:UCalgary2012 SurveyQuestion 2(graph 2) (new).PNG2012-10-03T08:03:53Z<p>Stephanie0101: </p>
<hr />
<div></div>Stephanie0101http://2012.igem.org/File:UCalgary2012_SurveyQuestion_2_(graph_1)_(new).PNGFile:UCalgary2012 SurveyQuestion 2 (graph 1) (new).PNG2012-10-03T08:02:54Z<p>Stephanie0101: </p>
<hr />
<div></div>Stephanie0101http://2012.igem.org/File:UCalgary2012_SurveyQuestion_1_(new).PNGFile:UCalgary2012 SurveyQuestion 1 (new).PNG2012-10-03T08:02:07Z<p>Stephanie0101: </p>
<hr />
<div></div>Stephanie0101http://2012.igem.org/Team:Calgary/Outreach/SparkTeam:Calgary/Outreach/Spark2012-10-03T07:11:37Z<p>Stephanie0101: </p>
<hr />
<div>{{Team:Calgary/TemplateOutreach|<br />
TITLE=Telus Spark Science Centre|<br />
<br />
CONTENT=<html><br />
<br />
</html>[[File:IGEMCalgary-Telus-Spark-Header.jpg]]<html><br />
<br />
<h3> </h3><br />
<h2>Sharing Synthetic Biology</h2><br />
<br />
<p>Telus Spark has given us the amazing opportunity to share and engage with the general public to demonstrate through hands on activities what biology is and how we can use synthetic biology to solve problems. The Science Centre brings together a huge range of people, from young children and their parents, to adults of all ages at their monthly Adults Only Nights. We have shown off our work and an exhibit meant to demonstrate the cool factor of synthetic biology. The first event we ran was Bacterial Art, and the second was an public beta test of our video game.<br />
<br />
<h2>Bacterial Art</h2><br />
<br />
<p>We have been able to let hundreds of people try their hand at making magnificent art by drawing bacteria containing different fluorescent biobricks onto agar plates. Stocks of bacteria with various colors of fluorescent protein plasmids were prepared. Visitors were got to use their imaginations and 'draw' the bacteria onto agar plates to make pieces of art. These agar plates contained a antibiotic to ensure the plasmid containing the fluorescent marker was not shed by the bacteria. These plates were then incubated overnight, before being photographed under a UV light and being posted to Telus Spark's <a href=https://www.facebook.com/media/set/?set=a.10152086146370538.896423.24253660537>facebook page</a> for the public to tag as theirs.<br />
<br />
<p>This event let us go hands on with the public with bacteria that we use everyday in our lab. We got to explain what the bacteria were, how we changed them, and a bit of the biology on how biobricks coding for fluorescent proteins work. Not only that we got to challenge some of the negative press that bacteria have by showing people that there are bacteria out their that do not cause disease and that can do amazing things.<br />
<br />
<p><br />
<br />
<h2>LAB ESCAPE Video Game Premiere</h2><br />
<br />
<p>Our second major event was the first public beta test of the <a href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game</a> we have been developing throughout the summer. We spent two days on a weekend (September 29-30, 2012) letting the general public try out our video game and learn a thing or two about synthetic biology. Players of the game gave us valuable feedback on places where we could improve the game, make it more clear, and most importantly how to make it more fun!<br />
<br />
<h2>Synthetic Biology Survey</h2><br />
<br />
<p><br />
<br />
</html>[[File:UCalgary2012_Survey_Question_1.PNG|500px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_2_(graph_1).PNG|500px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_2_(graph_2).PNG|500px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_3.PNG|500px|thumb|Caption|left]]<html><br />
<br />
</html><br />
}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Outreach/SparkTeam:Calgary/Outreach/Spark2012-10-03T07:07:58Z<p>Stephanie0101: </p>
<hr />
<div>{{Team:Calgary/TemplateOutreach|<br />
TITLE=Telus Spark Science Centre|<br />
<br />
CONTENT=<html><br />
<br />
</html>[[File:IGEMCalgary-Telus-Spark-Header.jpg]]<html><br />
<br />
<h3> </h3><br />
<h2>Sharing Synthetic Biology</h2><br />
<br />
<p>Telus Spark has given us the amazing opportunity to share and engage with the general public to demonstrate through hands on activities what biology is and how we can use synthetic biology to solve problems. The Science Centre brings together a huge range of people, from young children and their parents, to adults of all ages at their monthly Adults Only Nights. We have shown off our work and an exhibit meant to demonstrate the cool factor of synthetic biology. The first event we ran was Bacterial Art, and the second was an public beta test of our video game.<br />
<br />
<h2>Bacterial Art</h2><br />
<br />
<p>We have been able to let hundreds of people try their hand at making magnificent art by drawing bacteria containing different fluorescent biobricks onto agar plates. Stocks of bacteria with various colors of fluorescent protein plasmids were prepared. Visitors were got to use their imaginations and 'draw' the bacteria onto agar plates to make pieces of art. These agar plates contained a antibiotic to ensure the plasmid containing the fluorescent marker was not shed by the bacteria. These plates were then incubated overnight, before being photographed under a UV light and being posted to Telus Spark's <a href=https://www.facebook.com/media/set/?set=a.10152086146370538.896423.24253660537>facebook page</a> for the public to tag as theirs.<br />
<br />
<p>This event let us go hands on with the public with bacteria that we use everyday in our lab. We got to explain what the bacteria were, how we changed them, and a bit of the biology on how biobricks coding for fluorescent proteins work. Not only that we got to challenge some of the negative press that bacteria have by showing people that there are bacteria out their that do not cause disease and that can do amazing things.<br />
<br />
<p><br />
<br />
<h2>LAB ESCAPE Video Game Premiere</h2><br />
<br />
<p>Our second major event was the first public beta test of the <a href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game</a> we have been developing throughout the summer. We spent two days on a weekend (September 29-30, 2012) letting the general public try out our video game and learn a thing or two about synthetic biology. Players of the game gave us valuable feedback on places where we could improve the game, make it more clear, and most importantly how to make it more fun!<br />
<br />
<h2>Synthetic Biology Survey</h2><br />
<br />
<p><br />
<br />
</html>[[File:UCalgary2012_Survey_Question_1.PNG|500px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_2_(graph_1).PNG|400px|thumb|Caption|right]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_2_(graph_2).PNG|400px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_3.PNG|400px|thumb|Caption|right]]<html><br />
<br />
</html><br />
}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Outreach/SparkTeam:Calgary/Outreach/Spark2012-10-03T07:06:46Z<p>Stephanie0101: </p>
<hr />
<div>{{Team:Calgary/TemplateOutreach|<br />
TITLE=Telus Spark Science Centre|<br />
<br />
CONTENT=<html><br />
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</html>[[File:IGEMCalgary-Telus-Spark-Header.jpg]]<html><br />
<br />
<h3> </h3><br />
<h2>Sharing Synthetic Biology</h2><br />
<br />
<p>Telus Spark has given us the amazing opportunity to share and engage with the general public to demonstrate through hands on activities what biology is and how we can use synthetic biology to solve problems. The Science Centre brings together a huge range of people, from young children and their parents, to adults of all ages at their monthly Adults Only Nights. We have shown off our work and an exhibit meant to demonstrate the cool factor of synthetic biology. The first event we ran was Bacterial Art, and the second was an public beta test of our video game.<br />
<br />
<h2>Bacterial Art</h2><br />
<br />
<p>We have been able to let hundreds of people try their hand at making magnificent art by drawing bacteria containing different fluorescent biobricks onto agar plates. Stocks of bacteria with various colors of fluorescent protein plasmids were prepared. Visitors were got to use their imaginations and 'draw' the bacteria onto agar plates to make pieces of art. These agar plates contained a antibiotic to ensure the plasmid containing the fluorescent marker was not shed by the bacteria. These plates were then incubated overnight, before being photographed under a UV light and being posted to Telus Spark's <a href=https://www.facebook.com/media/set/?set=a.10152086146370538.896423.24253660537>facebook page</a> for the public to tag as theirs.<br />
<br />
<p>This event let us go hands on with the public with bacteria that we use everyday in our lab. We got to explain what the bacteria were, how we changed them, and a bit of the biology on how biobricks coding for fluorescent proteins work. Not only that we got to challenge some of the negative press that bacteria have by showing people that there are bacteria out their that do not cause disease and that can do amazing things.<br />
<br />
<p><br />
<br />
<h2>LAB ESCAPE Video Game Premiere</h2><br />
<br />
<p>Our second major event was the first public beta test of the <a href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game</a> we have been developing throughout the summer. We spent two days on a weekend (September 29-30, 2012) letting the general public try out our video game and learn a thing or two about synthetic biology. Players of the game gave us valuable feedback on places where we could improve the game, make it more clear, and most importantly how to make it more fun!<br />
<br />
<h2>Synthetic Biology Survey</h2><br />
<br />
<p><br />
<br />
</html>[[File:UCalgary2012_Survey_Question_1.PNG|400px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_2_(graph_1).PNG|400px|thumb|Caption|right]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_2_(graph_2).PNG|400px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_3.PNG|400px|thumb|Caption|right]]<html><br />
<br />
</html><br />
}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Outreach/SparkTeam:Calgary/Outreach/Spark2012-10-03T07:06:18Z<p>Stephanie0101: </p>
<hr />
<div>{{Team:Calgary/TemplateOutreach|<br />
TITLE=Telus Spark Science Centre|<br />
<br />
CONTENT=<html><br />
<br />
</html>[[File:IGEMCalgary-Telus-Spark-Header.jpg]]<html><br />
<br />
<h3> </h3><br />
<h2>Sharing Synthetic Biology</h2><br />
<br />
<p>Telus Spark has given us the amazing opportunity to share and engage with the general public to demonstrate through hands on activities what biology is and how we can use synthetic biology to solve problems. The Science Centre brings together a huge range of people, from young children and their parents, to adults of all ages at their monthly Adults Only Nights. We have shown off our work and an exhibit meant to demonstrate the cool factor of synthetic biology. The first event we ran was Bacterial Art, and the second was an public beta test of our video game.<br />
<br />
<h2>Bacterial Art</h2><br />
<br />
<p>We have been able to let hundreds of people try their hand at making magnificent art by drawing bacteria containing different fluorescent biobricks onto agar plates. Stocks of bacteria with various colors of fluorescent protein plasmids were prepared. Visitors were got to use their imaginations and 'draw' the bacteria onto agar plates to make pieces of art. These agar plates contained a antibiotic to ensure the plasmid containing the fluorescent marker was not shed by the bacteria. These plates were then incubated overnight, before being photographed under a UV light and being posted to Telus Spark's <a href=https://www.facebook.com/media/set/?set=a.10152086146370538.896423.24253660537>facebook page</a> for the public to tag as theirs.<br />
<br />
<p>This event let us go hands on with the public with bacteria that we use everyday in our lab. We got to explain what the bacteria were, how we changed them, and a bit of the biology on how biobricks coding for fluorescent proteins work. Not only that we got to challenge some of the negative press that bacteria have by showing people that there are bacteria out their that do not cause disease and that can do amazing things.<br />
<br />
<p><br />
<br />
<h2>LAB ESCAPE Video Game Premiere</h2><br />
<br />
<p>Our second major event was the first public beta test of the <a href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game</a> we have been developing throughout the summer. We spent two days on a weekend (September 29-30, 2012) letting the general public try out our video game and learn a thing or two about synthetic biology. Players of the game gave us valuable feedback on places where we could improve the game, make it more clear, and most importantly how to make it more fun!<br />
<br />
<h2>Synthetic Biology Survey</h2><br />
<br />
<p><br />
<br />
</html>[[File:UCalgary2012_Survey_Question_1.PNG|300px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_2_(graph_1).PNG|300px|thumb|Caption|right]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_2_(graph_2).PNG|400px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_3.PNG|400px|thumb|Caption|right]]<html><br />
<br />
</html><br />
}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Outreach/SparkTeam:Calgary/Outreach/Spark2012-10-03T07:04:32Z<p>Stephanie0101: </p>
<hr />
<div>{{Team:Calgary/TemplateOutreach|<br />
TITLE=Telus Spark Science Centre|<br />
<br />
CONTENT=<html><br />
<br />
</html>[[File:IGEMCalgary-Telus-Spark-Header.jpg]]<html><br />
<br />
<h3> </h3><br />
<h2>Sharing Synthetic Biology</h2><br />
<br />
<p>Telus Spark has given us the amazing opportunity to share and engage with the general public to demonstrate through hands on activities what biology is and how we can use synthetic biology to solve problems. The Science Centre brings together a huge range of people, from young children and their parents, to adults of all ages at their monthly Adults Only Nights. We have shown off our work and an exhibit meant to demonstrate the cool factor of synthetic biology. The first event we ran was Bacterial Art, and the second was an public beta test of our video game.<br />
<br />
<h2>Bacterial Art</h2><br />
<br />
<p>We have been able to let hundreds of people try their hand at making magnificent art by drawing bacteria containing different fluorescent biobricks onto agar plates. Stocks of bacteria with various colors of fluorescent protein plasmids were prepared. Visitors were got to use their imaginations and 'draw' the bacteria onto agar plates to make pieces of art. These agar plates contained a antibiotic to ensure the plasmid containing the fluorescent marker was not shed by the bacteria. These plates were then incubated overnight, before being photographed under a UV light and being posted to Telus Spark's <a href=https://www.facebook.com/media/set/?set=a.10152086146370538.896423.24253660537>facebook page</a> for the public to tag as theirs.<br />
<br />
<p>This event let us go hands on with the public with bacteria that we use everyday in our lab. We got to explain what the bacteria were, how we changed them, and a bit of the biology on how biobricks coding for fluorescent proteins work. Not only that we got to challenge some of the negative press that bacteria have by showing people that there are bacteria out their that do not cause disease and that can do amazing things.<br />
<br />
<p><br />
<br />
<h2>LAB ESCAPE Video Game Premiere</h2><br />
<br />
<p>Our second major event was the first public beta test of the <a href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game</a> we have been developing throughout the summer. We spent two days on a weekend (September 29-30, 2012) letting the general public try out our video game and learn a thing or two about synthetic biology. Players of the game gave us valuable feedback on places where we could improve the game, make it more clear, and most importantly how to make it more fun!<br />
<br />
<h2>Synthetic Biology Survey</h2><br />
<br />
<p><br />
<br />
</html>[[File:UCalgary2012_Survey_Question_1.PNG|400px|thumb|Caption|left]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_2_(graph_1).PNG|400px|thumb|Caption|right]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_2_(graph_2).PNG|400px|thumb|Caption|left]]<html><br />
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}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Outreach/SparkTeam:Calgary/Outreach/Spark2012-10-03T07:03:03Z<p>Stephanie0101: </p>
<hr />
<div>{{Team:Calgary/TemplateOutreach|<br />
TITLE=Telus Spark Science Centre|<br />
<br />
CONTENT=<html><br />
<br />
</html>[[File:IGEMCalgary-Telus-Spark-Header.jpg]]<html><br />
<br />
<h3> </h3><br />
<h2>Sharing Synthetic Biology</h2><br />
<br />
<p>Telus Spark has given us the amazing opportunity to share and engage with the general public to demonstrate through hands on activities what biology is and how we can use synthetic biology to solve problems. The Science Centre brings together a huge range of people, from young children and their parents, to adults of all ages at their monthly Adults Only Nights. We have shown off our work and an exhibit meant to demonstrate the cool factor of synthetic biology. The first event we ran was Bacterial Art, and the second was an public beta test of our video game.<br />
<br />
<h2>Bacterial Art</h2><br />
<br />
<p>We have been able to let hundreds of people try their hand at making magnificent art by drawing bacteria containing different fluorescent biobricks onto agar plates. Stocks of bacteria with various colors of fluorescent protein plasmids were prepared. Visitors were got to use their imaginations and 'draw' the bacteria onto agar plates to make pieces of art. These agar plates contained a antibiotic to ensure the plasmid containing the fluorescent marker was not shed by the bacteria. These plates were then incubated overnight, before being photographed under a UV light and being posted to Telus Spark's <a href=https://www.facebook.com/media/set/?set=a.10152086146370538.896423.24253660537>facebook page</a> for the public to tag as theirs.<br />
<br />
<p>This event let us go hands on with the public with bacteria that we use everyday in our lab. We got to explain what the bacteria were, how we changed them, and a bit of the biology on how biobricks coding for fluorescent proteins work. Not only that we got to challenge some of the negative press that bacteria have by showing people that there are bacteria out their that do not cause disease and that can do amazing things.<br />
<br />
<p><br />
<br />
<h2>LAB ESCAPE Video Game Premiere</h2><br />
<br />
<p>Our second major event was the first public beta test of the <a href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game</a> we have been developing throughout the summer. We spent two days on a weekend (September 29-30, 2012) letting the general public try out our video game and learn a thing or two about synthetic biology. Players of the game gave us valuable feedback on places where we could improve the game, make it more clear, and most importantly how to make it more fun!<br />
<br />
<h2>Synthetic Biology Survey</h2><br />
<br />
<p><br />
<br />
</html>[[File:UCalgary2012_Survey_Question_1.PNG|400px|thumb|Caption|center]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_2_(graph_1).PNG|400px|thumb|Caption|center]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_2_(graph_2).PNG|400px|thumb|Caption|center]]<html><br />
</html>[[File:UCalgary2012_Survey_Question_3.PNG|400px|thumb|Caption|center]]<html><br />
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</html><br />
}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Outreach/SparkTeam:Calgary/Outreach/Spark2012-10-03T06:57:07Z<p>Stephanie0101: </p>
<hr />
<div>{{Team:Calgary/TemplateOutreach|<br />
TITLE=Telus Spark Science Centre|<br />
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CONTENT=<html><br />
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</html>[[File:IGEMCalgary-Telus-Spark-Header.jpg]]<html><br />
<br />
<h3> </h3><br />
<h2>Sharing Synthetic Biology</h2><br />
<br />
<p>Telus Spark has given us the amazing opportunity to share and engage with the general public to demonstrate through hands on activities what biology is and how we can use synthetic biology to solve problems. The Science Centre brings together a huge range of people, from young children and their parents, to adults of all ages at their monthly Adults Only Nights. We have shown off our work and an exhibit meant to demonstrate the cool factor of synthetic biology. The first event we ran was Bacterial Art, and the second was an public beta test of our video game.<br />
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<h2>Bacterial Art</h2><br />
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<p>We have been able to let hundreds of people try their hand at making magnificent art by drawing bacteria containing different fluorescent biobricks onto agar plates. Stocks of bacteria with various colors of fluorescent protein plasmids were prepared. Visitors were got to use their imaginations and 'draw' the bacteria onto agar plates to make pieces of art. These agar plates contained a antibiotic to ensure the plasmid containing the fluorescent marker was not shed by the bacteria. These plates were then incubated overnight, before being photographed under a UV light and being posted to Telus Spark's <a href=https://www.facebook.com/media/set/?set=a.10152086146370538.896423.24253660537>facebook page</a> for the public to tag as theirs.<br />
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<p>This event let us go hands on with the public with bacteria that we use everyday in our lab. We got to explain what the bacteria were, how we changed them, and a bit of the biology on how biobricks coding for fluorescent proteins work. Not only that we got to challenge some of the negative press that bacteria have by showing people that there are bacteria out their that do not cause disease and that can do amazing things.<br />
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<h2>LAB ESCAPE Video Game Premiere</h2><br />
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<p>Our second major event was the first public beta test of the <a href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game</a> we have been developing throughout the summer. We spent two days on a weekend (September 29-30, 2012) letting the general public try out our video game and learn a thing or two about synthetic biology. Players of the game gave us valuable feedback on places where we could improve the game, make it more clear, and most importantly how to make it more fun!<br />
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<h2>Synthetic Biology Survey</h2><br />
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}}</div>Stephanie0101http://2012.igem.org/File:UCalgary2012_Survey_Question_3.PNGFile:UCalgary2012 Survey Question 3.PNG2012-10-03T06:53:10Z<p>Stephanie0101: </p>
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<div></div>Stephanie0101http://2012.igem.org/File:UCalgary2012_Survey_Question_1.PNGFile:UCalgary2012 Survey Question 1.PNG2012-10-03T06:49:08Z<p>Stephanie0101: </p>
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<div></div>Stephanie0101http://2012.igem.org/Team:Calgary/Notebook/CatecholDegradationTeam:Calgary/Notebook/CatecholDegradation2012-10-03T06:12:58Z<p>Stephanie0101: </p>
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<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decatecholization Journal|<br />
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CONTENT=<html><br />
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<h2>Week 1 and 2 (May 1-4 and May 7-11)</h2><br />
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<p>The Catechol degradation part of the project started off as general ring cleavage. The goal was to cleave both the aromatic and aliphatic rings contained in many toxic compounds in the tailings ponds. The first two weeks involved getting familiar with lab procedures and researching aromatic ring cleaving using intra- and extradiol dioxygenases from species of <i>Pseudomonas</i> and <i>Bacillus</i>. We also began literature searches on aliphatic ring cleavage performed by monooxygenases. The project was focused on ring cleavage from week 1-12 and catechol degradation the following weeks.</p><br />
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<h2>Week 3 (May 14 - May 18)</h2><br />
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<p>In the second week we continued to do more research on the degradation of alicyclic compounds and found two strains of bacteria that contained genes needed for this process. The first strain, <i>Thauera butanivorans</i>, contains the genes required to activate the ring by adding a hydroxyl group. The enzyme is called butane monooxygenase and is composed of three subunits, a hydroxylase, a reductase, and a regulatory component. The second strain, <i> Acinetobacter sp. </i> SE19, produces the enzyme needed to oxidize the alcohol, formed by butane monooxygenase, and to cleave the ring. There is a cluster of nine genes that perform this oxidation and cleavage but only six are involved directly.</p><br />
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<h2>Week 4 (May 22 - May 25)</h2><br />
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<p>We started looking into more organisms that can carry out cyclohexane degradation and found one called <i>Brachymonas petroleovorans</i>. This organism is capable of both hydroxylating cyclohexane and carrying out cyclohexanol oxidation. We were also hoping to find evidence that <i>Pseudomonas fluorescens</i>, Pf-5 produces enzymes that can cleave aliphatic rings, however, first we have to determine if Pf-5 can grow in the presence of butylcyclohexane. To do so we carried out a growth assays in LB and the compound butylcyclohexane, testing if it was toxic to the strain Pf-5. After 24 hours of incubation we used a spectrophotometer to take OD600 readings indicating cells density. The results suggested that the presence of butylcyclohexane did not have a detrimental effect on cell growth. We also looked up <i>Pseudomonas fluorescens</i> Pf-5 in the Pseudomonas Genome Database and found genes that code for proteins with similar functions to what we have found for aromatic and aliphatic ring cleavage. </p><br />
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<h2>Week 5 (May 28 - June 1)</h2><br />
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<p>This week we performed the growth/toxin survival assay with Pf-5 and butylcyclohexane using glass vials instead of plastic 15mL tubes. These vials allowed for more surface and better containment of the volatile compound. We carried out this assay using both LB and M9-MM (M9 minimal media). After 24 hours we took spectrophotometer readings at OD600 and obtained similar results as last week. The growth was considerably decreased in the M9-MM, however we are not sure whether the bacteria were able to use the compound as a carbon source because the MM contained glucose. The fact that the growth decreased showed that they might depend on the glucose in the media to grow. </p><br />
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<h2>Week 6 (June 4 - June 8)</h2><br />
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<p> This week the experiment of testing survival and growth was repeated with the intention of determining if butylcyclohexane could be metabolized by the cells. To conclude this, the cells needed to be cultured without glucose. We subcultured from Pf-5 in M9-MM into M9-MM without glucose to avoid carry-over and ensure that the bacteria use butylcyclohexane as their only carbon source. From these cultures we also obtained GC-MS (Gas Chromatography Mass Spectroscopy) readings and compared experimental values of the compound to standards made with water and butylcyclohexane. The Pf-5 samples demonstrated a decrease in amount of butylcyclohexane in the headspace (area above the liquid culture) compared to standards, indicating that the compound was being degraded by Pf-5. Furthermore, we made subcultures from original Pf-5 in M9-MM without glucose into M9-MM and toluene to test for pathways that degrade aromatic structures. </p><br />
<p>We also wanted to identify which genes of Pf-5 that were part of the pathway to degrade toluene and butylcycohexane. We started running blasts on the Pf-5 genome looking for genes that are homologous to ones that we have been looking at for the two ring cleavage pathways (aromatic and aliphatic). In addition we started a verification of <i>XylE</i> (aromatic ring cleavage enzyme) by transforming part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) from the parts registry. We performed two colony PCRs. Unfortunately none of them showed successful results and only the positive control showed an appropriate length DNA. </p><br />
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<h2>Week 7 (June 11 - June 15)</h2><br />
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<p> Continuing with the growth assays of Pf-5 in butylcyclohexane and toluene, we obtained more GC-MS readings. At this point the Pf-5 had been incubated with butylcyclohexane for 6 days and with toluene for 5 days. The amount of both butylcyclohexane and toluene decreased by a large amount in the experimental cultures but the amount of each in the abiotic standards also largely decreased by about the same amount. This lead us to conclude that the reason for decrease was not due to the bacteria metabolizing the compound, but due to unknown abiotic factors. To measure relative amounts of bacterial growth in these cultures we used a spectrophotometer at OD600 to read absorbance for both cultures. Optical density for both cultures was very low indicating very little bacterial growth. </p><br />
<p>The verification of <i>XylE</i> from the parts registry also continued. Three overnight cultures were miniprepped from part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. Unfortunately, the DNA concentrations were very high, likely due to genomic contamination of the miniprep. Also, a second part (<a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) containing <i>XylE</i> and an rbs site was transformed and a colony PCR was performed on part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. The gel did not indicate successful results, as again, only the positive control PCR. </p><br />
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<h2>Week 8 (June 18 - June 22)</h2><br />
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<p>This week we began a new growth assay of Pf-5, but instead of incubating it with butylcyclohexane, we incubated with naphthalene. Since naphthalene is not volatile, we no longer needed to sample the culture headspace but could directly sample remaining naphthalene in the culture itself. Like the previous experiment, we needed to establish rate of growth in M9-MM without glucose and with naphthalene as the sole carbon source. A spectrophotometer was used to measure bacterial growth. The original cultures were grown in LB from a Pf-5 culture in LB and subcultured into M9-MM without glucose. After 24 hours of growth these were subcultured into fresh M9-MM without glucose, again to prevent carryover of glucose from the LB. Positive controls were also made that consisted of M9-MM with glucose and Pf-5. Spectrophotometer readings of the subcultures were taken over a period of 8 days.</p><br />
<p>GC readings were taken of the Pf-5 incubated with butylcyclohexane after 11 days and with toluene after 10 days. The trend remained the same as the measurements taken after 5 and 6 days in the previous week. The amount of compound decreased in the experimental cultures but the amount of compound also decreased in the abiotic standards almost in equal proportions. With the results after 10 and 11 days we could see that the decrease was not due to the bacteria and we decided that the strain we were using did not contain the genes necessary for butylcyclohexane or toluene degradation.</p> <br />
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<p>Throughout the week we continued the verification of <i>XylE</i> by performing a second colony PCR from the transformation plate using part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. Again, the gel did not contain DNA of the right lengths and only the positive control worked. </p><br />
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<h2>Week 9 (June 25 - June 29)</h2><br />
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<p> After the 8 day incubation the readings of the naphthalene assay were graphed and there was a definite increase in the cell growth of the cultures containing naphthalene as a carbon source which indicated that the bacteria were most likely metabolizing the compound. A verification of another part, Alkane hydroxylase (<i>alkB2,rubB,rubA3,rubA4</i>) system (<a href=http://partsregistry.org/Part:BBa_K398014>K398014</a>), from the registry was started. The system consists of 4 genes that have been shown to hydroxylate C5-C8 cycloalkanes. We chose this part to assist in the degradation of cyclohexane and cyclopentane. The transformation was successful and the gel of the colony PCR showed one positive colony but since there was contamination in the gel we were not sure if we could conclude that the PCR had worked. Final GC readings on the butylcyclohexane and toluene cultures were taken and results were graphed. <br />
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</html>[[File:UCalgary2012ButCHgraph.PNG|frame|center]]<br />
[[File:UCalgary2012Toluenegraph.PNG|frame|center]] <br />
[[File:UCalgary2012Week9gelAHS.PNG|500px|thumb|Fig.3 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies 1-5 that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top band in lane 6 indicated successful amplification of the alkane hydroxylase system (2932 bp).|center]]<br />
[[File:UCalgary2012Naphthalenegraph.PNG|frame|center]]<html><br />
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<h2>Week 10 (July 2-July 6)</h2><br />
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<p> This week we performed another colony PCR of 5 colonies from the transformation plate of part <a href=http://partsregistry.org/Part:BBa_K398014>K398014</a> (alkane hydrolase construct: <i>alkB2,rubB,rubA3,rubA4</i>) and ran the products on a gel. We obtained 4 positive colonies and made overnight cultures. We decided to stop using this part for the project and therefore did not require a further miniprep on these overnight cultures. However we made an overnight culture of the positive colony from the alkane hydroxylase system gel (colony 5) and miniprepped the overnight culture. This resulted in a good concentration of 345.6 ng/μL as we verification of<i> XylE</i> by doing two more colony PCRs of each part (<a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) but only the positive control worked when we ran the gel. </p><br />
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</html>[[File:UCalgaryWeek10gelAHS.PNG|500px|thumb|Fig.5 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies A-E that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top bands in lanes 2-4 indicated successful amplification of the alkane hydroxylase system (2932 bp). There was also a band in the negative control indicating contamination so we cannot be positive that the cPCR worked.|center]]<html><br />
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<h2>Week 11 (July 9 - July 13)</h2><br />
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<p> Since we could not obtain any positive colonies from last weeks colony PCRs we began the transformation of each part again. The transformations were successful and when the colony PCR products were run on a gel there were two positive colonies for each part. Overnight cultures of the positive colonies were made. Minipreps were done on the overnight cultures and a restriction digest was performed on the products of the minipreps. The restriction digest products were run on a gel and there was one that looked like it had been successful but due to a poor quality ladder on this gel we could not make any final conclusions on the success of the restriction digest.</p><br />
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</html>[[File:UCalgaryWeek11gelXylE.PNG|500px|thumb|Fig.6 XylE Restriction Digest Gel. Both XylE parts from the registry were transformed into E.coli. One part contained the XylE gene and a native RBS site (J33204) and the other contained glucose repressible promoter (Pcst) and the XylE gene and a native RBS site (K118021). After running a colony PCR on the transformation plates two colonies from each plate looked like they contained the XylE gene. Overnight cultures of these colonies were made and restriction digests were ran on the mini-prepped cells. Lanes 2 and 3 contain the restriction digest product from colonies 2 and 4 on the J33204 plate and lanes 4 and 5 contain the product from colonies 3 and 5 from the k118021 plate. Because of the poor 1 Kb plus ladder it was difficult to tell what size the amplified DNA was so the gel was run again. |center]]<html><br />
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<h2>Week 12 (July 16 - July 20)</h2><br />
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<p> We began the week by rerunning the restriction digest products on a gel with a good ladder and obtained one positive result with two bands. One band was around 1000 bp for the part and the other was at about 2000 bp for the plasmid backbone. We made a streak plate of the colony that had worked for the restriction digest and sent the miniprep product for sequencing. The colony that worked was from the transformation plate of part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. We also repeated a colony PCR of the transformants from part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> using 10 colonies and all of them looked like positive colonies. Overnight culutres were made and a miniprep and restriction digest was completed.</p><br />
<br />
</html>[[File:UCalgary2012Week12agelXylE.PNG|500px|thumb|Fig.7 XylE Restriction Digest Gel. Both XylE parts from the registry were transformed into E.coli. One part contained the XylE gene and a native RBS site (J33204) and the other contained glucose repressible promoter (Pcst) and the XylE gene and a native RBS site (K118021). After running a colony PCR on the transformation plates two colonies from each plate looked like they contained the XylE gene. Overnight cultures of these colonies were made and restriction digests were ran on the mini-prepped cells. Lanes 2 and 3 contain the restriction digest product from colonies 2 and 4 on the J33204 plate and lanes 4 and 5 contain the product from colonies 3 and 5 from the k118021 plate. The two bands in 3rd lane after the 1 Kb plus ladder indicate that the restriction digest for that plasmid was successful. The top band around 2000 base pairs represented the pSB1A3 vector (2155 bp) and the bottom band around 1000 base pairs represented the XylE gene (1097 bp).|center]]<br />
[[File:UCalgary2012Week12bgelXylE.PNG|500px|thumb|Fig.8 Colony PCR gel of XylE with a native rbs site (J33204). Lanes 2-11 represent colonies A to J from the transformation plate. All of these lanes contain a band at the expected size of 958 bp. Lane 12 and 13 represent the positive and negative controls.|center]] <br />
[[File:UCalgary2012Week12cgelXylE.PNG|500px|thumb|Fig.9 Restriction digest gel of XylE gene with rbs site (J33204). Overnight cultures were made from colonies A,B,D,F and J from the Fig. 8 gel. These were mini-prepped and the restriction digest product was run on a gel. The digest of the plasmids from colonies B and D was successful which is shown by the bands at the expected size of 958 bp.|center]]<html><br />
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<h2>Week 13 (July 23 - July 27)</h2><br />
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<p>The colony PCR of part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i> with a native rbs site) was done again and all five colonies looked like they contained the plasmid after running the products on a gel. After miniprepping and running a restriction digest on these five colonies two of them were sent to sequencing. An assay using the compound catechol was also started this week. E.coli cells (top 10) transformed with the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) were used in this assay. Six overnight cultures were made, 3 using M9-MM and 3 using LB. These were spun down and resuspended in fresh M9-MM and LB and brought to a concentration of 0.1 M catechol by using a 1M stock solution. There seemed to be an initial colour change in the reactions from clear to yellow which was only visible in the cultures using M9-MM. All of the cultures were incubated overnight and they turned a black green colour. For this assay we should have used the supernatant because the reaction takes place outside of the cell so this is what we did next week.</p><br />
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<h2>Week 14 (July 30 - August 3)</h2><br />
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<p>The sequencing results for the part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> matched the part so a construction was started. The construction was a 3-way ligation putting the tetR promoter (<a href= http://partsregistry.org/Part:BBa_R0040>R0040</a>) and <i>XylE</i> (<a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) into a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. The ligation product was transformed into competent cells and a colony PCR was done on the transformants using both bio brick and <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> primers. The primers for <i>XylT</i>, which is a ferredoxin that allows for <i>XylE</i> to be activated after a reaction with catechol, arrived and a colony PCR on four different strains of Pseudomonas putida was done. None of the strains seemed to contain the TOL plasmid, which is where <i>XylT</i> is found in certain strains of P. putida, because no bands showed up in the gel. The catechol assay was continued this week. Six overnight cultures were made, 3 using M9-MM and 3 using LB. They were spun down and the supernatant was brought to a catechol concentration of 0.1M, 0.2 M and 0.5 M using a 1M stock solution. The solutions turned light yellow initially, but after a few hours they turned brown. The assay was repeated with the same procedure as above but a control, a colony that did not contain the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>, was added. After the catechol was added to the supernatants there was a colour change from clear to yellow in all of the tubes, even the control. For a negative control catechol was added to M9-MM and this solution became pink. The tubes were incubated on the bench and checked regularly. The colour changed from yellow to pink to brown when they were left on the bench overnight.</p><br />
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<h2>Week 17 (August 20 - August 24)</h2><br />
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<p>The Catechol assay was repeated with a new procedure. Overnight cultures were made in LB and washed the pellet in M9-MM without glucose for varying amounts of time. These cultures were spun down and the supernatant was brought to a concentration of 0.1 M. All of the supernatants became bright yellow except for the control which did not contain the <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. The colour change indicated that catechol was being converted to 2-HMS (2-Hydroxymuconate semialdehyde). The three way ligation didn’t work so a two-way ligation with <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> (tetR) in a <a href=http://partsregistry.org/Part:pSB1A3>pSB1A3</a> vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i>) in a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. One reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the insert, and another reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the insert and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the vector. Both ways worked and the new procedure for the catechol assay was done with the transformed cells and all of the supernatants turned bright yellow.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Fig.10 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added.|center]]<html><br />
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<h2>Week 18 (August 27 - August 31)</h2><br />
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<p>The sequencing results of the ligation were positive so assays were started with the cells transformed with the construct tetR-XylE. The assays involved the use of the petrobrick and a species of micrococcus. The purpose of these experiments was to see if any of the oxygen groups on the product of catechol degradation, which is 2-HMS, were removed.</p><br />
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</html>}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Notebook/CatecholDegradationTeam:Calgary/Notebook/CatecholDegradation2012-10-03T06:10:32Z<p>Stephanie0101: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decatecholization Journal|<br />
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CONTENT=<html><br />
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<h2>Week 1 and 2 (May 1-4 and May 7-11)</h2><br />
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<p>The Catechol degradation part of the project started off as general ring cleavage. The goal was to cleave both the aromatic and aliphatic rings contained in many toxic compounds in the tailings ponds. The first two weeks involved getting familiar with lab procedures and researching aromatic ring cleaving using intra- and extradiol dioxygenases from species of <i>Pseudomonas</i> and <i>Bacillus</i>. We also began literature searches on aliphatic ring cleavage performed by monooxygenases. The project was focused on ring cleavage from week 1-12 and catechol degradation the following weeks.</p><br />
<br />
<h2>Week 3 (May 14 - May 18)</h2><br />
<br />
<p>In the second week we continued to do more research on the degradation of alicyclic compounds and found two strains of bacteria that contained genes needed for this process. The first strain, <i>Thauera butanivorans</i>, contains the genes required to activate the ring by adding a hydroxyl group. The enzyme is called butane monooxygenase and is composed of three subunits, a hydroxylase, a reductase, and a regulatory component. The second strain, <i> Acinetobacter sp. </i> SE19, produces the enzyme needed to oxidize the alcohol, formed by butane monooxygenase, and to cleave the ring. There is a cluster of nine genes that perform this oxidation and cleavage but only six are involved directly.</p><br />
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<h2>Week 4 (May 22 - May 25)</h2><br />
<br />
<p>We started looking into more organisms that can carry out cyclohexane degradation and found one called <i>Brachymonas petroleovorans</i>. This organism is capable of both hydroxylating cyclohexane and carrying out cyclohexanol oxidation. We were also hoping to find evidence that <i>Pseudomonas fluorescens</i>, Pf-5 produces enzymes that can cleave aliphatic rings, however, first we have to determine if Pf-5 can grow in the presence of butylcyclohexane. To do so we carried out a growth assays in LB and the compound butylcyclohexane, testing if it was toxic to the strain Pf-5. After 24 hours of incubation we used a spectrophotometer to take OD600 readings indicating cells density. The results suggested that the presence of butylcyclohexane did not have a detrimental effect on cell growth. We also looked up <i>Pseudomonas fluorescens</i> Pf-5 in the Pseudomonas Genome Database and found genes that code for proteins with similar functions to what we have found for aromatic and aliphatic ring cleavage. </p><br />
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<h2>Week 5 (May 28 - June 1)</h2><br />
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<p>This week we performed the growth/toxin survival assay with Pf-5 and butylcyclohexane using glass vials instead of plastic 15mL tubes. These vials allowed for more surface and better containment of the volatile compound. We carried out this assay using both LB and M9-MM (M9 minimal media). After 24 hours we took spectrophotometer readings at OD600 and obtained similar results as last week. The growth was considerably decreased in the M9-MM, however we are not sure whether the bacteria were able to use the compound as a carbon source because the MM contained glucose. The fact that the growth decreased showed that they might depend on the glucose in the media to grow. </p><br />
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<h2>Week 6 (June 4 - June 8)</h2><br />
<br />
<p> This week the experiment of testing survival and growth was repeated with the intention of determining if butylcyclohexane could be metabolized by the cells. To conclude this, the cells needed to be cultured without glucose. We subcultured from Pf-5 in M9-MM into M9-MM without glucose to avoid carry-over and ensure that the bacteria use butylcyclohexane as their only carbon source. From these cultures we also obtained GC-MS (Gas Chromatography Mass Spectroscopy) readings and compared experimental values of the compound to standards made with water and butylcyclohexane. The Pf-5 samples demonstrated a decrease in amount of butylcyclohexane in the headspace (area above the liquid culture) compared to standards, indicating that the compound was being degraded by Pf-5. Furthermore, we made subcultures from original Pf-5 in M9-MM without glucose into M9-MM and toluene to test for pathways that degrade aromatic structures. </p><br />
<p>We also wanted to identify which genes of Pf-5 that were part of the pathway to degrade toluene and butylcycohexane. We started running blasts on the Pf-5 genome looking for genes that are homologous to ones that we have been looking at for the two ring cleavage pathways (aromatic and aliphatic). In addition we started a verification of <i>XylE</i> (aromatic ring cleavage enzyme) by transforming part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) from the parts registry. We performed two colony PCRs. Unfortunately none of them showed successful results and only the positive control showed an appropriate length DNA. </p><br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<br />
<p> Continuing with the growth assays of Pf-5 in butylcyclohexane and toluene, we obtained more GC-MS readings. At this point the Pf-5 had been incubated with butylcyclohexane for 6 days and with toluene for 5 days. The amount of both butylcyclohexane and toluene decreased by a large amount in the experimental cultures but the amount of each in the abiotic standards also largely decreased by about the same amount. This lead us to conclude that the reason for decrease was not due to the bacteria metabolizing the compound, but due to unknown abiotic factors. To measure relative amounts of bacterial growth in these cultures we used a spectrophotometer at OD600 to read absorbance for both cultures. Optical density for both cultures was very low indicating very little bacterial growth. </p><br />
<p>The verification of <i>XylE</i> from the parts registry also continued. Three overnight cultures were miniprepped from part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. Unfortunately, the DNA concentrations were very high, likely due to genomic contamination of the miniprep. Also, a second part (<a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) containing <i>XylE</i> and an rbs site was transformed and a colony PCR was performed on part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. The gel did not indicate successful results, as again, only the positive control PCR. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<br />
<p>This week we began a new growth assay of Pf-5, but instead of incubating it with butylcyclohexane, we incubated with naphthalene. Since naphthalene is not volatile, we no longer needed to sample the culture headspace but could directly sample remaining naphthalene in the culture itself. Like the previous experiment, we needed to establish rate of growth in M9-MM without glucose and with naphthalene as the sole carbon source. A spectrophotometer was used to measure bacterial growth. The original cultures were grown in LB from a Pf-5 culture in LB and subcultured into M9-MM without glucose. After 24 hours of growth these were subcultured into fresh M9-MM without glucose, again to prevent carryover of glucose from the LB. Positive controls were also made that consisted of M9-MM with glucose and Pf-5. Spectrophotometer readings of the subcultures were taken over a period of 8 days.</p><br />
<p>GC readings were taken of the Pf-5 incubated with butylcyclohexane after 11 days and with toluene after 10 days. The trend remained the same as the measurements taken after 5 and 6 days in the previous week. The amount of compound decreased in the experimental cultures but the amount of compound also decreased in the abiotic standards almost in equal proportions. With the results after 10 and 11 days we could see that the decrease was not due to the bacteria and we decided that the strain we were using did not contain the genes necessary for butylcyclohexane or toluene degradation.</p> <br />
<br />
<p>Throughout the week we continued the verification of <i>XylE</i> by performing a second colony PCR from the transformation plate using part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. Again, the gel did not contain DNA of the right lengths and only the positive control worked. </p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<br />
<p> After the 8 day incubation the readings of the naphthalene assay were graphed and there was a definite increase in the cell growth of the cultures containing naphthalene as a carbon source which indicated that the bacteria were most likely metabolizing the compound. A verification of another part, Alkane hydroxylase (<i>alkB2,rubB,rubA3,rubA4</i>) system (<a href=http://partsregistry.org/Part:BBa_K398014>K398014</a>), from the registry was started. The system consists of 4 genes that have been shown to hydroxylate C5-C8 cycloalkanes. We chose this part to assist in the degradation of cyclohexane and cyclopentane. The transformation was successful and the gel of the colony PCR showed one positive colony but since there was contamination in the gel we were not sure if we could conclude that the PCR had worked. Final GC readings on the butylcyclohexane and toluene cultures were taken and results were graphed. <br />
</p><br />
<br />
</html>[[File:UCalgary2012ButCHgraph.PNG|frame|center]]<br />
[[File:UCalgary2012_Toluene_assay.png|frame|center]] <br />
[[File:UCalgary2012Week9gelAHS.PNG|500px|thumb|Fig.3 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies 1-5 that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top band in lane 6 indicated successful amplification of the alkane hydroxylase system (2932 bp).|center]]<br />
[[File:UCalgary2012Naphthalene_assay.png|frame|center]]<html><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<br />
<p> This week we performed another colony PCR of 5 colonies from the transformation plate of part <a href=http://partsregistry.org/Part:BBa_K398014>K398014</a> (alkane hydrolase construct: <i>alkB2,rubB,rubA3,rubA4</i>) and ran the products on a gel. We obtained 4 positive colonies and made overnight cultures. We decided to stop using this part for the project and therefore did not require a further miniprep on these overnight cultures. However we made an overnight culture of the positive colony from the alkane hydroxylase system gel (colony 5) and miniprepped the overnight culture. This resulted in a good concentration of 345.6 ng/μL as we verification of<i> XylE</i> by doing two more colony PCRs of each part (<a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) but only the positive control worked when we ran the gel. </p><br />
<br />
</html>[[File:UCalgaryWeek10gelAHS.PNG|500px|thumb|Fig.5 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies A-E that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top bands in lanes 2-4 indicated successful amplification of the alkane hydroxylase system (2932 bp). There was also a band in the negative control indicating contamination so we cannot be positive that the cPCR worked.|center]]<html><br />
<br />
<h2>Week 11 (July 9 - July 13)</h2><br />
<br />
<p> Since we could not obtain any positive colonies from last weeks colony PCRs we began the transformation of each part again. The transformations were successful and when the colony PCR products were run on a gel there were two positive colonies for each part. Overnight cultures of the positive colonies were made. Minipreps were done on the overnight cultures and a restriction digest was performed on the products of the minipreps. The restriction digest products were run on a gel and there was one that looked like it had been successful but due to a poor quality ladder on this gel we could not make any final conclusions on the success of the restriction digest.</p><br />
<br />
</html>[[File:UCalgaryWeek11gelXylE.PNG|500px|thumb|Fig.6 XylE Restriction Digest Gel. Both XylE parts from the registry were transformed into E.coli. One part contained the XylE gene and a native RBS site (J33204) and the other contained glucose repressible promoter (Pcst) and the XylE gene and a native RBS site (K118021). After running a colony PCR on the transformation plates two colonies from each plate looked like they contained the XylE gene. Overnight cultures of these colonies were made and restriction digests were ran on the mini-prepped cells. Lanes 2 and 3 contain the restriction digest product from colonies 2 and 4 on the J33204 plate and lanes 4 and 5 contain the product from colonies 3 and 5 from the k118021 plate. Because of the poor 1 Kb plus ladder it was difficult to tell what size the amplified DNA was so the gel was run again. |center]]<html><br />
<br />
<h2>Week 12 (July 16 - July 20)</h2><br />
<br />
<p> We began the week by rerunning the restriction digest products on a gel with a good ladder and obtained one positive result with two bands. One band was around 1000 bp for the part and the other was at about 2000 bp for the plasmid backbone. We made a streak plate of the colony that had worked for the restriction digest and sent the miniprep product for sequencing. The colony that worked was from the transformation plate of part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. We also repeated a colony PCR of the transformants from part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> using 10 colonies and all of them looked like positive colonies. Overnight culutres were made and a miniprep and restriction digest was completed.</p><br />
<br />
</html>[[File:UCalgary2012Week12agelXylE.PNG|500px|thumb|Fig.7 XylE Restriction Digest Gel. Both XylE parts from the registry were transformed into E.coli. One part contained the XylE gene and a native RBS site (J33204) and the other contained glucose repressible promoter (Pcst) and the XylE gene and a native RBS site (K118021). After running a colony PCR on the transformation plates two colonies from each plate looked like they contained the XylE gene. Overnight cultures of these colonies were made and restriction digests were ran on the mini-prepped cells. Lanes 2 and 3 contain the restriction digest product from colonies 2 and 4 on the J33204 plate and lanes 4 and 5 contain the product from colonies 3 and 5 from the k118021 plate. The two bands in 3rd lane after the 1 Kb plus ladder indicate that the restriction digest for that plasmid was successful. The top band around 2000 base pairs represented the pSB1A3 vector (2155 bp) and the bottom band around 1000 base pairs represented the XylE gene (1097 bp).|center]]<br />
[[File:UCalgary2012Week12bgelXylE.PNG|500px|thumb|Fig.8 Colony PCR gel of XylE with a native rbs site (J33204). Lanes 2-11 represent colonies A to J from the transformation plate. All of these lanes contain a band at the expected size of 958 bp. Lane 12 and 13 represent the positive and negative controls.|center]] <br />
[[File:UCalgary2012Week12cgelXylE.PNG|500px|thumb|Fig.9 Restriction digest gel of XylE gene with rbs site (J33204). Overnight cultures were made from colonies A,B,D,F and J from the Fig. 8 gel. These were mini-prepped and the restriction digest product was run on a gel. The digest of the plasmids from colonies B and D was successful which is shown by the bands at the expected size of 958 bp.|center]]<html><br />
<br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<br />
<p>The colony PCR of part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i> with a native rbs site) was done again and all five colonies looked like they contained the plasmid after running the products on a gel. After miniprepping and running a restriction digest on these five colonies two of them were sent to sequencing. An assay using the compound catechol was also started this week. E.coli cells (top 10) transformed with the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) were used in this assay. Six overnight cultures were made, 3 using M9-MM and 3 using LB. These were spun down and resuspended in fresh M9-MM and LB and brought to a concentration of 0.1 M catechol by using a 1M stock solution. There seemed to be an initial colour change in the reactions from clear to yellow which was only visible in the cultures using M9-MM. All of the cultures were incubated overnight and they turned a black green colour. For this assay we should have used the supernatant because the reaction takes place outside of the cell so this is what we did next week.</p><br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<br />
<p>The sequencing results for the part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> matched the part so a construction was started. The construction was a 3-way ligation putting the tetR promoter (<a href= http://partsregistry.org/Part:BBa_R0040>R0040</a>) and <i>XylE</i> (<a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) into a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. The ligation product was transformed into competent cells and a colony PCR was done on the transformants using both bio brick and <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> primers. The primers for <i>XylT</i>, which is a ferredoxin that allows for <i>XylE</i> to be activated after a reaction with catechol, arrived and a colony PCR on four different strains of Pseudomonas putida was done. None of the strains seemed to contain the TOL plasmid, which is where <i>XylT</i> is found in certain strains of P. putida, because no bands showed up in the gel. The catechol assay was continued this week. Six overnight cultures were made, 3 using M9-MM and 3 using LB. They were spun down and the supernatant was brought to a catechol concentration of 0.1M, 0.2 M and 0.5 M using a 1M stock solution. The solutions turned light yellow initially, but after a few hours they turned brown. The assay was repeated with the same procedure as above but a control, a colony that did not contain the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>, was added. After the catechol was added to the supernatants there was a colour change from clear to yellow in all of the tubes, even the control. For a negative control catechol was added to M9-MM and this solution became pink. The tubes were incubated on the bench and checked regularly. The colour changed from yellow to pink to brown when they were left on the bench overnight.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>The Catechol assay was repeated with a new procedure. Overnight cultures were made in LB and washed the pellet in M9-MM without glucose for varying amounts of time. These cultures were spun down and the supernatant was brought to a concentration of 0.1 M. All of the supernatants became bright yellow except for the control which did not contain the <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. The colour change indicated that catechol was being converted to 2-HMS (2-Hydroxymuconate semialdehyde). The three way ligation didn’t work so a two-way ligation with <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> (tetR) in a <a href=http://partsregistry.org/Part:pSB1A3>pSB1A3</a> vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i>) in a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. One reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the insert, and another reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the insert and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the vector. Both ways worked and the new procedure for the catechol assay was done with the transformed cells and all of the supernatants turned bright yellow.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Fig.10 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added.|center]]<html><br />
<br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>The sequencing results of the ligation were positive so assays were started with the cells transformed with the construct tetR-XylE. The assays involved the use of the petrobrick and a species of micrococcus. The purpose of these experiments was to see if any of the oxygen groups on the product of catechol degradation, which is 2-HMS, were removed.</p><br />
<br />
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</html>}}</div>Stephanie0101http://2012.igem.org/File:UCalgary2012Naphthalenegraph.PNGFile:UCalgary2012Naphthalenegraph.PNG2012-10-03T06:08:12Z<p>Stephanie0101: </p>
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<div></div>Stephanie0101http://2012.igem.org/File:UCalgary2012Toluenegraph.PNGFile:UCalgary2012Toluenegraph.PNG2012-10-03T06:07:20Z<p>Stephanie0101: </p>
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<div></div>Stephanie0101http://2012.igem.org/File:UCalgary2012ButCHgraph.PNGFile:UCalgary2012ButCHgraph.PNG2012-10-03T06:06:06Z<p>Stephanie0101: </p>
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<div></div>Stephanie0101http://2012.igem.org/Team:Calgary/Notebook/CatecholDegradationTeam:Calgary/Notebook/CatecholDegradation2012-10-03T04:11:30Z<p>Stephanie0101: </p>
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<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decatecholization Journal|<br />
<br />
CONTENT=<html><br />
<br />
<h2>Week 1 and 2 (May 1-4 and May 7-11)</h2><br />
<br />
<p>The Catechol degradation part of the project started off as general ring cleavage. The goal was to cleave both the aromatic and aliphatic rings contained in many toxic compounds in the tailings ponds. The first two weeks involved getting familiar with lab procedures and researching aromatic ring cleaving using intra- and extradiol dioxygenases from species of <i>Pseudomonas</i> and <i>Bacillus</i>. We also began literature searches on aliphatic ring cleavage performed by monooxygenases. The project was focused on ring cleavage from week 1-12 and catechol degradation the following weeks.</p><br />
<br />
<h2>Week 3 (May 14 - May 18)</h2><br />
<br />
<p>In the second week we continued to do more research on the degradation of alicyclic compounds and found two strains of bacteria that contained genes needed for this process. The first strain, <i>Thauera butanivorans</i>, contains the genes required to activate the ring by adding a hydroxyl group. The enzyme is called butane monooxygenase and is composed of three subunits, a hydroxylase, a reductase, and a regulatory component. The second strain, <i> Acinetobacter sp. </i> SE19, produces the enzyme needed to oxidize the alcohol, formed by butane monooxygenase, and to cleave the ring. There is a cluster of nine genes that perform this oxidation and cleavage but only six are involved directly.</p><br />
<br />
<h2>Week 4 (May 22 - May 25)</h2><br />
<br />
<p>We started looking into more organisms that can carry out cyclohexane degradation and found one called <i>Brachymonas petroleovorans</i>. This organism is capable of both hydroxylating cyclohexane and carrying out cyclohexanol oxidation. We were also hoping to find evidence that <i>Pseudomonas fluorescens</i>, Pf-5 produces enzymes that can cleave aliphatic rings, however, first we have to determine if Pf-5 can grow in the presence of butylcyclohexane. To do so we carried out a growth assays in LB and the compound butylcyclohexane, testing if it was toxic to the strain Pf-5. After 24 hours of incubation we used a spectrophotometer to take OD600 readings indicating cells density. The results suggested that the presence of butylcyclohexane did not have a detrimental effect on cell growth. We also looked up <i>Pseudomonas fluorescens</i> Pf-5 in the Pseudomonas Genome Database and found genes that code for proteins with similar functions to what we have found for aromatic and aliphatic ring cleavage. </p><br />
<br />
<h2>Week 5 (May 28 - June 1)</h2><br />
<br />
<p>This week we performed the growth/toxin survival assay with Pf-5 and butylcyclohexane using glass vials instead of plastic 15mL tubes. These vials allowed for more surface and better containment of the volatile compound. We carried out this assay using both LB and M9-MM (M9 minimal media). After 24 hours we took spectrophotometer readings at OD600 and obtained similar results as last week. The growth was considerably decreased in the M9-MM, however we are not sure whether the bacteria were able to use the compound as a carbon source because the MM contained glucose. The fact that the growth decreased showed that they might depend on the glucose in the media to grow. </p><br />
<br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<br />
<p> This week the experiment of testing survival and growth was repeated with the intention of determining if butylcyclohexane could be metabolized by the cells. To conclude this, the cells needed to be cultured without glucose. We subcultured from Pf-5 in M9-MM into M9-MM without glucose to avoid carry-over and ensure that the bacteria use butylcyclohexane as their only carbon source. From these cultures we also obtained GC-MS (Gas Chromatography Mass Spectroscopy) readings and compared experimental values of the compound to standards made with water and butylcyclohexane. The Pf-5 samples demonstrated a decrease in amount of butylcyclohexane in the headspace (area above the liquid culture) compared to standards, indicating that the compound was being degraded by Pf-5. Furthermore, we made subcultures from original Pf-5 in M9-MM without glucose into M9-MM and toluene to test for pathways that degrade aromatic structures. </p><br />
<p>We also wanted to identify which genes of Pf-5 that were part of the pathway to degrade toluene and butylcycohexane. We started running blasts on the Pf-5 genome looking for genes that are homologous to ones that we have been looking at for the two ring cleavage pathways (aromatic and aliphatic). In addition we started a verification of <i>XylE</i> (aromatic ring cleavage enzyme) by transforming part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) from the parts registry. We performed two colony PCRs. Unfortunately none of them showed successful results and only the positive control showed an appropriate length DNA. </p><br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<br />
<p> Continuing with the growth assays of Pf-5 in butylcyclohexane and toluene, we obtained more GC-MS readings. At this point the Pf-5 had been incubated with butylcyclohexane for 6 days and with toluene for 5 days. The amount of both butylcyclohexane and toluene decreased by a large amount in the experimental cultures but the amount of each in the abiotic standards also largely decreased by about the same amount. This lead us to conclude that the reason for decrease was not due to the bacteria metabolizing the compound, but due to unknown abiotic factors. To measure relative amounts of bacterial growth in these cultures we used a spectrophotometer at OD600 to read absorbance for both cultures. Optical density for both cultures was very low indicating very little bacterial growth. </p><br />
<p>The verification of <i>XylE</i> from the parts registry also continued. Three overnight cultures were miniprepped from part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. Unfortunately, the DNA concentrations were very high, likely due to genomic contamination of the miniprep. Also, a second part (<a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) containing <i>XylE</i> and an rbs site was transformed and a colony PCR was performed on part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. The gel did not indicate successful results, as again, only the positive control PCR. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<br />
<p>This week we began a new growth assay of Pf-5, but instead of incubating it with butylcyclohexane, we incubated with naphthalene. Since naphthalene is not volatile, we no longer needed to sample the culture headspace but could directly sample remaining naphthalene in the culture itself. Like the previous experiment, we needed to establish rate of growth in M9-MM without glucose and with naphthalene as the sole carbon source. A spectrophotometer was used to measure bacterial growth. The original cultures were grown in LB from a Pf-5 culture in LB and subcultured into M9-MM without glucose. After 24 hours of growth these were subcultured into fresh M9-MM without glucose, again to prevent carryover of glucose from the LB. Positive controls were also made that consisted of M9-MM with glucose and Pf-5. Spectrophotometer readings of the subcultures were taken over a period of 8 days.</p><br />
<p>GC readings were taken of the Pf-5 incubated with butylcyclohexane after 11 days and with toluene after 10 days. The trend remained the same as the measurements taken after 5 and 6 days in the previous week. The amount of compound decreased in the experimental cultures but the amount of compound also decreased in the abiotic standards almost in equal proportions. With the results after 10 and 11 days we could see that the decrease was not due to the bacteria and we decided that the strain we were using did not contain the genes necessary for butylcyclohexane or toluene degradation.</p> <br />
<br />
<p>Throughout the week we continued the verification of <i>XylE</i> by performing a second colony PCR from the transformation plate using part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. Again, the gel did not contain DNA of the right lengths and only the positive control worked. </p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<br />
<p> After the 8 day incubation the readings of the naphthalene assay were graphed and there was a definite increase in the cell growth of the cultures containing naphthalene as a carbon source which indicated that the bacteria were most likely metabolizing the compound. A verification of another part, Alkane hydroxylase (<i>alkB2,rubB,rubA3,rubA4</i>) system (<a href=http://partsregistry.org/Part:BBa_K398014>K398014</a>), from the registry was started. The system consists of 4 genes that have been shown to hydroxylate C5-C8 cycloalkanes. We chose this part to assist in the degradation of cyclohexane and cyclopentane. The transformation was successful and the gel of the colony PCR showed one positive colony but since there was contamination in the gel we were not sure if we could conclude that the PCR had worked. Final GC readings on the butylcyclohexane and toluene cultures were taken and results were graphed. <br />
</p><br />
<br />
</html>[[File:UCalgary2012_Butchgraph.png|frame|center]]<br />
[[File:UCalgary2012_Toluene_assay.png|frame|center]] <br />
[[File:UCalgary2012Week9gelAHS.PNG|500px|thumb|Fig.3 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies 1-5 that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top band in lane 6 indicated successful amplification of the alkane hydroxylase system (2932 bp).|center]]<br />
[[File:UCalgary2012Naphthalene_assay.png|frame|center]]<html><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<br />
<p> This week we performed another colony PCR of 5 colonies from the transformation plate of part <a href=http://partsregistry.org/Part:BBa_K398014>K398014</a> (alkane hydrolase construct: <i>alkB2,rubB,rubA3,rubA4</i>) and ran the products on a gel. We obtained 4 positive colonies and made overnight cultures. We decided to stop using this part for the project and therefore did not require a further miniprep on these overnight cultures. However we made an overnight culture of the positive colony from the alkane hydroxylase system gel (colony 5) and miniprepped the overnight culture. This resulted in a good concentration of 345.6 ng/μL as we verification of<i> XylE</i> by doing two more colony PCRs of each part (<a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) but only the positive control worked when we ran the gel. </p><br />
<br />
</html>[[File:UCalgaryWeek10gelAHS.PNG|500px|thumb|Fig.5 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies A-E that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top bands in lanes 2-4 indicated successful amplification of the alkane hydroxylase system (2932 bp). There was also a band in the negative control indicating contamination so we cannot be positive that the cPCR worked.|center]]<html><br />
<br />
<h2>Week 11 (July 9 - July 13)</h2><br />
<br />
<p> Since we could not obtain any positive colonies from last weeks colony PCRs we began the transformation of each part again. The transformations were successful and when the colony PCR products were run on a gel there were two positive colonies for each part. Overnight cultures of the positive colonies were made. Minipreps were done on the overnight cultures and a restriction digest was performed on the products of the minipreps. The restriction digest products were run on a gel and there was one that looked like it had been successful but due to a poor quality ladder on this gel we could not make any final conclusions on the success of the restriction digest.</p><br />
<br />
</html>[[File:UCalgaryWeek11gelXylE.PNG|500px|thumb|Fig.6 XylE Restriction Digest Gel. Both XylE parts from the registry were transformed into E.coli. One part contained the XylE gene and a native RBS site (J33204) and the other contained glucose repressible promoter (Pcst) and the XylE gene and a native RBS site (K118021). After running a colony PCR on the transformation plates two colonies from each plate looked like they contained the XylE gene. Overnight cultures of these colonies were made and restriction digests were ran on the mini-prepped cells. Lanes 2 and 3 contain the restriction digest product from colonies 2 and 4 on the J33204 plate and lanes 4 and 5 contain the product from colonies 3 and 5 from the k118021 plate. Because of the poor 1 Kb plus ladder it was difficult to tell what size the amplified DNA was so the gel was run again. |center]]<html><br />
<br />
<h2>Week 12 (July 16 - July 20)</h2><br />
<br />
<p> We began the week by rerunning the restriction digest products on a gel with a good ladder and obtained one positive result with two bands. One band was around 1000 bp for the part and the other was at about 2000 bp for the plasmid backbone. We made a streak plate of the colony that had worked for the restriction digest and sent the miniprep product for sequencing. The colony that worked was from the transformation plate of part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. We also repeated a colony PCR of the transformants from part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> using 10 colonies and all of them looked like positive colonies. Overnight culutres were made and a miniprep and restriction digest was completed.</p><br />
<br />
</html>[[File:UCalgary2012Week12agelXylE.PNG|500px|thumb|Fig.7 XylE Restriction Digest Gel. Both XylE parts from the registry were transformed into E.coli. One part contained the XylE gene and a native RBS site (J33204) and the other contained glucose repressible promoter (Pcst) and the XylE gene and a native RBS site (K118021). After running a colony PCR on the transformation plates two colonies from each plate looked like they contained the XylE gene. Overnight cultures of these colonies were made and restriction digests were ran on the mini-prepped cells. Lanes 2 and 3 contain the restriction digest product from colonies 2 and 4 on the J33204 plate and lanes 4 and 5 contain the product from colonies 3 and 5 from the k118021 plate. The two bands in 3rd lane after the 1 Kb plus ladder indicate that the restriction digest for that plasmid was successful. The top band around 2000 base pairs represented the pSB1A3 vector (2155 bp) and the bottom band around 1000 base pairs represented the XylE gene (1097 bp).|center]]<br />
[[File:UCalgary2012Week12bgelXylE.PNG|500px|thumb|Fig.8 Colony PCR gel of XylE with a native rbs site (J33204). Lanes 2-11 represent colonies A to J from the transformation plate. All of these lanes contain a band at the expected size of 958 bp. Lane 12 and 13 represent the positive and negative controls.|center]] <br />
[[File:UCalgary2012Week12cgelXylE.PNG|500px|thumb|Fig.9 Restriction digest gel of XylE gene with rbs site (J33204). Overnight cultures were made from colonies A,B,D,F and J from the Fig. 8 gel. These were mini-prepped and the restriction digest product was run on a gel. The digest of the plasmids from colonies B and D was successful which is shown by the bands at the expected size of 958 bp.|center]]<html><br />
<br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<br />
<p>The colony PCR of part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i> with a native rbs site) was done again and all five colonies looked like they contained the plasmid after running the products on a gel. After miniprepping and running a restriction digest on these five colonies two of them were sent to sequencing. An assay using the compound catechol was also started this week. E.coli cells (top 10) transformed with the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) were used in this assay. Six overnight cultures were made, 3 using M9-MM and 3 using LB. These were spun down and resuspended in fresh M9-MM and LB and brought to a concentration of 0.1 M catechol by using a 1M stock solution. There seemed to be an initial colour change in the reactions from clear to yellow which was only visible in the cultures using M9-MM. All of the cultures were incubated overnight and they turned a black green colour. For this assay we should have used the supernatant because the reaction takes place outside of the cell so this is what we did next week.</p><br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<br />
<p>The sequencing results for the part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> matched the part so a construction was started. The construction was a 3-way ligation putting the tetR promoter (<a href= http://partsregistry.org/Part:BBa_R0040>R0040</a>) and <i>XylE</i> (<a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) into a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. The ligation product was transformed into competent cells and a colony PCR was done on the transformants using both bio brick and <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> primers. The primers for <i>XylT</i>, which is a ferredoxin that allows for <i>XylE</i> to be activated after a reaction with catechol, arrived and a colony PCR on four different strains of Pseudomonas putida was done. None of the strains seemed to contain the TOL plasmid, which is where <i>XylT</i> is found in certain strains of P. putida, because no bands showed up in the gel. The catechol assay was continued this week. Six overnight cultures were made, 3 using M9-MM and 3 using LB. They were spun down and the supernatant was brought to a catechol concentration of 0.1M, 0.2 M and 0.5 M using a 1M stock solution. The solutions turned light yellow initially, but after a few hours they turned brown. The assay was repeated with the same procedure as above but a control, a colony that did not contain the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>, was added. After the catechol was added to the supernatants there was a colour change from clear to yellow in all of the tubes, even the control. For a negative control catechol was added to M9-MM and this solution became pink. The tubes were incubated on the bench and checked regularly. The colour changed from yellow to pink to brown when they were left on the bench overnight.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>The Catechol assay was repeated with a new procedure. Overnight cultures were made in LB and washed the pellet in M9-MM without glucose for varying amounts of time. These cultures were spun down and the supernatant was brought to a concentration of 0.1 M. All of the supernatants became bright yellow except for the control which did not contain the <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. The colour change indicated that catechol was being converted to 2-HMS (2-Hydroxymuconate semialdehyde). The three way ligation didn’t work so a two-way ligation with <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> (tetR) in a <a href=http://partsregistry.org/Part:pSB1A3>pSB1A3</a> vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i>) in a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. One reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the insert, and another reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the insert and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the vector. Both ways worked and the new procedure for the catechol assay was done with the transformed cells and all of the supernatants turned bright yellow.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Fig.10 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added.|center]]<html><br />
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<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>The sequencing results of the ligation were positive so assays were started with the cells transformed with the construct tetR-XylE. The assays involved the use of the petrobrick and a species of micrococcus. The purpose of these experiments was to see if any of the oxygen groups on the product of catechol degradation, which is 2-HMS, were removed.</p><br />
<br />
<br />
</html>}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Notebook/CatecholDegradationTeam:Calgary/Notebook/CatecholDegradation2012-10-03T04:10:21Z<p>Stephanie0101: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decatecholization Journal|<br />
<br />
CONTENT=<html><br />
<br />
<h2>Week 1 and 2 (May 1-4 and May 7-11)</h2><br />
<br />
<p>The Catechol degradation part of the project started off as general ring cleavage. The goal was to cleave both the aromatic and aliphatic rings contained in many toxic compounds in the tailings ponds. The first two weeks involved getting familiar with lab procedures and researching aromatic ring cleaving using intra- and extradiol dioxygenases from species of <i>Pseudomonas</i> and <i>Bacillus</i>. We also began literature searches on aliphatic ring cleavage performed by monooxygenases. The project was focused on ring cleavage from week 1-12 and catechol degradation the following weeks.</p><br />
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<h2>Week 3 (May 14 - May 18)</h2><br />
<br />
<p>In the second week we continued to do more research on the degradation of alicyclic compounds and found two strains of bacteria that contained genes needed for this process. The first strain, <i>Thauera butanivorans</i>, contains the genes required to activate the ring by adding a hydroxyl group. The enzyme is called butane monooxygenase and is composed of three subunits, a hydroxylase, a reductase, and a regulatory component. The second strain, <i> Acinetobacter sp. </i> SE19, produces the enzyme needed to oxidize the alcohol, formed by butane monooxygenase, and to cleave the ring. There is a cluster of nine genes that perform this oxidation and cleavage but only six are involved directly.</p><br />
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<h2>Week 4 (May 22 - May 25)</h2><br />
<br />
<p>We started looking into more organisms that can carry out cyclohexane degradation and found one called <i>Brachymonas petroleovorans</i>. This organism is capable of both hydroxylating cyclohexane and carrying out cyclohexanol oxidation. We were also hoping to find evidence that <i>Pseudomonas fluorescens</i>, Pf-5 produces enzymes that can cleave aliphatic rings, however, first we have to determine if Pf-5 can grow in the presence of butylcyclohexane. To do so we carried out a growth assays in LB and the compound butylcyclohexane, testing if it was toxic to the strain Pf-5. After 24 hours of incubation we used a spectrophotometer to take OD600 readings indicating cells density. The results suggested that the presence of butylcyclohexane did not have a detrimental effect on cell growth. We also looked up <i>Pseudomonas fluorescens</i> Pf-5 in the Pseudomonas Genome Database and found genes that code for proteins with similar functions to what we have found for aromatic and aliphatic ring cleavage. </p><br />
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<h2>Week 5 (May 28 - June 1)</h2><br />
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<p>This week we performed the growth/toxin survival assay with Pf-5 and butylcyclohexane using glass vials instead of plastic 15mL tubes. These vials allowed for more surface and better containment of the volatile compound. We carried out this assay using both LB and M9-MM (M9 minimal media). After 24 hours we took spectrophotometer readings at OD600 and obtained similar results as last week. The growth was considerably decreased in the M9-MM, however we are not sure whether the bacteria were able to use the compound as a carbon source because the MM contained glucose. The fact that the growth decreased showed that they might depend on the glucose in the media to grow. </p><br />
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<h2>Week 6 (June 4 - June 8)</h2><br />
<br />
<p> This week the experiment of testing survival and growth was repeated with the intention of determining if butylcyclohexane could be metabolized by the cells. To conclude this, the cells needed to be cultured without glucose. We subcultured from Pf-5 in M9-MM into M9-MM without glucose to avoid carry-over and ensure that the bacteria use butylcyclohexane as their only carbon source. From these cultures we also obtained GC-MS (Gas Chromatography Mass Spectroscopy) readings and compared experimental values of the compound to standards made with water and butylcyclohexane. The Pf-5 samples demonstrated a decrease in amount of butylcyclohexane in the headspace (area above the liquid culture) compared to standards, indicating that the compound was being degraded by Pf-5. Furthermore, we made subcultures from original Pf-5 in M9-MM without glucose into M9-MM and toluene to test for pathways that degrade aromatic structures. </p><br />
<p>We also wanted to identify which genes of Pf-5 that were part of the pathway to degrade toluene and butylcycohexane. We started running blasts on the Pf-5 genome looking for genes that are homologous to ones that we have been looking at for the two ring cleavage pathways (aromatic and aliphatic). In addition we started a verification of <i>XylE</i> (aromatic ring cleavage enzyme) by transforming part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) from the parts registry. We performed two colony PCRs. Unfortunately none of them showed successful results and only the positive control showed an appropriate length DNA. </p><br />
<br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<br />
<p> Continuing with the growth assays of Pf-5 in butylcyclohexane and toluene, we obtained more GC-MS readings. At this point the Pf-5 had been incubated with butylcyclohexane for 6 days and with toluene for 5 days. The amount of both butylcyclohexane and toluene decreased by a large amount in the experimental cultures but the amount of each in the abiotic standards also largely decreased by about the same amount. This lead us to conclude that the reason for decrease was not due to the bacteria metabolizing the compound, but due to unknown abiotic factors. To measure relative amounts of bacterial growth in these cultures we used a spectrophotometer at OD600 to read absorbance for both cultures. Optical density for both cultures was very low indicating very little bacterial growth. </p><br />
<p>The verification of <i>XylE</i> from the parts registry also continued. Three overnight cultures were miniprepped from part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. Unfortunately, the DNA concentrations were very high, likely due to genomic contamination of the miniprep. Also, a second part (<a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) containing <i>XylE</i> and an rbs site was transformed and a colony PCR was performed on part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. The gel did not indicate successful results, as again, only the positive control PCR. </p><br />
<br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<br />
<p>This week we began a new growth assay of Pf-5, but instead of incubating it with butylcyclohexane, we incubated with naphthalene. Since naphthalene is not volatile, we no longer needed to sample the culture headspace but could directly sample remaining naphthalene in the culture itself. Like the previous experiment, we needed to establish rate of growth in M9-MM without glucose and with naphthalene as the sole carbon source. A spectrophotometer was used to measure bacterial growth. The original cultures were grown in LB from a Pf-5 culture in LB and subcultured into M9-MM without glucose. After 24 hours of growth these were subcultured into fresh M9-MM without glucose, again to prevent carryover of glucose from the LB. Positive controls were also made that consisted of M9-MM with glucose and Pf-5. Spectrophotometer readings of the subcultures were taken over a period of 8 days.</p><br />
<p>GC readings were taken of the Pf-5 incubated with butylcyclohexane after 11 days and with toluene after 10 days. The trend remained the same as the measurements taken after 5 and 6 days in the previous week. The amount of compound decreased in the experimental cultures but the amount of compound also decreased in the abiotic standards almost in equal proportions. With the results after 10 and 11 days we could see that the decrease was not due to the bacteria and we decided that the strain we were using did not contain the genes necessary for butylcyclohexane or toluene degradation.</p> <br />
<br />
<p>Throughout the week we continued the verification of <i>XylE</i> by performing a second colony PCR from the transformation plate using part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. Again, the gel did not contain DNA of the right lengths and only the positive control worked. </p><br />
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<h2>Week 9 (June 25 - June 29)</h2><br />
<br />
<p> After the 8 day incubation the readings of the naphthalene assay were graphed and there was a definite increase in the cell growth of the cultures containing naphthalene as a carbon source which indicated that the bacteria were most likely metabolizing the compound. A verification of another part, Alkane hydroxylase (<i>alkB2,rubB,rubA3,rubA4</i>) system (<a href=http://partsregistry.org/Part:BBa_K398014>K398014</a>), from the registry was started. The system consists of 4 genes that have been shown to hydroxylate C5-C8 cycloalkanes. We chose this part to assist in the degradation of cyclohexane and cyclopentane. The transformation was successful and the gel of the colony PCR showed one positive colony but since there was contamination in the gel we were not sure if we could conclude that the PCR had worked. Final GC readings on the butylcyclohexane and toluene cultures were taken and results were graphed. <br />
</p><br />
<br />
</html>[[File:UCalgary2012_Butchgraph.png|frame|center]]<br />
[[File:UCalgary2012_Toluene_assay.png|frame|center]] <br />
[[File:UCalgary2012Week9gelAHS.PNG|500px|thumb|Fig.3 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies 1-5 that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top band in lane 6 indicated successful amplification of the alkane hydroxylase system (2932 bp).|center]]<br />
[[File:UCalgary2012Naphthalene_assay.png|frame|center]]<html><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<br />
<p> This week we performed another colony PCR of 5 colonies from the transformation plate of part <a href=http://partsregistry.org/Part:BBa_K398014>K398014</a> (alkane hydrolase construct: <i>alkB2,rubB,rubA3,rubA4</i>) and ran the products on a gel. We obtained 4 positive colonies and made overnight cultures. We decided to stop using this part for the project and therefore did not require a further miniprep on these overnight cultures. However we made an overnight culture of the positive colony from the alkane hydroxylase system gel (colony 5) and miniprepped the overnight culture. This resulted in a good concentration of 345.6 ng/μL as we verification of<i> XylE</i> by doing two more colony PCRs of each part (<a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) but only the positive control worked when we ran the gel. </p><br />
<br />
</html>[[File:UCalgaryWeek10gelAHS.PNG|500px|thumb|Fig.5 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies A-E that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top bands in lanes 2-4 indicated successful amplification of the alkane hydroxylase system (2932 bp). There was also a band in the negative control indicating contamination so we cannot be positive that the cPCR worked.|center]]<html><br />
<br />
<h2>Week 11 (July 9 - July 13)</h2><br />
<br />
<p> Since we could not obtain any positive colonies from last weeks colony PCRs we began the transformation of each part again. The transformations were successful and when the colony PCR products were run on a gel there were two positive colonies for each part. Overnight cultures of the positive colonies were made. Minipreps were done on the overnight cultures and a restriction digest was performed on the products of the minipreps. The restriction digest products were run on a gel and there was one that looked like it had been successful but due to a poor quality ladder on this gel we could not make any final conclusions on the success of the restriction digest.</p><br />
<br />
</html>[[File:UCalgaryWeek11gelXylE.PNG|500px|thumb|Fig.6 XylE Restriction Digest Gel. Both XylE parts from the registry were transformed into E.coli. One part contained the XylE gene and a native RBS site (J33204) and the other contained glucose repressible promoter (Pcst) and the XylE gene and a native RBS site (K118021). After running a colony PCR on the transformation plates two colonies from each plate looked like they contained the XylE gene. Overnight cultures of these colonies were made and restriction digests were ran on the mini-prepped cells. Lanes 2 and 3 contain the restriction digest product from colonies 2 and 4 on the J33204 plate and lanes 4 and 5 contain the product from colonies 3 and 5 from the k118021 plate. Because of the poor 1 Kb plus ladder it was difficult to tell what size the amplified DNA was so the gel was run again. |center]]<html><br />
<br />
<h2>Week 12 (July 16 - July 20)</h2><br />
<br />
<p> We began the week by rerunning the restriction digest products on a gel with a good ladder and obtained one positive result with two bands. One band was around 1000 bp for the part and the other was at about 2000 bp for the plasmid backbone. We made a streak plate of the colony that had worked for the restriction digest and sent the miniprep product for sequencing. The colony that worked was from the transformation plate of part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. We also repeated a colony PCR of the transformants from part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> using 10 colonies and all of them looked like positive colonies. Overnight culutres were made and a miniprep and restriction digest was completed.</p><br />
<br />
</html>[[File:UCalgary2012Week12agelXylE.PNG|500px|thumb|Fig.7 XylE Restriction Digest Gel. Both XylE parts from the registry were transformed into E.coli. One part contained the XylE gene and a native RBS site (J33204) and the other contained glucose repressible promoter (Pcst) and the XylE gene and a native RBS site (K118021). After running a colony PCR on the transformation plates two colonies from each plate looked like they contained the XylE gene. Overnight cultures of these colonies were made and restriction digests were ran on the mini-prepped cells. Lanes 2 and 3 contain the restriction digest product from colonies 2 and 4 on the J33204 plate and lanes 4 and 5 contain the product from colonies 3 and 5 from the k118021 plate. The two bands in 3rd lane after the 1 Kb plus ladder indicate that the restriction digest for that plasmid was successful. The top band around 2000 base pairs represented the pSB1A3 vector (2155 bp) and the bottom band around 1000 base pairs represented the XylE gene (1097 bp).|center]]<br />
[[File:UCalgary2012Week12bgelXylE.PNG|500px|thumb|Fig.8 Colony PCR gel of XylE with a native rbs site (J33204). Lanes 2-11 represent colonies A to J from the transformation plate. All of these lanes contain a band at the expected size of 958 bp. Lane 12 and 13 represent the positive and negative controls.|center]] <br />
[[File:UCalgary2012Week12cgelXylE.PNG|500px|thumb|Fig.9 Restriction digest gel of XylE gene with rbs site (J33204). Overnight cultures were made from colonies A,B,D,F and J from the Fig. 8 gel. These were mini-prepped and the restriction digest product was run on a gel. The digest of the plasmids from colonies B and D was successful which is shown by the bands at the expected size of 958 bp.|center]]<html><br />
<br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<br />
<p>The colony PCR of part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i> with a native rbs site) was done again and all five colonies looked like they contained the plasmid after running the products on a gel. After miniprepping and running a restriction digest on these five colonies two of them were sent to sequencing. An assay using the compound catechol was also started this week. E.coli cells (top 10) transformed with the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) were used in this assay. Six overnight cultures were made, 3 using M9-MM and 3 using LB. These were spun down and resuspended in fresh M9-MM and LB and brought to a concentration of 0.1 M catechol by using a 1M stock solution. There seemed to be an initial colour change in the reactions from clear to yellow which was only visible in the cultures using M9-MM. All of the cultures were incubated overnight and they turned a black green colour. For this assay we should have used the supernatant because the reaction takes place outside of the cell so this is what we did next week.</p><br />
<br />
<h2>Week 14 (July 30 - August 3)</h2><br />
<br />
<p>The sequencing results for the part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> matched the part so a construction was started. The construction was a 3-way ligation putting the tetR promoter (<a href= http://partsregistry.org/Part:BBa_R0040>R0040</a>) and <i>XylE</i> (<a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) into a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. The ligation product was transformed into competent cells and a colony PCR was done on the transformants using both bio brick and <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> primers. The primers for <i>XylT</i>, which is a ferredoxin that allows for <i>XylE</i> to be activated after a reaction with catechol, arrived and a colony PCR on four different strains of Pseudomonas putida was done. None of the strains seemed to contain the TOL plasmid, which is where <i>XylT</i> is found in certain strains of P. putida, because no bands showed up in the gel. The catechol assay was continued this week. Six overnight cultures were made, 3 using M9-MM and 3 using LB. They were spun down and the supernatant was brought to a catechol concentration of 0.1M, 0.2 M and 0.5 M using a 1M stock solution. The solutions turned light yellow initially, but after a few hours they turned brown. The assay was repeated with the same procedure as above but a control, a colony that did not contain the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>, was added. After the catechol was added to the supernatants there was a colour change from clear to yellow in all of the tubes, even the control. For a negative control catechol was added to M9-MM and this solution became pink. The tubes were incubated on the bench and checked regularly. The colour changed from yellow to pink to brown when they were left on the bench overnight.</p><br />
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<h2>Week 17 (August 20 - August 24)</h2><br />
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<p>The Catechol assay was repeated with a new procedure. Overnight cultures were made in LB and washed the pellet in M9-MM without glucose for varying amounts of time. These cultures were spun down and the supernatant was brought to a concentration of 0.1 M. All of the supernatants became bright yellow except for the control which did not contain the <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. The colour change indicated that catechol was being converted to 2-HMS (2-Hydroxymuconate semialdehyde). The three way ligation didn’t work so a two-way ligation with <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> (tetR) in a <a href=http://partsregistry.org/Part:pSB1A3>pSB1A3</a> vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i>) in a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. One reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the insert, and another reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the insert and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the vector. Both ways worked and the new procedure for the catechol assay was done with the transformed cells and all of the supernatants turned bright yellow.</p><br />
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</html>[[https://2012.igem.org/File:UCalgary2012_Catechol_assay.jpg|500px|thumb||center]]<html><br />
<br />
Fig.10 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added.<br />
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<h2>Week 18 (August 27 - August 31)</h2><br />
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<p>The sequencing results of the ligation were positive so assays were started with the cells transformed with the construct tetR-XylE. The assays involved the use of the petrobrick and a species of micrococcus. The purpose of these experiments was to see if any of the oxygen groups on the product of catechol degradation, which is 2-HMS, were removed.</p><br />
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</html>}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Notebook/CatecholDegradationTeam:Calgary/Notebook/CatecholDegradation2012-10-03T04:08:11Z<p>Stephanie0101: </p>
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<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decatecholization Journal|<br />
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CONTENT=<html><br />
<br />
<h2>Week 1 and 2 (May 1-4 and May 7-11)</h2><br />
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<p>The Catechol degradation part of the project started off as general ring cleavage. The goal was to cleave both the aromatic and aliphatic rings contained in many toxic compounds in the tailings ponds. The first two weeks involved getting familiar with lab procedures and researching aromatic ring cleaving using intra- and extradiol dioxygenases from species of <i>Pseudomonas</i> and <i>Bacillus</i>. We also began literature searches on aliphatic ring cleavage performed by monooxygenases. The project was focused on ring cleavage from week 1-12 and catechol degradation the following weeks.</p><br />
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<h2>Week 3 (May 14 - May 18)</h2><br />
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<p>In the second week we continued to do more research on the degradation of alicyclic compounds and found two strains of bacteria that contained genes needed for this process. The first strain, <i>Thauera butanivorans</i>, contains the genes required to activate the ring by adding a hydroxyl group. The enzyme is called butane monooxygenase and is composed of three subunits, a hydroxylase, a reductase, and a regulatory component. The second strain, <i> Acinetobacter sp. </i> SE19, produces the enzyme needed to oxidize the alcohol, formed by butane monooxygenase, and to cleave the ring. There is a cluster of nine genes that perform this oxidation and cleavage but only six are involved directly.</p><br />
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<h2>Week 4 (May 22 - May 25)</h2><br />
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<p>We started looking into more organisms that can carry out cyclohexane degradation and found one called <i>Brachymonas petroleovorans</i>. This organism is capable of both hydroxylating cyclohexane and carrying out cyclohexanol oxidation. We were also hoping to find evidence that <i>Pseudomonas fluorescens</i>, Pf-5 produces enzymes that can cleave aliphatic rings, however, first we have to determine if Pf-5 can grow in the presence of butylcyclohexane. To do so we carried out a growth assays in LB and the compound butylcyclohexane, testing if it was toxic to the strain Pf-5. After 24 hours of incubation we used a spectrophotometer to take OD600 readings indicating cells density. The results suggested that the presence of butylcyclohexane did not have a detrimental effect on cell growth. We also looked up <i>Pseudomonas fluorescens</i> Pf-5 in the Pseudomonas Genome Database and found genes that code for proteins with similar functions to what we have found for aromatic and aliphatic ring cleavage. </p><br />
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<h2>Week 5 (May 28 - June 1)</h2><br />
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<p>This week we performed the growth/toxin survival assay with Pf-5 and butylcyclohexane using glass vials instead of plastic 15mL tubes. These vials allowed for more surface and better containment of the volatile compound. We carried out this assay using both LB and M9-MM (M9 minimal media). After 24 hours we took spectrophotometer readings at OD600 and obtained similar results as last week. The growth was considerably decreased in the M9-MM, however we are not sure whether the bacteria were able to use the compound as a carbon source because the MM contained glucose. The fact that the growth decreased showed that they might depend on the glucose in the media to grow. </p><br />
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<h2>Week 6 (June 4 - June 8)</h2><br />
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<p> This week the experiment of testing survival and growth was repeated with the intention of determining if butylcyclohexane could be metabolized by the cells. To conclude this, the cells needed to be cultured without glucose. We subcultured from Pf-5 in M9-MM into M9-MM without glucose to avoid carry-over and ensure that the bacteria use butylcyclohexane as their only carbon source. From these cultures we also obtained GC-MS (Gas Chromatography Mass Spectroscopy) readings and compared experimental values of the compound to standards made with water and butylcyclohexane. The Pf-5 samples demonstrated a decrease in amount of butylcyclohexane in the headspace (area above the liquid culture) compared to standards, indicating that the compound was being degraded by Pf-5. Furthermore, we made subcultures from original Pf-5 in M9-MM without glucose into M9-MM and toluene to test for pathways that degrade aromatic structures. </p><br />
<p>We also wanted to identify which genes of Pf-5 that were part of the pathway to degrade toluene and butylcycohexane. We started running blasts on the Pf-5 genome looking for genes that are homologous to ones that we have been looking at for the two ring cleavage pathways (aromatic and aliphatic). In addition we started a verification of <i>XylE</i> (aromatic ring cleavage enzyme) by transforming part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) from the parts registry. We performed two colony PCRs. Unfortunately none of them showed successful results and only the positive control showed an appropriate length DNA. </p><br />
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<h2>Week 7 (June 11 - June 15)</h2><br />
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<p> Continuing with the growth assays of Pf-5 in butylcyclohexane and toluene, we obtained more GC-MS readings. At this point the Pf-5 had been incubated with butylcyclohexane for 6 days and with toluene for 5 days. The amount of both butylcyclohexane and toluene decreased by a large amount in the experimental cultures but the amount of each in the abiotic standards also largely decreased by about the same amount. This lead us to conclude that the reason for decrease was not due to the bacteria metabolizing the compound, but due to unknown abiotic factors. To measure relative amounts of bacterial growth in these cultures we used a spectrophotometer at OD600 to read absorbance for both cultures. Optical density for both cultures was very low indicating very little bacterial growth. </p><br />
<p>The verification of <i>XylE</i> from the parts registry also continued. Three overnight cultures were miniprepped from part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. Unfortunately, the DNA concentrations were very high, likely due to genomic contamination of the miniprep. Also, a second part (<a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) containing <i>XylE</i> and an rbs site was transformed and a colony PCR was performed on part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. The gel did not indicate successful results, as again, only the positive control PCR. </p><br />
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<h2>Week 8 (June 18 - June 22)</h2><br />
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<p>This week we began a new growth assay of Pf-5, but instead of incubating it with butylcyclohexane, we incubated with naphthalene. Since naphthalene is not volatile, we no longer needed to sample the culture headspace but could directly sample remaining naphthalene in the culture itself. Like the previous experiment, we needed to establish rate of growth in M9-MM without glucose and with naphthalene as the sole carbon source. A spectrophotometer was used to measure bacterial growth. The original cultures were grown in LB from a Pf-5 culture in LB and subcultured into M9-MM without glucose. After 24 hours of growth these were subcultured into fresh M9-MM without glucose, again to prevent carryover of glucose from the LB. Positive controls were also made that consisted of M9-MM with glucose and Pf-5. Spectrophotometer readings of the subcultures were taken over a period of 8 days.</p><br />
<p>GC readings were taken of the Pf-5 incubated with butylcyclohexane after 11 days and with toluene after 10 days. The trend remained the same as the measurements taken after 5 and 6 days in the previous week. The amount of compound decreased in the experimental cultures but the amount of compound also decreased in the abiotic standards almost in equal proportions. With the results after 10 and 11 days we could see that the decrease was not due to the bacteria and we decided that the strain we were using did not contain the genes necessary for butylcyclohexane or toluene degradation.</p> <br />
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<p>Throughout the week we continued the verification of <i>XylE</i> by performing a second colony PCR from the transformation plate using part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. Again, the gel did not contain DNA of the right lengths and only the positive control worked. </p><br />
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<h2>Week 9 (June 25 - June 29)</h2><br />
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<p> After the 8 day incubation the readings of the naphthalene assay were graphed and there was a definite increase in the cell growth of the cultures containing naphthalene as a carbon source which indicated that the bacteria were most likely metabolizing the compound. A verification of another part, Alkane hydroxylase (<i>alkB2,rubB,rubA3,rubA4</i>) system (<a href=http://partsregistry.org/Part:BBa_K398014>K398014</a>), from the registry was started. The system consists of 4 genes that have been shown to hydroxylate C5-C8 cycloalkanes. We chose this part to assist in the degradation of cyclohexane and cyclopentane. The transformation was successful and the gel of the colony PCR showed one positive colony but since there was contamination in the gel we were not sure if we could conclude that the PCR had worked. Final GC readings on the butylcyclohexane and toluene cultures were taken and results were graphed. <br />
</p><br />
<br />
</html>[[File:UCalgary2012_Butchgraph.png|frame|center]]<br />
[[File:UCalgary2012_Toluene_assay.png|frame|center]] <br />
[[File:UCalgary2012Week9gelAHS.PNG|500px|thumb|Fig.3 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies 1-5 that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top band in lane 6 indicated successful amplification of the alkane hydroxylase system (2932 bp).|center]]<br />
[[File:UCalgary2012Naphthalene_assay.png|frame|center]]<html><br />
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<h2>Week 10 (July 2-July 6)</h2><br />
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<p> This week we performed another colony PCR of 5 colonies from the transformation plate of part <a href=http://partsregistry.org/Part:BBa_K398014>K398014</a> (alkane hydrolase construct: <i>alkB2,rubB,rubA3,rubA4</i>) and ran the products on a gel. We obtained 4 positive colonies and made overnight cultures. We decided to stop using this part for the project and therefore did not require a further miniprep on these overnight cultures. However we made an overnight culture of the positive colony from the alkane hydroxylase system gel (colony 5) and miniprepped the overnight culture. This resulted in a good concentration of 345.6 ng/μL as we verification of<i> XylE</i> by doing two more colony PCRs of each part (<a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) but only the positive control worked when we ran the gel. </p><br />
<br />
</html>[[File:UCalgaryWeek10gelAHS.PNG|500px|thumb|Fig.5 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies A-E that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top bands in lanes 2-4 indicated successful amplification of the alkane hydroxylase system (2932 bp). There was also a band in the negative control indicating contamination so we cannot be positive that the cPCR worked.|center]]<html><br />
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<h2>Week 11 (July 9 - July 13)</h2><br />
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<p> Since we could not obtain any positive colonies from last weeks colony PCRs we began the transformation of each part again. The transformations were successful and when the colony PCR products were run on a gel there were two positive colonies for each part. Overnight cultures of the positive colonies were made. Minipreps were done on the overnight cultures and a restriction digest was performed on the products of the minipreps. The restriction digest products were run on a gel and there was one that looked like it had been successful but due to a poor quality ladder on this gel we could not make any final conclusions on the success of the restriction digest.</p><br />
<br />
</html>[[File:UCalgaryWeek11gelXylE.PNG|500px|thumb|Fig.6 XylE Restriction Digest Gel. Both XylE parts from the registry were transformed into E.coli. One part contained the XylE gene and a native RBS site (J33204) and the other contained glucose repressible promoter (Pcst) and the XylE gene and a native RBS site (K118021). After running a colony PCR on the transformation plates two colonies from each plate looked like they contained the XylE gene. Overnight cultures of these colonies were made and restriction digests were ran on the mini-prepped cells. Lanes 2 and 3 contain the restriction digest product from colonies 2 and 4 on the J33204 plate and lanes 4 and 5 contain the product from colonies 3 and 5 from the k118021 plate. Because of the poor 1 Kb plus ladder it was difficult to tell what size the amplified DNA was so the gel was run again. |center]]<html><br />
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<h2>Week 12 (July 16 - July 20)</h2><br />
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<p> We began the week by rerunning the restriction digest products on a gel with a good ladder and obtained one positive result with two bands. One band was around 1000 bp for the part and the other was at about 2000 bp for the plasmid backbone. We made a streak plate of the colony that had worked for the restriction digest and sent the miniprep product for sequencing. The colony that worked was from the transformation plate of part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. We also repeated a colony PCR of the transformants from part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> using 10 colonies and all of them looked like positive colonies. Overnight culutres were made and a miniprep and restriction digest was completed.</p><br />
<br />
</html>[[File:UCalgary2012Week12agelXylE.PNG|500px|thumb|Fig.7 XylE Restriction Digest Gel. Both XylE parts from the registry were transformed into E.coli. One part contained the XylE gene and a native RBS site (J33204) and the other contained glucose repressible promoter (Pcst) and the XylE gene and a native RBS site (K118021). After running a colony PCR on the transformation plates two colonies from each plate looked like they contained the XylE gene. Overnight cultures of these colonies were made and restriction digests were ran on the mini-prepped cells. Lanes 2 and 3 contain the restriction digest product from colonies 2 and 4 on the J33204 plate and lanes 4 and 5 contain the product from colonies 3 and 5 from the k118021 plate. The two bands in 3rd lane after the 1 Kb plus ladder indicate that the restriction digest for that plasmid was successful. The top band around 2000 base pairs represented the pSB1A3 vector (2155 bp) and the bottom band around 1000 base pairs represented the XylE gene (1097 bp).|center]]<br />
[[File:UCalgary2012Week12bgelXylE.PNG|500px|thumb|Fig.8 Colony PCR gel of XylE with a native rbs site (J33204). Lanes 2-11 represent colonies A to J from the transformation plate. All of these lanes contain a band at the expected size of 958 bp. Lane 12 and 13 represent the positive and negative controls.|center]] <br />
[[File:UCalgary2012Week12cgelXylE.PNG|500px|thumb|Fig.9 Restriction digest gel of XylE gene with rbs site (J33204). Overnight cultures were made from colonies A,B,D,F and J from the Fig. 8 gel. These were mini-prepped and the restriction digest product was run on a gel. The digest of the plasmids from colonies B and D was successful which is shown by the bands at the expected size of 958 bp.|center]]<html><br />
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<h2>Week 13 (July 23 - July 27)</h2><br />
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<p>The colony PCR of part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i> with a native rbs site) was done again and all five colonies looked like they contained the plasmid after running the products on a gel. After miniprepping and running a restriction digest on these five colonies two of them were sent to sequencing. An assay using the compound catechol was also started this week. E.coli cells (top 10) transformed with the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) were used in this assay. Six overnight cultures were made, 3 using M9-MM and 3 using LB. These were spun down and resuspended in fresh M9-MM and LB and brought to a concentration of 0.1 M catechol by using a 1M stock solution. There seemed to be an initial colour change in the reactions from clear to yellow which was only visible in the cultures using M9-MM. All of the cultures were incubated overnight and they turned a black green colour. For this assay we should have used the supernatant because the reaction takes place outside of the cell so this is what we did next week.</p><br />
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<h2>Week 14 (July 30 - August 3)</h2><br />
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<p>The sequencing results for the part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> matched the part so a construction was started. The construction was a 3-way ligation putting the tetR promoter (<a href= http://partsregistry.org/Part:BBa_R0040>R0040</a>) and <i>XylE</i> (<a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) into a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. The ligation product was transformed into competent cells and a colony PCR was done on the transformants using both bio brick and <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> primers. The primers for <i>XylT</i>, which is a ferredoxin that allows for <i>XylE</i> to be activated after a reaction with catechol, arrived and a colony PCR on four different strains of Pseudomonas putida was done. None of the strains seemed to contain the TOL plasmid, which is where <i>XylT</i> is found in certain strains of P. putida, because no bands showed up in the gel. The catechol assay was continued this week. Six overnight cultures were made, 3 using M9-MM and 3 using LB. They were spun down and the supernatant was brought to a catechol concentration of 0.1M, 0.2 M and 0.5 M using a 1M stock solution. The solutions turned light yellow initially, but after a few hours they turned brown. The assay was repeated with the same procedure as above but a control, a colony that did not contain the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>, was added. After the catechol was added to the supernatants there was a colour change from clear to yellow in all of the tubes, even the control. For a negative control catechol was added to M9-MM and this solution became pink. The tubes were incubated on the bench and checked regularly. The colour changed from yellow to pink to brown when they were left on the bench overnight.</p><br />
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<h2>Week 17 (August 20 - August 24)</h2><br />
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<p>The Catechol assay was repeated with a new procedure. Overnight cultures were made in LB and washed the pellet in M9-MM without glucose for varying amounts of time. These cultures were spun down and the supernatant was brought to a concentration of 0.1 M. All of the supernatants became bright yellow except for the control which did not contain the <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. The colour change indicated that catechol was being converted to 2-HMS (2-Hydroxymuconate semialdehyde). The three way ligation didn’t work so a two-way ligation with <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> (tetR) in a <a href=http://partsregistry.org/Part:pSB1A3>pSB1A3</a> vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i>) in a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. One reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the insert, and another reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the insert and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the vector. Both ways worked and the new procedure for the catechol assay was done with the transformed cells and all of the supernatants turned bright yellow.</p><br />
<br />
</html>[[https://2012.igem.org/File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Fig.10 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added.|center]]<html><br />
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<h2>Week 18 (August 27 - August 31)</h2><br />
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<p>The sequencing results of the ligation were positive so assays were started with the cells transformed with the construct tetR-XylE. The assays involved the use of the petrobrick and a species of micrococcus. The purpose of these experiments was to see if any of the oxygen groups on the product of catechol degradation, which is 2-HMS, were removed.</p><br />
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</html>}}</div>Stephanie0101http://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradationTeam:Calgary/Project/OSCAR/CatecholDegradation2012-10-03T03:59:56Z<p>Stephanie0101: </p>
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<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Catechol Degradation|<br />
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CONTENT=<html><br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_OSCAR_Catechol_Low-Res.png" style="float: right; padding: 10px;"></img><br />
<p><b>****This section needs work. Why are we degrading catechol? What part did we use? What is the number?</b></p><br />
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<p>Catechol is a toxic compound found in tailings ponds that is a by-product of polyaromatic hydrocarbon metabolism. Catechol is toxic to a wide range of organisms from microorganisms to mammals (Schweigert <i>et al.</i>, 2001). The chemical properties of catechol allow it to react with biomolecules like DNA, proteins and membranes (Schweigert <i>et al.</i>, 2001). These interactions can cause serious damage including DNA breakage, enzyme inactivation and membrane uncoupling (Schweigert <i>et al.</i>, 2001). Catechol can be degraded by the enzyme catechol 2,3-dioxygenase encoded by the <i>xylE</i> gene on the Tol plasmid of <i>Pseudomonas putida</i> (Nakai <i>et al.</i>, 1983). The current iGEM Part repository has two BioBricks available of <i>xylE</i>. One contained <i>XylE</i> with its native ribosome-binding site (part: <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>), while the other part contained <i>XylE</i> under the glucose-repressible promoter cstA (Part: <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>). Given that <i>E. coli</i> is grown in the presence of glucose, we designed a new construct to keep <i>XylE</i> repressed by using the TetR promoter (Part:<a href= http://partsregistry.org/Part:BBa_R0040>R0040</a>).</p> <br />
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<h3></h3><br />
</html>[[File:UCalgary2010_R0040-XylE.png|400px|thumb|Fig.1 Genetic circuit for catechol degradation showing <i>XylE</i> biobricked under the TetR promoter|center]]<html><br />
<h3></h3><br />
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<p>Catechol 2,3-dioxygenase is an extradiol dioxygenase which cleaves catechol adjacent to the two hydroxyl groups. When this occurs 2-hydroxymuconate semialdehyde is produced, which is yellow in colour. This change in colour allows for visual assay to assess the activity of <i>XlyE</i>.</p><br />
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</html>[[File:UCalgary2012_Catechol_to_2-HMS.PNG|400px|thumb|Fig.2 Catechol 2,3-dioxygenase (<i>XlyE</i>) converts catechol to 2-Hydroxymuconate semialdehyde in the presence of oxygen. Adapted from Shu ''et al''., 1995.|center]]<html><br />
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<p>The visual assays were performed with <i>E.coli</i> cells transformed with <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> as well as with <i>E.coli</i> cells transformed with the newly constructed part (<a href=http://partsregistry.org/Part:BBa_K902048 >K902048</a>) by bringing the supernatant of an overnight culture to a concentration of 0.1 M of catechol. When the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> was used the pellet was first washed in M9-MM and spun down before catechol was added to the supernatant. This was done to avoid the glucose in the LB from repressing the cstA promoter (<a href=http://partsregistry.org/Part:BBa_K118011 >K118011</a>). The catechol was added to the supernatant because the reaction takes place outside of the cell. Within minutes of the addition of catechol to the supernatant, the solution turned from the pale yellow of LB to a bright yellow. This assay was completed by following the previous assay done by the 2008 Edinburgh iGEM team.</p><br />
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</html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Fig.3 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added. |center]]<html><br />
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<p><b>****This section needs work. Why are we degrading catechol? What part did we use? What is the number?</b></p><br />
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<p>Catechol is a toxic compound found in tailings ponds that is a by-product of polyaromatic hydrocarbon metabolism. Catechol is toxic to a wide range of organisms from microorganisms to mammals (Schweigert <i>et al.</i>, 2001). The chemical properties of catechol allow it to react with biomolecules like DNA, proteins and membranes (Schweigert <i>et al.</i>, 2001). These interactions can cause serious damage including DNA breakage, enzyme inactivation and membrane uncoupling (Schweigert <i>et al.</i>, 2001). Catechol can be degraded by the enzyme catechol 2,3-dioxygenase encoded by the <i>xylE</i> gene on the Tol plasmid of <i>Pseudomonas putida</i> (Nakai <i>et al.</i>, 1983). The current iGEM Part repository has two BioBricks available of <i>xylE</i>. One contained <i>XylE</i> with its native ribosome-binding site (part: <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>), while the other part contained <i>XylE</i> under the glucose-repressible promoter cstA (Part: <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>). Given that <i>E. coli</i> is grown in the presence of glucose, we designed a new construct to keep <i>XylE</i> repressed by using the TetR promoter (Part:<a href= http://partsregistry.org/Part:BBa_R0040>R0040</a>).</p> <br />
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</html>[[File:UCalgary2010_R0040-XylE.png|400px|thumb|Fig.1 Genetic circuit for catechol degradation showing <i>XylE</i> biobricked under the TetR promoter|center]]<html><br />
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<p>Catechol 2,3-dioxygenase is an extradiol dioxygenase which cleaves catechol adjacent to the two hydroxyl groups. When this occurs 2-hydroxymuconate semialdehyde is produced, which is yellow in colour. This change in colour allows for visual assay to assess the activity of <i>XlyE</i>.</p><br />
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</html>[[File:UCalgary2012_Catechol_to_2-HMS.PNG|400px|thumb|Fig.2 Catechol 2,3-dioxygenase (<i>XlyE</i>) converts catechol to 2-Hydroxymuconate semialdehyde in the presence of oxygen. Adapted from Shu ''et al''., 1995.|center]]<html><br />
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<p>The visual assays were performed with <i>E.coli</i> cells transformed with <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> as well as with <i>E.coli</i> cells transformed with the newly constructed part (<a href=http://partsregistry.org/Part:BBa_K902048 >K902048</a>) by bringing the supernatant of an overnight culture to a concentration of 0.1 M of catechol. When the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> was used the pellet was first washed in M9-MM and spun down before catechol was added to the supernatant. This was done to avoid the glucose in the LB from repressing the cstA promoter (<a href=http://partsregistry.org/Part:BBa_K118011 >K118011</a>). The catechol was added to the supernatant because the reaction takes place outside of the cell. Within minutes of the addition of catechol to the supernatant, the solution turned from the pale yellow of LB to a bright yellow. This assay was completed by following the previous assay done by the 2008 Edinburgh iGEM team.</p><br />
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</html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Fig.3 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene. This supernatant remained clear when the catechol was added. |center]]<html><br />
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