http://2012.igem.org/wiki/index.php?title=Special:Contributions/Rsaer&feed=atom&limit=50&target=Rsaer&year=&month=2012.igem.org - User contributions [en]2024-03-28T10:26:40ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:British_Columbia/DataTeam:British Columbia/Data2012-10-04T03:50:38Z<p>Rsaer: </p>
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<font face=arial narrow size=5><b>Our System</b></font></br></br><font face=arial narrow><br />
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<p>We have designed a tunable microbial consortium to distribute the 4S biodesulfurization pathway, responsible for the bio-desulfurization of DBT, as a metabolic network.</p><br />
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<h2>There are two major genetic circuits contained within our system:</h2><br><br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/ProjectConsortia"> first</a> is responsible for tuning the relative populations of the bacteria within the consortium. It is composed of different fluorescence markers under constitutive promoters, used to differentiate member of the population. As well as an amino acid biosynthesis genes under a inducible promoter, used to regulate the bacterial populations within the consortium.</p><br />
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<p>The <a href="https://2012.igem.org/Team:British_Columbia/Desulfurization"> second</a> codes for the distributed 4S pathway, and was created by splitting the <i>dsz</i> operon into each member species of our tunable consortium.</p><br />
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<p align=center><img src="https://static.igem.org/mediawiki/2012/3/3f/Data_Page_diagram_.png"></p><br />
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<font face=arial narrow size=4><b>Data for our Favourite New Parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000"><b>Main Page</a> - Strong Constitutive Promoter-ECFP generator, BBa_K804000:</b> This is an Enhanced Cyan Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses ECFP (BBa_E0420). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, ECFP excites at 439nm and emits at 476nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001"><b>Main Page</a> - Strong constitutive promoter-EYFP generator, BBa_K804001:</b> This is an Enhanced Yellow Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses EYFP (BBa_E0430). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, EYFP excites at 514nm and emits at 527nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804012"><b>Main Page</a> - Rhamnose Inducible TrpA coding gene, BBa_K804012:</b> This part contains a rhamnose inducible (pRha) TrpA coding gene. Upon induction with rhamnose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
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<font face=arial narrow size=4><b>Data for Pre-Existing Parts</b></font></br></br><font face=arial narrow><br />
<b><a href="http://partsregistry.org/Part:BBa_K902065:Experience">Experience</a> - Rhamnose inducible, glucose repressible promoter (pRha), BBa_K902065 (Calgary, iGEM 2012):</b> We placed the Rhamnose promoter upstream of our TrpA and MetA coding genes. In the respective auxotrophs, induction by arabinose resulted in growth compared to a negative control as measured by a plate reader.</br><br />
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<b><a href="http://partsregistry.org/Part:BBa_E0030:Experience">Experience</a> - Enhanced yellow fluorescent protein, BBa_E0030 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001">BBa_K804001</a>) and measured fluorescence and OD by a plate reader.</br><br />
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<b><a href="http://partsregistry.org/Part:BBa_E0020:Experience">Experience</a> - Engineered cyan fluorescent protein, BBa_E0020 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000">BBa_K804000</a>) and measured fluorescence and OD by a plate reader.</br><br />
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<font face=arial narrow size=4><b>We've also characterized the following parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804007"><b>Main Page</a> - Constitutive ECFP generator - Arabinose inducible TrpA coding gene, BBa_K804007:</b> This part contains a constitutive ECFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804009"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible TrpA coding gene, BBa_K804009:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804011"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible MetA coding gene, BBa_K804011:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804002"><b>Main Page</a> - TrpA coding gene, BBa_K804002:</b> This is the TrpA coding gene. It codes for the alpha subunit of tryptophan synthase (TSase α), and functions as both a binding site for indole-3-glycerol-phosphate (InGP) and can catalyze the cleavage of InGP to indole and glyceraldehyde-3-phosphate. This is the same part as BBa_K187028, but is in the pSB1C3 plasmid.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804003"><b>Main Page</a> - TyrA coding gene, BBa_K804003:</b> This is a TyrA coding gene for both the tyrosine and phenylalanine bio-synthetic pathways. TyrA expresses a bifunctional chorismate mutase/prehenate dehydrogenase which catalyzes the conversion of chorismate into prephenate and NAD+-dependent oxidative decarboxylation of prephanate.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804004"><b>Main Page</a> - MetA coding gene, BBa_K804004:</b> This is a MetA coding gene for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><b>Main Page</a> - DszC coding gene, BBa_K804005:</b> This is a DszC coding gene from the DszABC operon in the 4S pathway of <i>Rhodococcus erythropolis</i> IGTS8. It encodes one of the three biodesulfurizing enzymes in the DszABC operon. DszC enzymes have been shown to catalyze the oxidation of dibenzothiophene (DBT)to dibenzothiophene-5-oxide (DBTO) in the first reaction and then from DBTO to DBT sulfoxide (DBTO2) in the second reaction, both in the presence of NADH, oxygen, FMN and flavin reductases.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804006"><b>Main Page</a> - DszD coding gene, BBa_K804006:</b> This is a DszD coding gene isolated from <i>Rhodococcus erythropolis</i> IGTS8, which encodes for a NADH:FMN oxidoreductase to enhance the activities of DszA and DszC in the DszABC operon.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804008"><b>Main Page</a> - Arabinose inducible TrpA coding gene, BBa_K804008:</b> This part contains an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804010"><b>Main Page</a> - Arabinose inducible MetA coding gene, BBa_K804010:</b> This part contains an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804013"><b>Main Page</a> - Rhamnose Inducible MetA coding gene, BBa_K804013:</b> This part contains a rhamnose inducible (pRha) MetA coding gene. Upon induction with rhamnose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
</html></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PartsTeam:British Columbia/Parts2012-10-04T03:45:05Z<p>Rsaer: </p>
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<h1><b><div style="text-align: center;"><font face=arial size="5">Parts Submitted to the Registry</font></div></b></h1><br />
<br /><groupparts>iGEM012 British_Columbia</groupparts></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PartsTeam:British Columbia/Parts2012-10-04T03:44:33Z<p>Rsaer: </p>
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<h1><b><div style="text-align: center;"><font size="5">Parts Submitted to the Registry</font></div></b></h1><br />
<br /><groupparts>iGEM012 British_Columbia</groupparts></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PartsTeam:British Columbia/Parts2012-10-04T03:43:46Z<p>Rsaer: </p>
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<h1><b><div style="text-align: center;">Parts Submitted to the Registry</div></b></h1><br />
<br /><groupparts>iGEM012 British_Columbia</groupparts></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PartsTeam:British Columbia/Parts2012-10-04T03:43:26Z<p>Rsaer: </p>
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<h1><b><br />Parts Submitted to the Registry</div></b></h1><br />
<br /><groupparts>iGEM012 British_Columbia</groupparts></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PartsTeam:British Columbia/Parts2012-10-04T03:42:43Z<p>Rsaer: </p>
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<h1>Parts Submitted to the Registry</h1><br />
<br /><groupparts>iGEM012 British_Columbia</groupparts></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PartsTeam:British Columbia/Parts2012-10-04T03:42:04Z<p>Rsaer: </p>
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<br /><groupparts>iGEM012 British_Columbia</groupparts></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PartsTeam:British Columbia/Parts2012-10-04T03:41:50Z<p>Rsaer: </p>
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<groupparts>iGEM012 British_Columbia</groupparts></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PartsTeam:British Columbia/Parts2012-10-04T03:41:06Z<p>Rsaer: </p>
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<groupparts>iGEM012 British_Columbia</groupparts></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/ConsortiaTeam:British Columbia/Consortia2012-10-04T03:38:45Z<p>Rsaer: </p>
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<font face=arial narrow size=5><b>Modeling Microbial Consortia: The Auxotroph Approach</b></font></br><font face=arial narrow><br />
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<iframe width="640" height="480" src="https://static.igem.org/mediawiki/2012/1/1f/Model_animation.swf" frameborder="0" allowfullscreen></iframe><br />
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To gain a predictive understanding of consortium dynamics in the wet lab, we chose to model a system of simple auxotrophic interdependence. This system also enables us to establish a rapid foundation for which to conduct preliminary model refinements. </br></br><br />
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The three interdependent <i>E. coli</i> auxotrophs studied in our project are <i>ΔtrpA</i>, <i>ΔmetA</i>, and <i>ΔtyrA</i>. These three strains are deficient in the production of the amino acids tryptophan, methionine, and tyrosine, respectively. Thus, in order for each strain to survive, they must exist in the context of a consortium. Our model is designed to predict the growth and survival of such a system, and is based on the measured amino acid excretion rates under basal genomic expression. The model is written such that it can be easily updated when specific tuning of the amino acid production rates is desired (for example, by introducing induction systems for amino acid production <i>in trans</i>), rendering it ideal for modeling tuned and un-tuned consortium dynamics. </br></br><br />
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The key assumption for this model is that the tryptophan, tethionine, and tyrosine are the only growth limiting substrates for the <i>ΔtrpA</i>, <i>ΔmetA</i>, and <i>ΔtyrA</i> <i>E. coli</i> knock outs respectively.</br></br><br />
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One important equation which has been incorporated into our model is the Monod equation. This equation is most commonly used to model microbial growth and has been shown to fit a large variety of empirical data[1]. In addition, it shares the key assumption described above. This equation relates the specific growth rate (µ<sub>g</sub>, the increase in cell mass per unit time) as function of the maximum growth rate (µ<sub>m</sub>), and the concentration of a limiting substrate (S). The equation is written as follows:</br></br><br />
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<p align=center><img src="https://static.igem.org/mediawiki/2012/b/b1/British_Columbia_2012_MonodEquation.png"> </p><br />
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where K<sub>S</sub> is the limiting substrate concentration when the specific growth rate is at half maximum (K<sub>S</sub> = S when µ<sub>g</sub> = µ<sub>m</sub>/2). K<sub>S</sub> is also known as the <i>saturation constant</i> or <i>half-velocity constant</i>[1]. </br></br><br />
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<font face=arial narrow size=4><b>Equations and Constants</b></font></br><font face=arial narrow></br><br />
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Applying the Monod kinetic model, we sought out to measure three variables: the concentration of tryptophan, methionine, and tyrosine in the media available for each specific auxotroph. We were also interested in how these concentrations vary with respect to time. </br></br><br />
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As there is very limited initial availability of each amino acid in the media, <a href="https://2012.igem.org/Team:British_Columbia/ProjectConsortia">the ability of each auxotroph to grow in co-culture</a> demonstrated that the amino acids necessary for growth are being produced and exported from the cell, and that each auxotroph is feeding on the amino acids produced by the other cells in the consortium. For the sake of the model, we assumed that the environmentally released tryptophan, methionine, and tyrosine were only consumed by the respective auxotrophic strain, and that use of this amino acid was funnelled only into cell growth (i.e. increases in amino acid consumption are attributed to changes in cell growth and not other factors, such as cell maintenance).</br></br><br />
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Combining the above assumptions with Monod kinetics, we proposed the following fundamental equations for our consortium model. In this model, each auxotrophic strain is denoted with a superscript minus (i.e. <i>ΔtrpA</i>, <i>ΔmetA</i>, and <i>ΔtyrA</i> are denoted trpA<sup>-</sup>, metA<sup>-</sup>, and tryA<sup>-</sup>, respectively); [Trp], [Met], and [Tyr] represent the concentrations of tryptophan, methionine, and tyrosine; ODtrpA<sup>-</sup>, ODmetA<sup>-</sup>, and ODtyrA<sup>-</sup> represent the optical densities of the <i>ΔtrpA</i>, <i>ΔmetA</i>, and <i>ΔtyrA</i> strains; a1, a2, and a3 are the consumption constants with respect to changes in ODtrpA<sup>-</sup>, ODmetA<sup>-</sup>, and ODtyrA<sup>-</sup>, respectively; µtrpA<sup>-</sup>, µmetA<sup>-</sup>, µtyrA<sup>-</sup> are the specific growth rates of the <i>ΔtrpA</i>, <i>ΔmetA</i>, and <i>ΔtyrA</i> strains; µMaxtrpA<sup>-</sup>, µMaxmetA<sup>-</sup>, µMaxtyrA<sup>-</sup> are the maximum growth rates of the <i>ΔtrpA</i>, <i>ΔmetA</i>, and <i>ΔtyrA</i> strains; and KstrpA<sup>-</sup>, KsmetA<sup>-</sup>, KStyrA<sup>-</sup> are the <i>half-velocity constants</i> of the <i>ΔtrpA</i>, <i>ΔmetA</i>, and <i>ΔtyrA</i> strains. The terms and constants of this equation will be discussed in further detail later.</br></br><br />
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<p align=center><img src="https://static.igem.org/mediawiki/2012/f/f1/British_Columbia_2012_governing_Equation.png"></p><br />
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Please note that: equation (7), (8), and (9) can be substituted into equation (4), (5), and (6) directly, and equation (4), (5), (6) after the substitution can be further substituted into equation (1), (2), and (3) to simplify the code further. The overall three equations shown later in the Matlab Code section is based on the described substitution. It is indicated here for viewers' convenience of following the Matlab code later. </br></br><br />
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In the above equations, some of the key constants for the Monod Kinetics are obtained through analyzing the growth curves in a 96-well plate, with varying amino acid concentrations. Characteristic growth rates were observed and plotted: </br></br><br />
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<p align=center><img src="https://static.igem.org/mediawiki/2012/3/3d/GrowthRatesAA_university_of_british_columbia.png"></br><b>Figure 1: Monoculture Growth Rates at various Limiting Amino Acid Concentrations</br></br><br />
<img src="https://static.igem.org/mediawiki/2012/0/05/GrowthRatesAA_LOG_university_of_british_columbia.png"></br><br />
Figure 2: Log Scale of Monoculture Growth Rates at various Limiting Amino Acid Concentrations</b></br></br><br />
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Note: we used lower amino acid concentrations as well, however we found out that when concentration is below 1E<sup>-7</sup> M, we observed no significant overnight growth of the cells. For a detailed protocol, please visit <a href="https://2012.igem.org/Team:British_Columbia/Protocols/Monod">here</a>.</br></br><br />
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Based on Figures 1 and 2, the key constants, such as maximum growth rates, and half-velocity constants, for each auxotroph were obtained [1]:</br></br><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/b/b8/British_Columbia_2012_constants_from_GrowthRates.png" width=400></p><br />
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As discussed in the beginning of this section, we assumed that the amino acid consumption is solely dependent on the cell growth rate. In order to find out the consumption rate constants, (a1, a2, and a3), we need to find out a way to measure the amino acid concentrations in the media with respect to the change of its OD for each auxotroph. To do this, we need to find a way to measure media amino acid concentrations. With no good access to HPLC, we noticed in previous experiments that each of the knockout monocultures will reach to a certain final OD at a different known concentration of amino acid. In addition, recently published paper: ''A Programmable <i>Escherichia coli</i> Consortium via Tunable Symbiosis'', Kerner A et. al (2012) confirmed that there is indeed a relationship between the final auxotroph ODs with the environmental amino acid concentrations [2]. We thus generated a calibration curve to relate the limiting amino acid concentrations to their final OD readings. We based this on our own wet lab data shown in the figures below: </br></br><br />
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<p align=center><img src="https://static.igem.org/mediawiki/2012/f/f0/ODcalibrationTrp_university_of_british_columbia.png"></br><b><br />
Figure 3: TrpA Auxotroph Final OD and Trp Concentration Calibration Curve</b></br></br><br />
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<img src="https://static.igem.org/mediawiki/2012/b/be/ODcalibrationMet_university_of_british_columbia.png"><br />
</br><b>Figure 4: MetA Auxotrph Final OD and Met Concentration Calibration Curve</b></br></br><br />
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<img src="https://static.igem.org/mediawiki/2012/8/8e/ODcalibrationTyr_university_of_british_columbia.png"><br />
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Figure 5: TyrA Auxotroph Final OD and Tyr Concentration Calibration Curve</b></p><br />
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Note: all the data are the result of triplicate measurements, and a detailed protocol can be found <a href="https://2012.igem.org/Team:British_Columbia/Protocols/AArate">here</a>. We then tested the accuracy of one of the calibration curves. The blue dot in Figure 5 is a data value we obtained at a known Tyrosine concentration (5E<sup>-5</sup> M), and it is reasonably close to the predicted curve. This test point suggests that this calibration curve is relevant to our wet lab results, however more experimental data is necessary to confirm the accuracy of the curve.</br></br><br />
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We then sought out to find out how the amino acid in the environment is consumed with respect to cell growth. </br></br><br />
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The three auxotrophs were grown in 50 ml flasks with known initial Tryptophan, Methionine, and Tyrosine concentrations (5E<sup>-5</sup> M), respectively, and 5ml of cell free supernatants at various ODs was isolated. These supernatants served as new 96-well plate media for each knock out cultures with newly added M9-Glucose shots (2% glucose in the well) to ensure the amino acids of interest remained as the limiting substrate for all knock outs. A detailed protocol can be found <a href="https://2012.igem.org/Team:British_Columbia/Protocols/AArate">here</a>. </br></br><br />
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The cells were grown in a 96-well plate for 15 hours at 37 <sup>o</sup>C, and growth curve data was obtained using a plate reader (most of the monocultures reached stationary phase at this point). </br></br><br />
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Based on the calibration curves (Figure 3, 4, 5), the amino acid concentrations in the wells were calculated from the final OD readings, and when corrected with a dilution factor of 5/4 (as we added in the 5X, it was 1/5 of the total well media volume, as described <a href="https://2012.igem.org/Team:British_Columbia/Protocols/AArate">here</a>), we obtained the resultant amino acid concentrations in the supernatant at various auxotroph monoculture densities. Again, with the assumption that the only change in the amino acid concentration in the media is due to cell growth, and as they are specific knock outs, they were not able to release any amino acid into the environment, each auxotrophs' consumption constants with respect to cell growth rate (a1, a2, a3) can be calculated. Their values are as follows: </br></br><br />
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<p align=center><img src="https://static.igem.org/mediawiki/2012/1/1f/ConsumptionRateConstants_university_of_british_columbia.png"></p><br />
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Now that we have our consupmtion constants, we were interested in finding out how the other two auxotrophs will contribute to changes in environmental amino acid concentrations. </br></br><br />
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With regards to the behaviour of auxotrophs in coculture under changing amino acid concentrations, the closest assumption we can make is that each auxotroph will behave similarly to the conditions of monoculture. Thus, we used the same calibration curve for relating the final OD to the limiting substrate amino acid concentration in the media. The difference here is that, instead of finding out the corresponding limiting amino acid concentration, e.g., <i>ΔmetA</i> media for [Met] measurements, we are interested in measuring the other two non-limiting amino acid concentrations of each knocked out <i>E. coli</i> auxotrophs, e.g. <i>ΔmetA</i> media for [Trp] and [Tyr] concentrations, at various OD readings [2]. We were then able to derive six functions that relate the change of all amino acid concentrations with respect to time (e.g. d[Trp]/dt), based on two auxotrophs' optical densities (e.g. ODmetA<sup>-</sup> and ODtyrA<sup>-</sup>), as shown below: </br></br><br />
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<p align=center><img src="https://static.igem.org/mediawiki/2012/b/b7/Equation10-15_data_analyzed_data_university_of_british_columbia.png"><br />
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Note: These equations can now be directly substituted into equations (1), (2), and (3), in order to simplify the Matlab Code section.</br></br><br />
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One shortcoming in the use of these equations is their dependency on the accuracy of the calibration curves (Figure 3, 4, and 5), as well as the OD readings, especially when OD readings are low and noisy (< 0.1, nearing the limits of the plate reader's accuracy). </br></br><br />
<br />
One extreme example is equation (15). The raw data are shown below: </br></br><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/2/2c/TyrA_in_metAknockoutssupernatants_university_of_british_columbia.png" width=800><br />
</br><b>Figure 6: <i>ΔtyrA</i> auxotroph growth curve in various <i>ΔmetA</i> ODs cell free supernatants in 96-well plate reader environment.</b></p><br />
<br />
Note: As discussed before, this may lead to inaccuracies in calculation from low OD readings. All data sets are labelled correspondingly, and triplicate measurements are noted by wells 1, 2, and 3. As we can see, there are some distinct differences in the OD readings among the three different supernatant data sets; however, when corrected by focusing on the difference between the initial reading and the final reading with a base adjustment of 0.025 (the lower value used for all the calibration curves), they average roughly the same, with an average OD of 0.03847, 0.04043, and 0.04218, respectively, for ODmet 0.219, 0.546, and 0.764. </br></br><br />
<br />
<br />
To illustrate this point more clearly, here is a comparison with another set of growth curve data run in the same plate.</br></br><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/0/05/TyrA_and_trpA_in_metAknockoutssupernatants_university_of_british_columbia.png" width=800></br><b><br />
Figure 7: Comparison between <i>ΔtrpA</i> and <i>ΔtyrA</i> auxotroph growth curve in the same supernatants of <i>ΔmetA</i> at its various optical density media.</p></b><br />
<br />
Note that all three of the trpA<sup>-</sup> sets of growth curve grew to a much higher OD (reaching about 0.17) at the end of 15 hours and did not reach stationary phase, whereas all tyrA<sup>-</sup> sets grew significantly less, no significant change in OD at the end of the experiment.</br></br><br />
<br />
<br />
<font face=arial narrow size=4><b>Matlab Code</b></font></br><font face=arial narrow></br><br />
<br />
Now with all the key functions and constants approximated, we are able to encode our model into Matlab, where the six key ODE functions can be solved and output the our prediction of population dynamics. </br></br><br />
<br />
The code is shown below, which contains a detailed step by step instruction for future iGEM teams or related scientific researches use, as well as the clarifications of our own code. It is also written is a way that it can be directly copied and pasted into Matlab blank m.files with all the comments in Matlab commenting format already, so that the coder can read the coded instructions and explanations in the coding command window without having to switching between, for example, the matlab and browser windows. </br></br><br />
<br />
Of course, copying and pasting the code here is welcomed! </br></br><br />
<br />
<bCode begins here:</b> </br></br><br />
<br />
% input the following commands, which are all in <b>bold</b>, into MATLAB command window, and <b>not the m.file</b>, before you call the ODE function. </br></br><br />
<br />
%firstly, define a time array, set the upper limit to be the time (hr) you want this model to predict. For this model to be accurate, it is advised not to use extreme values. </br></br><br />
<br />
%in our cases, the upper limit is set to 12 as some of the modelling equations are based on 11 hour culture supernatant data. Thus accuracy is expected within 12 hours. </br></br><br />
<br />
%<b> >> xspan = [0 12]</b>; </br></br><br />
<br />
%in order to set up the initial conditions, where they corespond to the initial values of y(1), y(2), y(3)..., to y(6) in our case, and y(n) if you have <i>n</i> number of ODE equations </br></br><br />
<br />
% >><b> ynot = [1E-7 1E-7 1E-7 0.01 0.01 0.01]';</b> </br></br><br />
<br />
% the first three values are the initial values for y(1), y(2), and y(3) respectively, which in out cases are the initial amino acid concentrations, and as we are adding the cells directly into the M9 media without supplementing any amino acids, it is believed to be at very low concentrations, and thus is set to 1E-7 mole/L. </br></br><br />
<br />
% the remaining three values are the initial conditions for y(4), y(5), and y(6), which are the initial cell ODs in out case, and they are all set to 0.01, which is based on the lower values of the OD plate readings after correcting with blanks. </br></br><br />
<br />
%Then RUN THE FUNCTION </br></br><br />
<br />
% >><b>[X,Y] = ode45(@UBCiGEM2012_ConsortiaModel,xspan,ynot);</b> </br></br> <br />
<br />
% this takes the function name in your m.file, the range of your interest, and the initial conditions which is just defined, respectively </br></br><br />
<br />
%Note: have to make sure that the name is exactly the same as the function name in your m.file for it to be able to run correctly. </br></br><br />
<br />
<br />
<b>%the following code is for the actual mfile.</b> </br></br><br />
<br />
% 1) Declare the name of the function: </br></br><br />
<br />
function dy = UBCiGEM2012_ConsortiaModel(t,y); </br></br><br />
<br />
% 2) define all your ODE variables clearly for your own understanding as they can get quite confusing if you don't. </br></br><br />
<br />
% in our case: </br></br><br />
<br />
<b>% y(1) = [Trp]; </br></br><br />
<br />
% dy(1) = d[Trp]/dt; </br></br><br />
<br />
% y(2) = [Met]; </br></br><br />
<br />
% dy(2) = d[Met]/dt; </br></br><br />
<br />
% y(3) = [Tyr]; </br></br><br />
<br />
% dy(3) = d[Tyr]/dt; </br></br><br />
<br />
% y(4) = [OD_trpA-]; </br></br><br />
<br />
% dy(4) = d[OD_trpA-]/dt; </br></br><br />
<br />
% y(5) = [OD_tyrA-]; </br></br><br />
<br />
% dy(5) = d[OD_tyrA-]/dt; </br></br><br />
<br />
% y(6) = [OD_metA-]; </br></br><br />
<br />
% dy(6) = d[OD_metA-]/dt; </br></br></b><br />
<br />
% 3) define your constants first that will not depend on variables </br></br><br />
<br />
% in our case, it was all measured through our own wet lab data and is explained in the previous section </br></br><br />
<br />
%define maximum growth rate constants </br></br><br />
<br />
u_maxtrpA = 0.23; % units = hr-1 </br></br><br />
<br />
u_maxmetA = 0.34; % units = hr-1 </br></br><br />
<br />
u_maxtyrA = 0.28; % units = hr-1 </br></br><br />
<br />
<br />
%define half-velocity constants </br></br><br />
<br />
Ks_trpA = 3.30E-7; % units = M </br></br><br />
<br />
Ks_metA = 5.10E-6; % units = M </br></br><br />
<br />
Ks_tyrA = 8.50E-7; % units = M </br></br><br />
<br />
<br />
%define the consumption rate constants </br></br><br />
<br />
a1 = 4.08E-4; %units= M/OD </br></br><br />
<br />
a2 = 1.44E-4; %units= M/OD </br></br><br />
<br />
a3 = 1.23E-5; %units= M/OD </br></br><br />
<br />
<br />
% 4) write out ODE CODE, suggest to write out all equations on paper first, as they are a lot more clear than coding them directly, and will reduce the chance for you to make error why coding significantly. </br></br><br />
<br />
dy = zeros(6,1); </br></br><br />
<br />
dy(1) = ((1e-8)*(exp(1))^(16.586*y(4))) + ((-0.023*(y(5))^2)+0.021*y(5)-3.5e-3) - a1*(u_maxtrpA*(y(1))/(Ks_trpA+y(1)))*y(4); </br></br><br />
<br />
dy(2) = ((9e-6)*(y(6))^2-(7e-6)*y(6)+1e-6) + (((2e-6)*(y(4))^2)-(2e-6)*y(4)+9e-7) - a2*(u_maxmetA*(y(2))/(Ks_metA+y(2)))*y(5); </br></br><br />
<br />
dy(3) = ((3e-7)*(y(4))^2-(8e-7)*y(4)+4e-7) + (((1e-6)*(y(5))^2)-(2e-6)*y(5)+6e-7) - a3*(u_maxtyrA*(y(3))/(Ks_tyrA+y(3)))*y(6); </br></br><br />
<br />
dy(4) = (u_maxtrpA*y(1))/(Ks_trpA+y(1))*(y(4)); </br></br><br />
<br />
dy(5) = (u_maxmetA*y(2))/(Ks_metA+y(2))*(y(5)); </br></br><br />
<br />
dy(6) = (u_maxtyrA*y(3))/(Ks_tyrA+y(3))*(y(6)); </br></br><br />
<br />
% note: <B>For easy coding and checking purposes, all this equations are exactly the same as the one shown in equation (1), (2), and (3) for dy(1), dy(2), and dy(3) respectively, with also the same arrangements of each equation term within them.</b> </br></br><br />
<br />
% and <b>Please also note:</b> these equation has been derived with direct simple substitution of equations, where equation (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14) and (15) have been substituted into the equation (1), (2) and (3). </br></br><br />
<br />
%if you want, you can choose to plot diagrams and display the predicting dynamics within the m.file or manually plot the arrays of your interests in command window </br></br><br />
<br />
%For example, for this project, we are interesting in predicting the ODs of each auxotrophs, thus, we manually plotted values in y(4), y(5), and y(6) (which again, are the OD values of each auxotrophs) agaisnt time, by typing the following in the command window after run the ODE45 functions: </br></br><br />
<br />
<b>%>>plot(X,Y(:,4:6))<br />
<br />
</br></br><br />
End of the m.file </br></br><br />
<br />
Note: I will indicate here that the direct translation of the previous discussed equations with simple substitutions at either the beginning or the end of this section. </br></br><br />
</b><br />
<font face=arial narrow size=4><b>Simulation Results, Analysis and Future Work</b></font></br><font face=arial narrow><br />
</br><br />
When the Matlab Code was run, it generated a graphic result predicting the population dynamics without inducing, shown below: </br></br><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/c/c8/12hours_nomordificationprediction_university_of_british_columbia.png" width=700></br><br />
<br />
<b>Figure 8. Population Dynamics Prediction within 12-hour Confidence Range</br></br><br />
<img src="https://static.igem.org/mediawiki/2012/c/cc/16hours_nomordificationprediction_university_of_british_columbia.png" width=700></br><br />
<br />
<br />
<br />
Figure 9. Population Dynamics Prediction for 16 hours</br></p></b><br />
<br />
As we can see here, the prediction is no longer reasonable as the <i>ΔtyrA</i> OD shoots off into 1.8 after 16 hours of incubation. </br></br><br />
<br />
However, as we explained in the end of Equation and Constants section, equation (15) can be an outlier equation for this modelling, and the [Tyr] concentration is proven to be so low in the environment that we can barely observe any growth. Thus, we were very interested in finding out how the predictions would be different if we removed equation (15) from the simulation code, and assumed the environmental tyrosine concentration had no relationship with <i>ΔmetA</i> growth. The result of both 12 and 16 hour predictions are shown in Figures 10, and 11. </br></br><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/97/12hours_Withmordificationpredictionno32_university_of_british_columbia.png" width=700></br><b><br />
Figure 10. Population Dynamics 12-hour Prediction after eliminating one potential inaccurate function, equation (15)</br></br><br />
<img src="https://static.igem.org/mediawiki/2012/5/5c/16hours_Withmordificationpredictionno32_university_of_british_columbia.png" width=700></br><br />
Figure 11. Population Dynamics 16-hour Prediction after eliminating one potential inaccurate function, equation (15)</b></br></p><br />
<br />
From these figures, it was not surprising to see that the prediction becomes reasonable once more. This indicates that the attempt of generating the functions to describe d[Tyr]/dt as a function of ODmetA<sup>-</sup> from small OD readings was detrimental accuracy of the model. Assuming this, the growth related tyrosine production or consumption from the environment was so insignificant that it was assumed to be zero (d[Tyr]/dt= f(ODmetA<sup>-</sup>)tyr=0). </br></br><br />
<br />
Also, please note that the 12 hour predictions are similar independent of the inclusion of equation 15. This shows that within 12 hours, our model works accurately despite some of the equations losing accuracy at later time points.</br></br><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/5/5a/12hours_comparisionWithwithoutmordificationpredictionno32_university_of_british_columbia.png" width=700></br><br />
<b><br />
Figure 12. Population Dynamics 12-hour predictions with and without eliminating one potential inaccurate function, equation (15)</b></br></p><br />
<br />
For our <b>future work</b>, we can refine our model if we have more experimental data. This can be accomplished by running the <a href="https://2012.igem.org/Team:British_Columbia/Protocols/AArate">amino acid</a> experiment data points of interest. Alternatively, we can use more direct methods of amino acid measurement, like HPLC analysis.</br></br><br />
<br />
This code can also be adapted to predict the population dynamics of our cocultures when tuning effects are introduced. This is accomplished by by generating six new functions to describe the amino acid dynamics of each monoculture. We can then use this model to decide the amount of tuning (by adding in different amount of inducer) needed to obtain a desired population of each auxotroph.</br></br><br />
<br />
<font face=arial narrow size=4><b>References</b></font></br><font face=arial narrow></br><br />
<br />
[1] Shuler, ML; Kargi, F; Bioprocess Engineering Basic Concepts, 2002 2nd edition. Prentice Hall PTR.</br></br><br />
<br />
[2] Kerner A; Park J; Williams A; Lin XN; A Programmable Escherichia coli Consortium via Tunable Symbiosis, 2012. PLoS ONE 7(3): e34032. doi:10.1371/journal.pone.0034032</div>Rsaerhttp://2012.igem.org/Team:British_Columbia/ConsortiaTunabilityTeam:British Columbia/ConsortiaTunability2012-10-04T03:35:49Z<p>Rsaer: </p>
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[[File:Joe1Team_british_columbia_2012.png|centre|400x500px]]<br />
<br />
<br />
'''July 15'''<br />
<br />
An attempt at doing an experiment to calibrate the fluorescent proteins for the plate reader (looking for quenching, linear relationship between OD and fluorescence, etc) was done on Sunday, but as it turns out, we had the wrong promoter for a fluorescent protein, and the wrong antibiotic resistance for one of the other proteins. Given this, we gave up, and decided to do it later with the proper strains. <br />
<br />
Several new combinations of fluorescent genes, antibiotic resistances, and Keio strains were made. We used the wrong ones last weekend, but now, we are going to test TyrA- +GFP, TrpA- +RFP, MetA- +YFP, and also wildtype with the fluorescent genes. Recently, we switched backbones for the 13M RFP to Psb1C3, for we do not know the provenance of our current psb1C3 with rfp (we suspect it might be on the lac operon, as per the registry guidelines), and we also put the GFP on the psb1C3 instead of a kanamycin resistant plasmid, because we were transforming into kanamycin resistant auxotrophs. <br />
<br />
Just for qualitative fun, we are trying to grow the ArgC- and the ArgE- together in M9 to see if anything results, with negative controls for both. We also tried growing a wildtype with a TrpB auxotroph with rfp to look for qualitative red fluorescence. The fluorescence calibration experiment must be done before we can do these sorts of things quantitatively. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 19'''<br />
<br />
We tested the growth rate of our 3 main auxotrophs in various (log scale from 100 pM to 10 mM) concentrations of the amino acids to do monod kinetics, which will allow us to predict growth rates at various concentrations of the amino acids. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 20'''<br />
<br />
Obtained the data from the 3 main auxotrophs growth rate dependences of amino acid concentration. <br />
<br />
Obtained the reading of fluorescence-population correlation plate. <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''July 22'''<br />
<br />
Joe did 5 more fluorescence-population correlation plate, though found out that the yfp is not expressing correctly. <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''July 23'''<br />
<br />
Analyzed the data, and found out that the growth rate and the amino acid (Tyr) concentration has a correlation as follows: <br />
<br />
[[File:growth-rate at different try concentration_british_columbia_2012.png|650px]]<br />
*Figure 4. cell growth-rate at different Tyr concentrations<br />
<br />
[[File:growth-rate at different tyr concentration log scale_british_columbia_2012.png|650px]]<br />
<br />
*Figure 5. Log scale of cell growth-rate at different Tyr concentrations <br />
<br />
It was found that the cells depleted the nutrient quickly at low Tyr concentration, and for future experiments it is suggested to use lower initial cell concentrations to extend the time of its exponential growth phase. Also, the wild type has almost the exact growth rate as the autotroph when the the Try concentration is exceptionally high (0.01 M). <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''July 26'''<br />
<br />
The growth rate and the amino acids (Met and Trp) concentration has a correlation as follows: <br />
<br />
[[File:growth-rate at different met concentration_british_columbia_2012.png|650px]]<br />
<br />
*Figure 6. cell growth-rate at different Met concentrations<br />
<br />
[[File:growth-rate at different Met concentration log scale_british_columbia_2012.png|650px]]<br />
<br />
*Figure 7. Log scale of cell growth-rate at different Met concentrations <br />
<br />
[[File:growth-rate at different trp concentration_british_columbia_2012.png|650px]]<br />
<br />
*Figure 8. cell growth-rate at different Trp concentrations<br />
<br />
[[File:growth-rate at different trp concentration log scale_british_columbia_2012.png|650px]]<br />
<br />
*Figure 9. Log scale of cell growth-rate at different Trp concentrations <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''August 3rd'''<br />
<br />
Started to work on the data obtained on July 28th plate readings. <br />
<br />
Eventually arrived at the following graphs of growth rates observations, when n=3<br />
<br />
[[file: GrowthRatesAA_university_of_british_columbia.png|650px]]<br />
<br />
[[file: GrowthRatesAA_LOG_university_of_british_columbia.png|650px]]<br />
<br />
From these graphs, the maximum growth rate, and Ks values that are used in Monod Kinetics can be determined. <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''August 12'''<br />
<br />
With a set of the primers that arrived, we were able to PCR amplify certain parts that will be able to be Gibson assembled. On the plasmid that has RFP (a modified BBa_K093012, placed on the Psb1C3), we wanted to put arabinose inducible MetA or arabinose inducible TyrA. However, one of the primers necessary to amplify the plasmid itself, which would be necessary to incorporate the proper homologous sequence for Gibson assembly, did not arrive. Additionally, the PCR to amplify the MetA gene did not work the first time, but has been repeated. The arabinose promoter was successfully PCR'd out of the E. coli genome for both cases. <br />
For the plasmid with YFP, the BBa_I13973, we wanted to put rhamnose inducible TrpA or TyrA after the YFP gene. We received all the primers necessary for this, but the PCR for the plasmid backbone gave several bands and was repeated at a higher annealing temperature. The results are not yet known. The rhamnose promoter was successfully PCR'd out of the genome, as was the TrpA gene. However, we lacked one of the promoters for the TyrA gene. Since there were several bands for the YFP plasmid, we did not do a Gibson assembly. Additionally, it is possible that the primers were designed for the pSB1C3 rather than the pSB1A2, but that should have little effect due to the similarities on the ends of the two plasmids. A simple digest and ligation should be able to move the part from the one plasmid to the other.<br />
We also wanted to put a IPTG inducible (BBa_K091111, LacIQ) metA gene after a GFP. The PCR for the MetA gene and the GFP plasmid were successful (although the GFP plasmid appeared as a fairly weak band on the gel), but the PCR for the LacIQ promoter failed. The initial DNA for this PCR was added directly from the parts distribution kit, and as such the composition and concentration of exactly what was added was unknown. The PCR was repeated as a cPCR on the genome, so as to at least get a usable part with the right overhang. <br />
<br />
In an effort to see how much amino acid each type of cell in the co-culture will produce, we have been harvesting supernatants of our normal cultures as per the protocol in Kerner et al. Once we have supernatants of the cultures at various ODs, we are going to grow the auxotrophs in this supernatant, and then look at the growth rate and final OD. <br />
<br />
Joe has found inconsistent results with his fluorescent proteins on the various promoters, and we have plans to standardize the promoter and rbs for the fluorescent proteins that will be used. Additionally, there is some overlap between YFP and GFP that is proving difficult to account for, so we are going to try to switch one to either BFP or CFP. <br />
<br />
We also wanted to grow three of the consortia together to see what sort of final OD they reach, and have been monitoring it sporadically for the past 24 hours. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''August 13'''<br />
<br />
Having harvested the supernatant of the various cultures at various ODs, we filter sterilized it with a 0.2 uM filter to remove any cells that may have still remained. Now that we have the sterilized supernatant, we are going to follow the protocol in Kerner et al 2012, and add 1/5 5X M9 media, and grow different auxotrophic cultures, noting the OD and growth rate. From this, we will try to calculate, or at least approximate, the amino acid export rate of the cells. <br />
<br />
The data that we collected regarding the growth curves of the auxotrophs and the mixture are very different from what the plate reader previously showed. We think this may be because of a lack of correction for path length by the plate reader, but aren't entirely sure. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''August 15'''<br />
<br />
After two days of shaking at 37° in 1 mM IPTG, the supposed IPTG inducible metA constructs in the metA auxotrophs were unable to grow. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''August 16'''<br />
<br />
The supposed metA constructs still appears unable to complement a metA knockout. It has been suggested that the concentration of metA may have been too high, and this would have led to a severe detriment to the cell, and it has also been suggested that the IPTG, being a rather old stock, was no longer good. Another step that we are going to take is to see if the expression of the construct can be seen on an sds page under induction.<br />
<br />
Concnerning the other constructs, the PCR for the YFP has yet to give us a single solid band, so we are trying to move it into the psb1c3 plasmid in case there are some plasmid specific sequences that were giving us poor results. The ligation and transformation has been done, and we are waiting for colonies. The PCR products necessary for the gibson assembly of the GFP-IPTG-TrpA gene were also all available, and that gibson assembly has been done. However, due to the failure of several gels, there was very little product left, and PCR purification would likely result in unusuable quantities, so we tried a Gibson assembly with the straight PCR product. <br />
<br />
Another PCR was done to generate some of the other parts required for assembly of other parts. The PCR for the arabinose promoter (using a new primer) has not yet resulted in a band. <br />
<br />
There are two distinct bands on the RFP PCR, one at the correct length, the other much lower. Several other products were also generated today, but there are still two essential parts, that of YFP and that of the new arabinose promoter, which have not yet been successfully PCR'd. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''August 17'''<br />
<br />
The Gibson assembled parts using unpurified PCR products did not yield any colonies when transformed.<br />
<br />
The plans for the metA construct induction were further discussed. Overnight colonies will be made of the construct and a negative control, then they will be diluted in 1 in 100 LB, allowed to grow to 0.4 OD, induced with 0.1 mM new IPTG, and harvested at a certain OD following this, then run on an SDS PAGE. This will test to see if the IPTG really would stimulate the production of the metA gene. The negative controls were not in place yet. Four potential constructs have been grown up overnight, and are labeled 1-4 on tape in the right section of the left shaker. <br />
<br />
The data from the supernatant growth experiment was partially analyzed. It appears that 14 hours was not sufficient to reach a final OD in some cases, but there was growth in many of the wells. It was also suggested that we could just test the relevant amino acid concentration using HPLC.<br />
<br />
From our previous experiments concerning the consortia syntrophy, we saw that three cultures inoculated straight from LB into minimal M9 led to a mixed culture OD that reached about 1.3, similar to 0.1 mM (saturating) Met for metA-, and 0.1 mM Trp for TrpA-. We suspect that this is due to syntrophy, but have yet to do the relevant tests to see if 1 in 100 LB actually does supply significant amounts of amino acids. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<div style="text-align: center;"><h2>[https://2012.igem.org/Team:British_Columbia/ConsortiaTunability Back to top]</h2></div></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/ConsortiaDynamicsTeam:British Columbia/ConsortiaDynamics2012-10-04T03:35:13Z<p>Rsaer: </p>
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<br />
'''June 15'''<br />
<br />
The Amp plates made the day before worked. However, the Kan plates (negative control, EPI300 pIJ790, and DH5a pIJ790) showed unexpected results. The negative control, which only had untransformed K12 cells plated, had more colonies growing than those from the plates with resistance-transformed EPI300 pIJ790 and DH5a pIJ790. Even then, there were only a few small colonies on the EPI300 pIJ790 plate and none at all on the DH5a pIJ790 plate. It looks like the Kan plates may not be working, and it is possible that the EPI300 and DH5a cells with the pIJ790 plasmid are not competent.<br />
<br />
- [[User:Tingchiawong|Tingchiawong]]<br />
<br />
<br />
'''June 18'''<br />
Yesterday's plates did not grow, but the experiment was repeated today.<br />
<br />
1) A gel from yesterday's PCR was shown to have some products that worked, while others did not. The MetA, TrpA, TrpB, and ArgE biobrick PCR's worked fine, but the ArgE-Kan cassette and the ArgC biobrick PCRs did not, and were not repeated today. From personal correspondence with Joe, his PCR was not working either, despite trying different cycles and conditions. He suspects that there may be something wrong with the template, given the inconsistent performance of the Kan plates. <br />
<br />
2) The biobrick PCRs that did work were digested with EcoRI and PstI, as was the psb1C3 linearized plasmid backbone. <br />
it was unknown whether or not the plasmid had any methylation, so DpnI was used only in the PCR product digestion. The procedure will be uploaded to the wiki in the near future. A gel was made showing the ligation products.<br />
<br />
3) The biobrick ligation products were used to transform K12 cells and were plated appropriately. The successful amino acid-antibiotic cassettes were also transformed into EPI300, DH5a, and BL21 cells with the appropriate recombineering plasmid, and plated. To date, all the tet resistance strains have been plated, as well as Mehul's Amp resistant strain. The kanamycin resistant strains remain recalcitrant, perhaps due to, again, the inconsistent performance of the kanamycin plates. <br />
<br />
It should be noted that several of the transformations sparked during the electroporation procedure. Some success has been reported with sparked cultures, so they were plated anyways. <br />
<br />
As an update, here is a table showing what has been done so far. <br />
<br />
{| class="wikitable"<br />
|-<br />
| Gene||Type||PCR||Ligation||Transformation<br />
|-<br />
|TrpA+Tet resistance||Recombineering||Successful||NA||?<br />
|-<br />
|TrpB+Kan resistance||Recombineering||Unsuccessful||NA||No<br />
|-<br />
|ArgE+Tet resistance||Recombineering||Successful||NA||?<br />
|-<br />
|ArgC+Kan resistance||Recombineering||Unsuccessful||NA||No<br />
|-<br />
|TyrA+Tet resistance||Recombineering||Successful||NA||?<br />
|-<br />
|MetA+Amp resistance||Recombineering||Successful||NA||?<br />
|-<br />
|MetA||Biobrick||Successful||Done||?<br />
|-<br />
|ArgE||Biobrick||Successful||Done||?<br />
|-<br />
|TrpA||Biobrick||Successful||Done||?<br />
|-<br />
|TrpB||Biobrick||Successful||Done||?<br />
|-<br />
|TyrA||Biobrick||Successful||Done||?<br />
|-<br />
|ArgC||Biobrick||Unsuccessful||No||No<br />
|}<br />
<br />
It should be noted that when a ligation has been done, it does not mean that it was successful. We will not know until we see growth on the plates and a cPCR of the colonies has been done. Even then, it would be useful to have it sequenced. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
Worked with Jacob on steps 3 and 4 listed above. I made the gel, practiced loading samples into wells (with Ruichen), and learned that the machine used to see the results is finicky. Unfortunately, the 1 kb reference ladder did not work (unexpectedly and for unknown reasons).<br />
<br />
(Step 3) Transformation and plating of the biobrick PCR products were the last things we did today. Of the 5, only TrpB sparked during electroporation. 1 uL of PCR product was used to transform cells.<br />
<br />
(Step 4) Of the successful amino acid/antibiotic cassettes, Jacob and I transformed genes "4", "7", "12" (Tet resistant) and "M3" (Amp resistant)- names of the genes to follow - into EPI300, DH5a, and BL21 all with the recombineering plasmid pIJ790. The ones that sparked during electroporation are: 4 (EPI300), 4 (DH5a), 7 (BL21), and M3 (BL21). Quite a mix, but the addition of DMSO and MgCl<sub>2</sub> to boost PCR efficiency increased the salt concentration of the solution and may have caused sparking. Gene 4 had both compounds added and 7 had DMSO (unsure about M3 - that was Mehul). Only 1 uL of PCR product was used to transform cells to reduce chances of sparking, which may have helped.<br />
<br />
- [[User:Grace.yi|Grace.yi]]<br />
<br />
<br />
'''June 19'''<br />
<br />
Designed primers for constitutively expressed GFP, RFP, and YFP to be put in the PCC1FOS vector. This is to allow us to see consortia dynamics by a standard curve of fluorescence. An EcoRI site was added just before the start of the promoter, and a BamHI site was added to the 5' end of the reverse complement. The melting temperature was normalized to about 59°C, and the primers were checked for any secondary structure. <br />
<br />
There were no colonies on the plates transformed with any putative biobricks, so the procedure was attempted again.<br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
After consultation, we moved the IGTS8, IGTS9, and Rhodococcus JHV1 strains that were growing on terrific broth and also ones growing on LB from the 37C shaker to one at 30C.<br />
<br />
- [[User:Tingchiawong|Tingchiawong]]<br />
<br />
<br />
'''June 21'''<br />
<br />
The culture has been proven to be transformed successfully with the 3I plasmid! They grew colonies on the Chlor plate and not the Kan plate. <br />
<br />
Learned how to miniprep<br />
<br />
Checked out some cool posters for the microbiology Conference, and found that there is a assay of interest. The assay (2,6 DCPIP) helps organisms to grow on a diesel contaminated soil. <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
'''June 22'''<br />
<br />
Transformed with the overnight (Room temperature) ligation, using 1 uL ligation mix and 1800V. Joe then plated the cells after recovery. <br />
<br />
Found a MetA biobrick colony on the appropriate plate from 2 days ago. Decided to grow it up in culture and run a PCR to confirm that it was successful. <br />
<br />
Had previously grown up 10 mL of each fluorescent strain (GFP, RFP, YFP) to use for fluorescence calibration, but the plate reader became unavailable. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
We have been having issues with having no colonies grow on plates with cells transformed with PCR products ligated into vectors. One possible cause is the transformation itself. All of Joe's transformation resulted in a spark. He had added 5 uL ligated to competent cells. The high salt concentration may have heavily influenced the sparking. So, I redid some of the transformations in a smaller volume of ligate. I transformed Joe's ligated 3:3 dszB, 3:3 dszC, and 3:1 dszB plasmids into EPI300 cells. Added 0.5 uL ligate to 40 uL competent cells and electroporated them. The 3:1 dszB cells sparked during electroporation. The cells were left to recover undisturbed at 37C for an hour before plating 50 uL transformants onto Chlor plates.<br />
<br />
- [[User:Tingchiawong|Tingchiawong]]<br />
<br />
<br />
'''June 23'''<br />
<br />
Made competent ''Pseudomonas putida'' cells using the [[Competent Cell Production]] protocol.<br />
<br />
We had trouble getting the cells transformed with the ligated amino acid pathway genes to grow when plated. We're unsure if the problem lies in the digest, the ligation, or the transformation. Thus, we decided to redo all these steps.<br />
<br />
Re-digested the "M," "TA," "TB," "TyrA," and "ArgC" PCR products provided by Jacob. Made a master mix of 5 uL NEB buffer 2, 0.5 uL BSA, 0.5 uL EcoRI HF, 0.5 uL PstI, and 18.5 uL dH2O. Incubated each of the mixtures of 4 uL master mix plus 4 uL PCR product in a Thermocycler for ____.<br />
<br />
Re-ligated digest of "M," "TA," "TB," "TyrA," and "ArgC." We suspect that it may be the ligase we used that was at fault for the undesired results seen in previous experiments. So, we decided to add in more ligase and add in ligase from 2 different sources/tubes. We added 1 uL pSBIC3 EP, 4.0 uL dH2O, 1 uL T4 ligase buffer, and 0.5 uL from each tube of T4 ligase were added to 3 uL of the respective digest products. A control was also set up so that there was only plasmid DNA added. The ligate mixtures were placed in the Thermocycler to incubate at 16C for 30 minutes and inactive at 65C for 20 minutes.<br />
<br />
John miniprepped MetA and ArgE, which are being prepared as potential biobricks.<br />
<br />
A new set of PCR reactions was set up for MetA (miniprepped potential biobrick), TyrA (colony PCR), ArgE (miniprepped potential biobrick), and yddG (colony PCR). Made a ligation master mix by combining 60 uL Phusion 5X buffer, 147 uL dH2O, 15 uL DMSO, 12 uL MgCl2, and 18 uL 10 mM dNTP.<br />
<br />
- [[User:Tingchiawong|Tingchiawong]]<br />
<br />
<br />
'''June 25'''<br />
<br />
Designed primers for SDM of dszB gene<br />
<br />
PCRed yddG and Try A with Marianne <br />
<br />
Had a Skype meeting with Calgary iGEM team<br />
<br />
-[[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''June 26'''<br />
<br />
Designed primers for SDM of dszC gene<br />
<br />
Joined Jacob and Joe for transformation of Heat(chemical) competent cells: <br />
<br />
{| class="wikitable"<br />
|-<br />
| Plasmid transformed||Volume of Ligation Mixture used (µL) <br />
|-<br />
|dszB, dszC (3 to 3 ratio, Old and New)||3<br />
|-<br />
|MetA, TrpA, TrpB, TryA, ArgC, positive and negative control||7<br />
|}<br />
<br />
-[[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''June 27'''<br />
<br />
Run the gel of Try A and yddg genes at 95 Volts for 1 hr and 30 min, with 1Kb ladder. <br />
<br />
-[[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''June 30'''<br />
<br />
We picked another candidate colony from the ArgE plate because the PCR of the plasmid between VF2 and VR (standard biobrick sequencing primers) was only about 200 bp, while we were expecting about 1.2 kb. We got the same result from our potential MetA colony. We received new ligase, and if that doesn't work, we are going to try the gibson assembly method. Using a previous restriction digest, we attempted another ligation of our new potential biobricks.<br />
<br />
The first set of chemically competent cells did not grow anything. The procedure was repeated and completed today. The cells were transformed with a known plasmid and appropriately plated. <br />
<br />
Rafael gave us 4 new electroshock cuvettes to temporarily replace the ones that we had destroyed. Joe used 3 of them to test his knockout, and as of today, there is one candidate colony growing on a 1/4 antibiotic concentration plate. A battery of tests have yet to be run to confirm that it is a knockout. <br />
<br />
Yesterday, we went to Dr. Ramey, who had taught students who made a successful knockout using the lambda red system. They used a different recombineering plasmid than we did, and we received this plasmid. It was, however, growing in a strain that we were unfamiliar with, and we decided to isolate the plasmid and transform our Epi300 cells with it. <br />
It is currently growing in a culture at 30°.<br />
<br />
We received only the TrpB gene from the registry, and successfully grew it on a plate. We are now growing a culture to isolate the plasmid and if Joe's knockout is indeed a knockout, we can characterize the existing biobrick part by complementation. <br />
<br />
We ordered the Gibson assembly kit, and it ran upwards of 700 dollars for 50 reactions. <br />
<br />
We made Tet, Kan, and Amp plates with 1/2 and 1/4 antibiotic concentration to try growing our knockouts. There was no growth from a resistance-less K12 cell on any of the plates tested. <br />
<br />
Our PCR for the yddg and tyrA genes was unsuccessful.<br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 1'''<br />
<br />
There are many (hundreds) of colonies on Joe's recombineering plates, and the concentration of colonies goes up as the concentration of antibiotic goes down. It appears that the addition of arabinose to the culture did nothing to increase recombineering efficiency. There are several large, well defined colonies on the full antibiotic plate. It appears that 2 days are required for sufficient growth at 30°.<br />
<br />
I PCR purified a couple of other knockouts (ArgE Tet and TrpB Kan) and transformed them, reusing the electroshock cuvettes we borrowed from Rafael. A pack of 50 was ordered so we could do more than 4 transformations per day. They are currently growing in the 30°C room on the second floor.<br />
<br />
We only had one chlor plate left, and 4 potential biobricks which were to be put on the chloramphenicol resistant pSB1C3 plasmid. Since there was only one plate (and one electroshock cuvette), only the MetA biobrick ligation product was transformed. <br />
<br />
The pKD46 recombineering vector that we received from Dr. Ramey grew well overnight at 30°C, and was miniprepped, and then transformed into the Epi300 strain. The recovery period was done at 30°C as well. If the transformation is successful, Ting will make electrocompetent cells out of the Epi300+pKD46. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 7'''<br />
<br />
In the past couple of days, we have found candidate knockouts for all relevant genes but trpA, and attempted to confirm that they were all actual knockouts by PCR. Unfortunately, none of the PCR's worked. We suspect that it was because of a bad enzyme, so some of the reactions were repeated. Currently, the results are inconclusive as to whether or not the knockouts are actually knockouts. We also ordered the relevant knockouts from the Keio collection.<br />
<br />
Colonies with potential biobricks of trpA, tyrA, argE, metA, and dszD were successfully grown. This time, chemically competent cells were used, and they were plated on 1/2 concentration chlor plates. We also transformed and grew out the previously characterized minC biobrick.<br />
<br />
To physiologically characterize the knockouts, we made M9 plates, and plan to set up an amino acid gradient. Sadly, we ran out of M9 salts before we could make tet plates, so some knockouts can't be tested in this manner yet. We also made spec plates to grow some things from the registry, but a lawn grew on the negative control. Looking into the literature, it looks like the concentrated antibiotic we were using was not the correct strength. <br />
<br />
PCR of the yddg gene failed yet again. It is possible that the primers are not correct. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 9'''<br />
<br />
One of our potential knockouts and a positive control were grown on the M9 plates. There was growth on both plates, although the growth on the positive control was much faster and greater than the potential knockout. Since the potential knockout was able to grow on the amino acid-less M9 media, it appears to be not auxotrophic. Characterization with PCR continues to be difficult, and it was suggested that we simply sequence the strains. <br />
<br />
The plates with the potential knockouts were placed in the 37° incubator overnight to cure the plasmid.<br />
<br />
The potential biobricks were successfully miniprepped, and a PCR was performed to isolate what was actually between the VF2 and the VR primers in the plasmid. The product will then be run on a gel, and if it appears the correct length, will be sent for sequencing.<br />
<br />
Joe made M9 plates with an amino acid, to further test his knockout. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 10'''<br />
<br />
The PCR to isolate and amplify the insert from the potential biobrick showed many bands, characteristic of non-specific primer binding. As another test to see if the insert was correct, we cut with EcoRI and PstI to look for the appropriate insert size, and indeed found that there was an insert at about the right size. <br />
<br />
Cameron reports knockouts from Yale.<br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 15'''<br />
<br />
Sent the potential biobricks for sequencing last Friday, ordered primers for mutagenesis on DszC, ordered primers for attachment of fluorescent genes to amino acid genes, received all but yddg knockouts from Yale, found that the Arg knockouts work as described. <br />
<br />
An attempt at doing an experiment to calibrate the fluorescent proteins for the plate reader (looking for quenching, linear relationship between OD and fluorescence, etc) was done on Sunday, but as it turns out, we had the wrong promoter for a fluorescent protein, and the wrong antibiotic resistance for one of the other proteins.<br />
<br />
Competent cells were made from each of the knockouts, and they worked well. The yddg strain was ordered through collaboration with Calgary as well as the keio collection proper in Japan. <br />
<br />
The sequencing results from our amino acid biobricks came back, and it looks like only MetA was successful. It should be noted that the melting temperatures of the Vf2 and the VR primers are 60°, for cPCR validation of biobricks.<br />
<br />
Several new combinations of fluorescent genes, antibiotic resistances, and Keio strains were made. We used the wrong ones last weekend, but now, we are going to test TyrA- +GFP, TrpA- +RFP, MetA- +YFP, and also wildtype with the fluorescent genes. Recently, we switched backbones for the 13M RFP to Psb1C3, for we do not know the provenance of our current psb1C3 with rfp (we suspect it might be on the lac operon, as per the registry guidelines), and we also put the GFP on the psb1C3 instead of a kanamycin resistant plasmid, because we were transforming into kanamycin resistant auxotrophs. <br />
<br />
Just for qualitative fun, we are trying to grow the ArgC- and the ArgE- together in M9 to see if anything results, with negative controls for both. We also tried growing a wildtype with a TrpB auxotroph with rfp to look for qualitative red fluorescence. The fluorescence calibration experiment must be done before we can do these sorts of things quantitatively. <br />
<br />
We miniprepped 4/6 of the biobricks that we ordered from the registry. We intend on sequencing them in the near future. We, lacking LB amp plates at the time, plated one on an M9 plate, but we now think that the strain the registry sent us is auxotrophic for something. We replated on LB amp after making new plates. The last one that we ordered did not arrive. <br />
<br />
Joe made M9 plates with and without 1 mM arginine, and it was found that the two arginine auxotrophs grew as expected. <br />
<br />
We bought a fridge for our new office, but have yet to fill it. If anyone with extra snacks reads this and wants to help, our fridge is currently underfilled. <br />
<br />
Overnight cultures have been set up to make competent cells out of DH5a, K12, and IGTS8.<br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 17'''<br />
<br />
Today, we made dh5a and k12 competent cells. We also ran a cPCR on our biobrick colonies that turned out to not be biobricks.<br />
<br />
As said yesterday, we grew the two arg auxotrophs together, and they grew substantially more (~0.15 OD600) than the negative control. The wt grew to an OD of about 1. We are growing them for another night to see if the OD of the mix increases at all. <br />
<br />
Since sequencing confirmed a MetA biobrick, we transformed K12 cells with the remaining stock of our plasmid, so we could amplify it. <br />
<br />
All relevant transformed strains from yesterday grew well on the plates. We are now ready to properly do the fluorescence calibration experiment, but due to time constraints, we are going to do it on Thursday.<br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
[[File:Pseudomonas Crystals.jpg|650px]]<br />
<br />
<br />
'''July 19'''<br />
<br />
We tested the growth rate of our 3 main auxotrophs in various (log scale from 100 pM to 10 mM) concentrations of the amino acids to do monod kinetics. We also miniprepped our metA biobrick (so we have enough to send to the registry and use for experiments), new potential candidates for other biobricks (tyrA and ArgC), our construct which is identical to 13M on plate 3, except on a different plasmid, and a biobrick that we received from the registry. Sequencing to follow. <br />
<br />
Joe has, to the best of my knowledge, identified potential biobrick colonies for the DszB,C, and D. Again, sequencing to follow. <br />
<br />
It appears that Joe and Grace were attempting the fluorescence calibration experiment, but things apparently were going wrong, and we have not yet analyzed the data completely. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 20'''<br />
<br />
Obtained the data from the 3 main auxotrophs growth rate dependences of amino acid concentration. <br />
<br />
Obtained the reading of fluorescence-population correlation plate. <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''July 22'''<br />
<br />
Joe did 5 more fluorescence-population correlation plate, though found out that the yfp is not expressing correctly. <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''July 23'''<br />
<br />
Analyzed the data, and found out that the growth rate and the amino acid (Tyr) concentration has a correlation as follows: <br />
<br />
[[File:growth-rate at different try concentration_british_columbia_2012.png|650px]]<br />
*Figure 4. cell growth-rate at different Tyr concentrations<br />
<br />
[[File:growth-rate at different tyr concentration log scale_british_columbia_2012.png|650px]]<br />
<br />
*Figure 5. Log scale of cell growth-rate at different Tyr concentrations <br />
<br />
It was found that the cells depleted the nutrient quickly at low Tyr concentration, and for future experiments it is suggested to use lower initial cell concentrations to extend the time of its exponential growth phase. Also, the wild type has almost the exact growth rate as the autotroph when the the Try concentration is exceptionally high (0.01 M). <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''July 26'''<br />
<br />
The growth rate and the amino acids (Met and Trp) concentration has a correlation as follows: <br />
<br />
[[File:growth-rate at different met concentration_british_columbia_2012.png|650px]]<br />
<br />
*Figure 6. cell growth-rate at different Met concentrations<br />
<br />
[[File:growth-rate at different Met concentration log scale_british_columbia_2012.png|650px]]<br />
<br />
*Figure 7. Log scale of cell growth-rate at different Met concentrations <br />
<br />
[[File:growth-rate at different trp concentration_british_columbia_2012.png|650px]]<br />
<br />
*Figure 8. cell growth-rate at different Trp concentrations<br />
<br />
[[File:growth-rate at different trp concentration log scale_british_columbia_2012.png|650px]]<br />
<br />
*Figure 9. Log scale of cell growth-rate at different Trp concentrations <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''August 3rd'''<br />
<br />
Started to work on the data obtained on July 28th plate readings. <br />
<br />
Eventually arrived at the following graphs of growth rates observations, when n=3<br />
<br />
[[file: GrowthRatesAA_university_of_british_columbia.png|650px]]<br />
<br />
[[file: GrowthRatesAA_LOG_university_of_british_columbia.png|650px]]<br />
<br />
From these graphs, the maximum growth rate, and Ks values that are used in Monod Kinetics can be determined. <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''August 12'''<br />
<br />
With a set of the primers that has just arrived, we were able to PCR amplify certain parts that will be able to be Gibson assembled. On the plasmid that has RFP (a modified BBa_K093012, placed on the Psb1C3), we wanted to put arabinose inducible MetA or arabinose inducible TyrA. However, one of the primers necessary to amplify the plasmid itself, which would be necessary to incorporate the proper homologous sequence for Gibson assembly, did not arrive. Additionally, the PCR to amplify the MetA gene did not work the first time, but has been repeated. The arabinose promoter was successfully PCR'd out of the E. coli genome for both cases. <br />
For the plasmid with YFP, the BBa_I13973, we wanted to put rhamnose inducible TrpA or TyrA after the YFP gene. We received all the primers necessary for this, but the PCR for the plasmid backbone gave several bands and was repeated at a higher annealing temperature. The results are not yet known. The rhamnose promoter was successfully PCR'd out of the genome, as was the TrpA gene. However, we lacked one of the promoters for the TyrA gene. Since there were several bands for the YFP plasmid, we did not do a Gibson assembly. Additionally, it is possible that the primers were designed for the pSB1C3 rather than the pSB1A2, but that should have little effect due to the similarities on the ends of the two plasmids. A simple digest and ligation should be able to move the part from the one plasmid to the other.<br />
We also wanted to put a IPTG inducible (BBa_K091111, LacIQ) metA gene after a GFP. The PCR for the MetA gene and the GFP plasmid were successful (although the GFP plasmid appeared as a fairly weak band on the gel), but the PCR for the LacIQ promoter failed. The initial DNA for this PCR was added directly from the parts distribution kit, and as such the composition and concentration of exactly what was added was unknown. The PCR was repeated as a cPCR on the genome, so as to at least get a usable part with the right overhang. <br />
<br />
In an effort to see how much amino acid each type of cell in the co-culture will produce, we have been harvesting supernatants of our normal cultures as per the protocol in Kerner et al. Once we have supernatants of the cultures at various ODs, we are going to grow the auxotrophs in this supernatant, and then look at the growth rate and final OD. The list of which supernatants we have can be found in the lab. <br />
<br />
Joe has found inconsistent results with his fluorescent proteins on the various promoters, and we have plans to standardize the promoter and rbs for the fluorescent proteins that will be used. Additionally, there is some overlap between YFP and GFP that is proving difficult to account for, so we are going to try to switch one to either BFP or CFP. <br />
<br />
We also wanted to grow three of the consortia together to see what sort of final OD they reach, and have been monitoring it sporadically for the past 24 hours. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''August 13'''<br />
<br />
Having harvested the supernatant of the various cultures at various ODs, we filter sterilized it with a 0.2 uM filter to remove any cells that may have still remained. Now that we have the sterilized supernatant, we are going to follow the protocol in Kerner et al 2012, and add 1/5 5X M9 media, and grow different auxotrophic cultures, noting the OD and growth rate. From this, we will try to calculate, or at least approximate, the amino acid export rate of the cells. <br />
<br />
There were colonies on the MinC Intein plate, the ArgE BB plate, and the GFP construct LB plate, but nothing on the GFP construct M9 plate. This may have been because there was no lactose or IPTG to induce the metA gene. Interestingly, there was a green fluorescent patch, but it was rather weak and had no distinct colonies. We plan to pick several colonies from the LB plate and try growing them in M9 culture with and without IPTG overnight. We also plan to do a cPCR on the MinC plate using the intein primers. There were no colonies on the DszA or yddg plate. We have had no luck with electroporating cells with ligation mixtures, although we have had some success with heat shock competent cells. We think there may be something in the ligation mixture that is interfering with the electroporation, and could probably be fixed by purifying the ligation mix. <br />
<br />
The data that we collected regarding the growth curves of the auxotrophs and the mixture are very different from what the plate reader previously showed. We think this may be because of a lack of correction for path length by the plate reader, but aren't entirely sure. <br />
<br />
We also did a PCR on the TyrA BB product, the previous failed YFP plasmid, and several other previously failed PCRs. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''August 15'''<br />
<br />
After two days of shaking at 37° in 1 mM IPTG, the supposed IPTG inducible metA constructs in the metA auxotrophs were unable to grow. <br />
<br />
The gels of the PCR products that we have been getting lately have not been working properly. We think that there is a problem with the buffer due to how many times it has been reusued, or possibly by the new TBE that was recently made. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''August 16'''<br />
<br />
The supposed metA constructs still appears unable to complement a metA knockout. It has been suggested that the concentration of metA may have been too high, and this would have led to a severe detriment to the cell, and it has also been suggested that the IPTG, being a rather old stock, was no longer good. Another step that we are going to take is to see if the expression of the construct can be seen on an sds page under induction.<br />
<br />
Despite, or perhaps because of this failure, we are moving on, trying to make new constructs that will be used for tunability. The YFP has yet to give us a solid band, so we are trying to move it into the psb1c3 plasmid in case there are some plasmid specific sequences that were giving us poor results. The ligation and transformation has been done, and we are waiting for colonies. The PCR products necessary for the gibson assembly of the GFP-IPTG-TrpA gene were also all available, and that gibson assembly has been done. However, due to the failure of several gels, there was very little product left, and PCR purification would likely result in unusuable quantities, so we tried a Gibson assembly with the straight PCR product, and we will see if it works. <br />
<br />
Another PCR was done to generate some of the other parts required for assembly of other parts. The new forward primer for the arabinose promoter does not seem to be working, although the old one worked well. We recently ran out of phusion polymerase, and have resorted to using an eclectic mix of collected polymerases in our PCR box. <br />
<br />
There are two distinct bands on the RFP PCR, one at the correct length, the other much lower. Seems like a perfect chance for gel extraction. Several other products were also generated today, but there are still two essential parts, that of YFP and that of the new arabinose promoter, which have not yet been successfully PCR'd. <br />
<br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''August 17'''<br />
<br />
The plans for the metA construct induction were further discussed. Overnight colonies will be made of the construct and a negative control, then they will be diluted in 1 in 100 LB, allowed to grow to 0.4 OD, induced with 0.1 mM new IPTG, and harvested at a certain OD following this, then run on an SDS PAGE. This will test to see if the IPTG really would stimulate the production of the metA gene. The negative controls were not in place yet. Four potential constructs have been grown up overnight, and are labeled 1-4 on tape in the right section of the left shaker. Is there some label on the metA protein that we could use to do a Western? <br />
<br />
The data from the supernatant growth experiment was partially analyzed. It appears that 14 hours was not sufficient to reach a final OD in some cases, but there was growth in many of the wells. It was also suggested that we could just test the relevant amino acid concentration using HPLC. One thing that I was wary about concerning this is that the HPLC would only pick up pure amino acid, and if there was, for instance, a short protein fragment, it could be used for growth, but not show up on the HPLC. Any thoughts?<br />
<br />
From our previous experiments concerning the consortia syntrophy, we saw that three cultures inoculated straight from LB into minimal M9 led to a mixed culture OD that reached about 1.3, similar to 0.1 mM (saturating) Met for metA-, and 0.1 mM Trp for TrpA-. We suspect that this is due to syntrophy, but have yet to do the relevant tests to see if 1 in 100 LB actually does supply significant amounts of amino acids. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<div style="text-align: center;"><h2>[https://2012.igem.org/Team:British_Columbia/ConsortiaDynamics Back to top]</h2></div></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/BiobrickConstructionTeam:British Columbia/BiobrickConstruction2012-10-04T03:34:31Z<p>Rsaer: </p>
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<br />
<br />
'''June 15'''<br />
<br />
1)Miniprepped the resistance cassette cultures. Previously, to get the part for the template for the PCR, we just picked a colony, but we thought that it would be beneficial to have a plasmid stock in case we needed to repeat the PCR. <br />
<br />
2)PCR with new (biobrick) primers was attempted. We received primers for the Dsz genes and also for the amino acid genes. We did not, however, receive the DszA forward primer, and decided to hold off on working with the Dsz genes until later. We performed a cPCR for all the genes we had, and ran the results on the gel.<br />
<br />
3)Ran an important gel. Below are the results from yesterday's and today's PCR. Cameron made the gel and taped the edges of the gel box to prevent the gel from escaping. The gel itself was made to 1% agarose in 0.5% TBE, and run on a 50 well gel box. 3 uL of loading buffer were used for 8 uL (10 uL for yesterday's products) sample to increase contrast, and various ladders were tested. The negative control PCR tube for today's PCR popped open during the reaction, and evaporated.<br />
<br />
# Broad Range Ladder<br />
# TrpA (biobricks)+DMSO<br />
# TrpA <br />
# TrpB +DMSO<br />
# TrpB <br />
# ArgC +DMSO<br />
# ArgC <br />
# ArgE +DMSO<br />
# ArgE<br />
# MetA +DMSO<br />
# MetA<br />
# TyrA +DMSO<br />
# TyrA <br />
# empty<br />
# 1kb ladder<br />
# Mehul's Stuff metA p1002 Kan<br />
# Mehul's Stuff metA p1002 Kan<br />
# Mehul's Stuff metA p1002 Kan<br />
# Mehul's Stuff metA p1002 Kan<br />
# empty<br />
# Ruichen's Stuff<br />
# Ruichen's Stuff<br />
# Ruichen's Stuff<br />
# Ruichen's Stuff<br />
# Ruichen's Stuff<br />
# empty<br />
# 1kb + Ladder<br />
# TrpA tet <br />
# TrpA tet +DMSO<br />
# TrpA tet +MgCl2<br />
# TrpA tet +DMSO+MgCl2<br />
# ArgE tet <br />
# ArgE tet +DMSO<br />
# ArgE tet +MgCl2<br />
# ArgE tet +DMSO+MgCl2<br />
# TyrA tet <br />
# TyrA tet +DMSO<br />
# TyrA tet +MgCl2<br />
# TyrA tet +DMSO+MgCl2<br />
<br />
[[File:June15igemmyriad.png |650px]]<br />
* Figure 1. Myriad Gel <br />
<br />
As you can see, we have successful bands for tyrA (from genome, biobrick primers) with DMSO, MetA with kanamycin resistance (for knockouts), trpA with tetracycline resistance, ArgE with tetracycline resistance, and tyrA with tetracyline resistance. The 1 kb+ ladder appears the most useful in this experiment. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
The Amp plates made the day before worked. However, the Kan plates (negative control, EPI300 pIJ790, and DH5a pIJ790) showed unexpected results. The negative control, which only had untransformed K12 cells plated, had more colonies growing than those from the plates with resistance-transformed EPI300 pIJ790 and DH5a pIJ790. Even then, there were only a few small colonies on the EPI300 pIJ790 plate and none at all on the DH5a pIJ790 plate. It looks like the Kan plates may not be working, and it is possible that the EPI300 and DH5a cells with the pIJ790 plasmid are not competent.<br />
<br />
- [[User:Tingchiawong|Tingchiawong]]<br />
<br />
<br />
'''June 16'''<br />
<br />
The time I came in Joe was making the PCR gel. <br />
<br />
The cultures with 3I plasmid that left for growth in the 37 degree room actually did turn a bit cloudy (6:00 p.m.).<br />
<br />
Their ODs were then measured with a blank LB for reference, and the detailed info will be updated. <br />
<br />
For the remaining cultures, 100µl of each culture was tested on Chlor plates with an control of K12 (20µl) to see if they are actually the cell cultures that of our interest, and also testing the chlor plates that are newly made, and the remaining cultures were stored in the 4 degree room for potential future usage. <br />
<br />
The plates were left in the 37 degree room, and will be collected by Joe and stored in the 4 degree room. <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''June 17'''<br />
<br />
As shown above in the gel image, there is still some PCR that needs to be done. It appears apparent that increased concentrations of DMSO and magnesium chloride help the reaction, so the appropriate amount of each was added to the mastermix. The reactions were as follows: TrpB with Kan cassette, TrpA, TrpB, ArgC, and MetA. <br />
<br />
Since we were planning to put these into biobricks, it was imperative that we made sure that there were no restriction sites. To do this, we used the NCBI data for the gene sequence and ran it through Nebcutter, searching for all illegal sites. No illegal sites were found in TrpB, TyrA, TrpA, ArgC, or MetA. Lucky.<br />
<br />
As shown above, a reaction for the tyrA gene worked. Using this PCR product and the rfp plasmid, we digested with EcoRI and PstI and ligated with T4 DNA ligase. The resultant product was used to transform Epi300 cells. Tomorrow, we will check for white colonies among the red ones, which would suggest that the ligation was successful. A colony PCR will be done to confirm.<br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''June 18'''<br />
<br />
1) A gel from yesterday's PCR was shown to have some products that worked, while others did not. The MetA, TrpA, TrpB, and ArgE biobrick PCR's appear to have product.<br />
<br />
2) The biobrick PCRs that did work were digested with EcoRI and PstI, as was the pSB1C3 linearized plasmid backbone. <br />
It was unknown whether or not the plasmid had any methylation, so DpnI was used only in the PCR product digestion. The procedure will be uploaded to the wiki in the near future. A gel was made showing the ligation products.<br />
<br />
3) The biobrick ligation products were used to transform K12 cells and were plated appropriately.<br />
<br />
It should be noted that several of the transformations sparked during the electroporation procedure. Some success has been reported with sparked cultures, so they were plated anyways. <br />
<br />
As an update, here is a table showing what has been done so far. <br />
<br />
{| class="wikitable"<br />
|-<br />
| Gene||Type||PCR||Ligation||Transformation<br />
|-<br />
|TrpA+Tet resistance||Recombineering||Successful||NA||?<br />
|-<br />
|TrpB+Kan resistance||Recombineering||Unsuccessful||NA||No<br />
|-<br />
|ArgE+Tet resistance||Recombineering||Successful||NA||?<br />
|-<br />
|ArgC+Kan resistance||Recombineering||Unsuccessful||NA||No<br />
|-<br />
|TyrA+Tet resistance||Recombineering||Successful||NA||?<br />
|-<br />
|MetA+Amp resistance||Recombineering||Successful||NA||?<br />
|-<br />
|MetA||Biobrick||Successful||Done||?<br />
|-<br />
|ArgE||Biobrick||Successful||Done||?<br />
|-<br />
|TrpA||Biobrick||Successful||Done||?<br />
|-<br />
|TrpB||Biobrick||Successful||Done||?<br />
|-<br />
|TyrA||Biobrick||Successful||Done||?<br />
|-<br />
|ArgC||Biobrick||Unsuccessful||No||No<br />
|}<br />
<br />
It should be noted that when a ligation has been done, it does not mean that it was successful. We will not know until we see growth on the plates and a cPCR of the colonies has been done. Even then, it would be useful to have it sequenced. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
Worked with Jacob on steps 3 and 4 listed above. I made the gel, practiced loading samples into wells (with Ruichen), and learned that the machine used to see the results is finnicky. Unfortunately, the 1 kb reference ladder did not work (unexpectedly and for unknown reasons).<br />
<br />
(Step 3) Transformation and plating of the biobrick PCR products were the last things we did today. Of the 5, only TrpB sparked during electroporation. 1 uL of PCR product was used to transform cells.<br />
<br />
- [[User:Grace.yi|Grace.yi]]<br />
<br />
<br />
'''June 19'''<br />
<br />
There were no colonies on the plates transformed with any putative biobricks, so the procedure was attempted again.<br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''June 20'''<br />
<br />
Plated the cultures with 3I plasmid on Kan for control and Chlor for testing purpose. <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
Still no biobrick colonies. Tried again with an altered ligation protocol. Also ordered a variety of kill switches from the registry, along with Joe's Panamanian Pseudomonas Rhamnosyltransferase. Repeated PCR on things that had previously failed, and got the final amino acid biobrick. It should be noted that I ran reactions with either PFU or phusion, and only the phusion worked. This may have been because of the low volume (0.5 uL) used.<br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''June 21'''<br />
<br />
The culture has been proven to be transformed successfully with the 3I plasmid! They grew colonies on the Chlor plate and not the Kan plate. <br />
<br />
Learned how to miniprep<br />
<br />
Checked out some cool posters for the microbiology Conference, and found that there is a assay of interest. The assay (2,6 DCPIP) helps organisms to grow on a diesel contaminated soil. <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
All amino acids have been PCR'd out of the genome, and several colonies appeared on the ArgE ligation transformation plate. One colony was selected and grown in culture. The rest of the biobrick ligations were redone and allowed to ligate overnight. <br />
<br />
The digests of the products were done with EcoRI and PstI in buffer 3, as opposed to the EcoRI-HF and PstI in buffer 2 we did previously. This was done because PstI has 100% activity in buffer 3, as opposed to 75% activity in buffer 2. Also, we learned that EcoRI-HF has 0% activity in buffer 3. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
'''June 22'''<br />
<br />
Transformed with the overnight (Room temperature) ligation, using 1 uL ligation mix and 1800V. Joe then plated the cells after recovery. <br />
<br />
Found a MetA biobrick colony on the appropriate plate from 2 days ago. Decided to grow it up in culture and run a PCR to confirm that it was successful. <br />
<br />
Wanted to design mutagenesis primers, but couldn't remember what software to use. For future reference:<br />
<br />
NCBI for FASTA<br />
<br />
Nebcutter to find sites<br />
<br />
Virtual Ribosome for associated peptide<br />
<br />
Wikipedia for codon table<br />
<br />
PrimerX for basic design.<br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
The IGTS8 and IGTS9 cell cultures in both terrific broth and LB grew dense and cloudy at 30C. Glycerol stocks will be made out of these.<br />
<br />
We have been having issues with having no colonies grow on plates with cells transformed with PCR products ligated into vectors. One possible cause is the transformation itself. All of Joe's transformation resulted in a spark. He had added 5 uL ligated to competent cells. The high salt concentration may have heavily influenced the sparking. So, I redid some of the transformations in a smaller volume of ligate. I transformed Joe's ligated 3:3 dszB, 3:3 dszC, and 3:1 dszB plasmids into EPI300 cells. Added 0.5 uL ligate to 40 uL competent cells and electroporated them. The 3:1 dszB cells sparked during electroporation. The cells were left to recover undisturbed at 37C for an hour before plating 50 uL transformants onto Chlor plates.<br />
<br />
- [[User:Tingchiawong|Tingchiawong]]<br />
<br />
<br />
'''June 23'''<br />
<br />
Made competent ''Pseudomonas putida'' cells using the [[Competent Cell Production]] protocol.<br />
<br />
We had trouble getting the cells transformed with the ligated amino acid pathway genes to grow when plated. We're unsure if the problem lies in the digest, the ligation, or the transformation. Thus, we decided to redo all these steps.<br />
<br />
Re-digested the "M," "TA," "TB," "TyrA," and "ArgC" PCR products provided by Jacob. Made a master mix of 5 uL NEB buffer 2, 0.5 uL BSA, 0.5 uL EcoRI HF, 0.5 uL PstI, and 18.5 uL dH2O. Incubated each of the mixtures of 4 uL master mix plus 4 uL PCR product in a Thermocycler for ____.<br />
<br />
Re-ligated digest of "M," "TA," "TB," "TyrA," and "ArgC." We suspect that it may be the ligase we used that was at fault for the undesired results seen in previous experiments. So, we decided to add in more ligase and add in ligase from 2 different sources/tubes. We added 1 uL pSBIC3 EP, 4.0 uL dH2O, 1 uL T4 ligase buffer, and 0.5 uL from each tube of T4 ligase were added to 3 uL of the respective digest products. A control was also set up so that there was only plasmid DNA added. The ligate mixtures were placed in the Thermocycler to incubate at 16C for 30 minutes and inactive at 65C for 20 minutes.<br />
<br />
John miniprepped MetA and ArgE, which are being prepared as potential biobricks.<br />
<br />
A new set of PCR reactions was set up for MetA (miniprepped potential biobrick), TyrA (colony PCR), ArgE (miniprepped potential biobrick), and yddG (colony PCR). Made a ligation master mix by combining 60 uL Phusion 5X buffer, 147 uL dH2O, 15 uL DMSO, 12 uL MgCl2, and 18 uL 10 mM dNTP.<br />
<br />
- [[User:Tingchiawong|Tingchiawong]]<br />
<br />
<br />
'''June 25'''<br />
<br />
Designed primers for SDM of dszB gene<br />
<br />
PCRed yddG and Tyr A with Marianne <br />
<br />
Had a Skype meeting with Calgary iGEM team<br />
<br />
-[[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''June 26'''<br />
<br />
Designed primers for SDM of dszC gene<br />
<br />
Joined Jacob and Joe for transformation of Heat(chemical) competent cells: <br />
<br />
{| class="wikitable"<br />
|-<br />
| Plasmid transformed||Volume of Ligation Mixture used (µL) <br />
|-<br />
|dszB, dszC (3 to 3 ratio, Old and New)||3<br />
|-<br />
|MetA, TrpA, TrpB, TryA, ArgC, positive and negative control||7<br />
|}<br />
<br />
-[[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''June 27'''<br />
<br />
Run the gel of Tyr A and yddg genes at 95 Volts for 1 hr and 30 min, with 1Kb ladder. <br />
<br />
-[[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''June 30'''<br />
Yesterday, we went to Dr. Ramey, who had taught students who made a successful knockout using the lambda red system. They used a different recombineering plasmid than we did, and we received this plasmid. It was, however, growing in a strain that we were unfamiliar with, and we decided to isolate the plasmid and transform our Epi300 cells with it. <br />
It is currently growing in a culture at 30°.<br />
<br />
We received only the TrpA gene from the registry, and successfully grew it on a plate. We are now growing a culture to isolate the plasmid and if Joe's knockout is indeed a knockout, we can characterize the existing biobrick part by complementation. <br />
<br />
We ordered the Gibson assembly kit, and it ran upwards of 700 dollars for 50 reactions. <br />
<br />
Joe successfully PCR'd out the DszD gene from Rhodococcus genomic DNA that he isolated.<br />
<br />
Our further attempts at PCR for the yddg and tyrA genes was unsuccessful.<br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 1'''<br />
<br />
There are many (hundreds) of colonies on Joe's recombineering plates, and the concentration of colonies goes up as the concentration of antibiotic goes down. It appears that the addition of arabinose to the culture did nothing to increase recombineering efficiency. There are several large, well defined colonies on the full antibiotic plate. It appears that 2 days are required for sufficient growth at 30°.<br />
<br />
With this success in mind, I PCR purified a couple of other knockouts (ArgE Tet and TrpB Kan) and transformed them, reusing the electroshock cuvettes we borrowed from Rafael. A pack of 50 was ordered so we could do more than 4 transformations per day. <br />
<br />
We only had one chlor plate left, and 4 potential biobricks which were to be put on the chloramphenicol resistant pSB1C3 plasmid. Since there was only one plate (and one electroshock cuvette), only the MetA biobrick ligation product was transformed. <br />
<br />
The pKD46 recombineering vector that we received from Dr. Ramey grew well overnight at 30°C, and was miniprepped, and then transformed into the Epi300 strain. The recovery period was done at 30°C as well. If the transformation is successful, Ting will make electrocompetent cells out of the Epi300+pKD46. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 7'''<br />
<br />
Colonies with potential biobricks of trpA, tyrA, argE, metA, and dszD were successfully grown. This time, chemically competent cells were used, and they were plated on 1/2 concentration chlor plates. We also transformed and grew out the previously characterized minC biobrick.<br />
<br />
PCR of the yddg gene failed yet again. It is possible that the primers are not correct. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 9'''<br />
<br />
The potential biobricks were successfully miniprepped, and a PCR was performed to isolate what was actually between the VF2 and the VR primers in the plasmid. The product will then be run on a gel, and if it appears the correct length, will be sent for sequencing.<br />
<br />
Our Pseudomonas competent cells appear to work (conferring antibiotic resistance), but they are not expressing rfp. <br />
<br />
We made spec plates, but a lawn grew with our transformed cells. Interestingly, on top of this lawn grew several well defined colonies. We attempted to grow these colonies (and the proper negative controls) in varying amounts of spectinomycin. It is possible the plates had insufficient spec, or that the strain we are working with is naturally resistant (there was a lawn, but no large colonies, on the negative control).<br />
<br />
Several more biobricks from the registry were transformed.<br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 15'''<br />
<br />
Sent the potential biobricks for sequencing last Friday, ordered primers for mutagenesis on DszC, ordered primers for attachment of fluorescent genes to amino acid genes, received all but yddg knockouts from Yale, found that the Arg knockouts work as described. <br />
<br />
An attempt at doing an experiment to calibrate the fluorescent proteins for the plate reader (looking for quenching, linear relationship between OD and fluorescence, etc) was done on Sunday, but as it turns out, we had the wrong promoter for a fluorescent protein, and the wrong antibiotic resistance for one of the other proteins. <br />
<br />
Recently, there was a meeting with the kill switch team from Calgary. Primers to create a biobrick with an intein in it for temperature specific activity were ordered. <br />
<br />
Competent cells were made from each of the knockouts, and they worked well. The yddg strain was ordered through collaboration with Calgary as well as the keio collection proper in Japan. <br />
<br />
Our Gibson assembly kit arrived.<br />
<br />
The sequencing results from our amino acid biobricks came back, and it looks like only MetA was successful. It should be noted that the melting temperatures of the Vf2 and the VR primers are 60°, for cPCR validation of biobricks.<br />
<br />
Several new combinations of fluorescent genes, antibiotic resistances, and Keio strains were made. We used the wrong ones last weekend, but now, we are going to test TyrA- +GFP, TrpA- +RFP, MetA- +YFP, and also wildtype with the fluorescent genes. Recently, we switched backbones for the 13M RFP to Psb1C3, for we do not know the history of our current psb1C3 with rfp (we suspect it might be on the lac promoter, as per the registry guidelines), and we also put the GFP on the psb1C3 instead of a kanamycin resistant plasmid, because we were transforming into kanamycin resistant auxotrophs. <br />
<br />
We are trying to grow the ArgC- and the ArgE- together in M9 to see if anything results, with negative controls for both. We also trying to grow the wildtype with the TrpB auxotroph with rfp to look for qualitative red fluorescence. <br />
<br />
We miniprepped 4/6 of the biobricks that we ordered from the registry. We intend on sequencing them in the near future. We, lacking LB amp plates at the time, plated one on an M9 plate, but we now think that the strain the registry sent us is auxotrophic for something. We replated on LB amp after making new plates. The last one that we ordered did not arrive. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 17'''<br />
<br />
Today, we made dh5a and k12 competent cells. We also ran a cPCR on our biobrick colonies, and the results showed that they did not have an insert of the proper length.<br />
<br />
As said yesterday, we grew the two arg auxotrophs together, and they grew substantially more (~0.15 OD600) than the negative control. The wt grew to an OD of about 1. We are growing them for another night to see if the OD of the mix increases at all. <br />
<br />
Since sequencing confirmed a MetA biobrick, we transformed K12 cells with the remaining stock of our plasmid, so we could amplify it. <br />
<br />
All relevant transformed strains from yesterday grew well on the plates.<br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
[[File:Pseudomonas Crystals.jpg|650px]]<br />
<br />
<br />
'''July 19'''<br />
We miniprepped our metA biobrick (so we have enough to send to the registry and use for experiments), as well as new potential candidates for other biobricks (tyrA and ArgC).<br />
<br />
We have, in the past few days, designed a plasmid that will aid in the testing of biobricks. <br />
<br />
Concerning the kill switch plasmid parts, we want to take the psb1C3 plasmid, add a constitutively expressed LacI gene, and a lac promoter and rbs immediately before the cut sites. This way, a toxic protein can be well controlled. In fact, while we are designing this for toxic proteins, it could have uses in general protein production. Previous attempts for using the lac promoter on a high copy plasmid have failed leaky expression) due to the lack of the LacI protein produced in the genome. This new plasmid design remedies this, and will be accomplished by Gibson assembly. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''July 23'''<br />
<br />
Analyzed the data, and found out that the growth rate and the amino acid (Tyr) concentration has a correlation as follows: <br />
<br />
[[File:growth-rate at different try concentration_british_columbia_2012.png|650px]]<br />
*Figure 4. cell growth-rate at different Tyr concentrations<br />
<br />
[[File:growth-rate at different tyr concentration log scale_british_columbia_2012.png|650px]]<br />
<br />
*Figure 5. Log scale of cell growth-rate at different Tyr concentrations <br />
<br />
It was found that the cells depleted the nutrient quickly at low Tyr concentration, and for future experiments it is suggested to use lower initial cell concentrations to extend the time of its exponential growth phase. Also, the wild type has almost the exact growth rate as the autotroph when the the Try concentration is exceptionally high (0.01 M). <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''July 26'''<br />
<br />
The growth rate and the amino acids (Met and Trp) concentration has a correlation as follows: <br />
<br />
[[File:growth-rate at different met concentration_british_columbia_2012.png|650px]]<br />
<br />
*Figure 6. cell growth-rate at different Met concentrations<br />
<br />
[[File:growth-rate at different Met concentration log scale_british_columbia_2012.png|650px]]<br />
<br />
*Figure 7. Log scale of cell growth-rate at different Met concentrations <br />
<br />
[[File:growth-rate at different trp concentration_british_columbia_2012.png|650px]]<br />
<br />
*Figure 8. cell growth-rate at different Trp concentrations<br />
<br />
[[File:growth-rate at different trp concentration log scale_british_columbia_2012.png|650px]]<br />
<br />
*Figure 9. Log scale of cell growth-rate at different Trp concentrations <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''August 3rd'''<br />
<br />
Started to work on the data obtained on July 28th plate readings. <br />
<br />
Eventually arrived at the following graphs of growth rates observations, when n=3<br />
<br />
[[file: GrowthRatesAA_university_of_british_columbia.png|650px]]<br />
<br />
[[file: GrowthRatesAA_LOG_university_of_british_columbia.png|650px]]<br />
<br />
From these graphs, the maximum growth rate, and Ks values that are used in Monod Kinetics can be determined. <br />
<br />
- [[User:Ruichen|Ruichen]]<br />
<br />
<br />
'''August 12'''<br />
<br />
A large set of primers arrived, which gave us a lot to do.<br />
<br />
With a set of the primers that arrived, we were able to PCR amplify certain parts that will be able to be Gibson assembled to create new biobricks. On the plasmid that has RFP (a modified BBa_K093012, placed on the Psb1C3), we wanted to put arabinose inducible MetA or arabinose inducible TyrA. However, one of the primers necessary to amplify the plasmid itself, which would be necessary to incorporate the proper homologous sequence for Gibson assembly, did not arrive. Additionally, the PCR to amplify the MetA gene did not work the first time, but has been repeated. The arabinose promoter was successfully PCR'd out of the E. coli genome for both cases. <br />
For the plasmid with YFP, the BBa_I13973, we wanted to put rhamnose inducible TrpA or TyrA after the YFP gene. We received all the primers necessary for this, but the PCR for the plasmid backbone gave several bands and was repeated at a higher annealing temperature. The results are not yet known. The rhamnose promoter was successfully PCR'd out of the genome, as was the TrpA gene. However, we lacked one of the promoters for the TyrA gene. Since there were several bands for the YFP plasmid, we did not do a Gibson assembly. Additionally, it is possible that the primers were designed for the pSB1C3 rather than the pSB1A2, but that should have little effect due to the similarities on the ends of the two plasmids. A simple digest and ligation should be able to move the part from the one plasmid to the other.<br />
We also wanted to put a IPTG inducible (BBa_K091111, LacIQ) metA gene after a GFP. The PCR for the MetA gene and the GFP plasmid were successful (although the GFP plasmid appeared as a fairly weak band on the gel), but the PCR for the LacIQ promoter failed. The initial DNA for this PCR was added directly from the parts distribution kit, and as such the composition and concentration of exactly what was added was unknown. The PCR was repeated as a cPCR on the genome, so as to at least get a usable part with the right overhang. <br />
<br />
The final primers for the gibson assembly of the kill switch primer also arrived. There are four parts to this plasmid: the plasmid backbone, the lacI promoter, the constitutively expressed lacI gene, and the constitutively expressed RFP for easy selection. The plasmid backbone, the promoter, and the constitutively expressed RFP PCR products has been ready for several weeks, but we lacked the primers for the constituvely expressed lacI gene until now. To generate the DNA for the constitutively expressed lacI gene, we took BBa_K081005 (const. promoter+rbs) cut with only SpeI and BBa_I732100 (lacI) cut with only XbaI, and ligated the two together, and then did a PCR on the ligation. It worked beautifully. <br />
<br />
The primers also arrived to isolate the VMA Sce-I intein, and Alina provided yeast genomic DNA. The PCR was done successfully. <br />
<br />
A previous potential part, ArgE, was restriction confirmed, and will be sent for sequencing at the soonest convenience.<br />
<br />
The PCR for yddg was finally a success. The part has been restriction digested, along with the pSB1C3 plasmid, and a ligation will follow. <br />
<br />
We finally received the DszA forward primer, and did a restriction digest on it as well. It will be ligated as well in the near future. The DszA gene has several Pst1 sites, which limits some of the reactions we can do with it, but we can still place things before it by cutting with EcoRI and/or XbaI. <br />
<br />
Joe has found inconsistent results with his fluorescent proteins on the various promoters, and we have plans to standardize the promoter and rbs for the fluorescent proteins that will be used. Additionally, there is some overlap between YFP and GFP that is proving difficult to account for, so we are going to try to switch one to either BFP or CFP. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''August 13'''<br />
<br />
There were colonies on the MinC Intein plate, the ArgE BB plate, and the GFP construct LB plate, but nothing on the GFP construct M9 plate. This may have been because there was no lactose or IPTG to induce the metA gene. Interestingly, there was a green fluorescent patch, but it was rather weak and had no distinct colonies. We plan to pick several colonies from the LB plate and try growing them in M9 culture with and without IPTG overnight. We also plan to do a cPCR on the MinC plate using the intein primers. There were no colonies on the DszA or yddg biobrick plate. We have had no luck with electroporating cells with ligation mixtures, although we have had some success with heat shock competent cells. We think there may be something in the ligation mixture that is interfering with the electroporation, and could probably be fixed by purifying the ligation mix. <br />
<br />
We also did a PCR for the TyrA BB product, the previous failed YFP plasmid, and several other previously failed PCRs. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''August 15'''<br />
<br />
After two days of shaking at 37° in 1 mM IPTG, the supposed IPTG inducible metA constructs in the metA auxotrophs were unable to grow. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''August 16'''<br />
<br />
The supposed metA constructs still appears unable to complement a metA knockout. It has been suggested that the concentration of metA may have been too high, and this would have led to a severe detriment to the cell, and it has also been suggested that the IPTG, being a rather old stock, was no longer good. Another step that we are going to take is to see if the expression of the construct can be seen on an sds page under induction.<br />
<br />
The YFP PCR for gibson assembly has yet to give us a solid band, so we are trying to move it into the psb1c3 plasmid in case there are some plasmid specific sequences that were giving us poor results. The ligation and transformation has been done, and we are waiting for colonies. The PCR products necessary for the gibson assembly of the GFP-IPTG-TrpA gene were also all available, and that gibson assembly has been done. However, due to the failure of several gels, there was very little product left, and PCR purification would likely result in unusuable quantities, so we tried a Gibson assembly with the straight PCR product, and we will see if it works. <br />
<br />
All of the parts necessary for the assembly of the kill switch plasmid have been successfully PCR'd.<br />
<br />
Another PCR was done to generate some of the other parts required for assembly of other parts. <br />
<br />
A cPCR of the colonies with the MinC protein with the Sce VME intein showed that the gene for the intein was present in at least 2 colonies.<br />
<br />
There are two distinct bands on the RFP PCR, one at the correct length, the other much lower. <br />
<br />
The cPCR on the DszA colonies gave no result. This may have been due to an unoptimized Taq protocol,and might be worth repeating. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<br />
'''August 17'''<br />
<br />
The plans for the metA BB construct induction were further discussed. Overnight colonies will be made of the construct and a negative control, then they will be diluted in 1 in 100 LB, allowed to grow to 0.4 OD, induced with 0.1 mM new IPTG, and harvested at a certain OD following this, then run on an SDS PAGE. This will test to see if the IPTG really would stimulate the production of the metA<br />
The cultures for the MinteinC (As it shall henceforth be known) grew well, but were not miniprepped. Once they are miniprepped, then site directed mutagenesis must be done. The cultures of the potential yddg biobricks grew well, and will be confirmed either by PCR of the plasmid using VR and VF2, or by restriction digest. <br />
<br />
- [[User:jacobtoth|Jacob Toth]]<br />
<br />
<div style="text-align: center;"><h2>[https://2012.igem.org/Team:British_Columbia/BiobrickConstruction Back to top]</h2></div></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/LabNotebookTeam:British Columbia/LabNotebook2012-10-04T03:33:44Z<p>Rsaer: </p>
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'''Our new home at the Life Sciences Institute, UBC'''<br />
<br />
[[File:Ubclsi.jpg | center]]<br />
<br />
<br />
[[File:UBCiGEMatwork.jpg | center]]<br />
<br />
Our team is hosted by [http://www.cmde.science.ubc.ca/hallam/index.php Steven Hallam's laboratory].<br />
<br />
<br />
'''August 2''' <br />
<br />
Field trip to Chevron Burnaby site!<br />
<br />
[[file:Ubchev.jpg | center]]<br />
<br />
[[file:Ubcigemchevcollage.jpg | center]]<br />
<br />
[[file:Ubcchev.jpg | center]]<br />
<br />
<br />
<br />
<br />
'''Sept 15-16'''<br />
<br />
Traveling to Edmonton for aGEM!<br />
<br />
Our team attended the Alberta Genetically Engineered Machines (aGEM) competition alongside the 2012 University of Alberta, University of Calgary and Lethbridge University iGEM teams. aGEM consisted of a jamboree-style presentation by each team and feedback from a judging panel. We obtained a lot of constructive advice from the panel regarding our presentation. Overall, the judges found our project to be very exciting and applicable industrially, and thought that we needed to present our story more coherently and confidently! We are looking forward to meeting with the teams from Alberta again at the West Americas regionals and hopefully the Worlds Championship.<br />
<br />
[[file:Ubcagem1.jpg | center]]<br />
<br />
[[file:Ubcagem2.jpg | center]]<br />
<br />
[[file:Ubcagem3.jpg | center]]<br />
<br />
<div style="text-align: center;"><h2>[https://2012.igem.org/Team:British_Columbia/LabNotebook Back to top]</h2></div></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/LabNotebookTeam:British Columbia/LabNotebook2012-10-04T03:31:45Z<p>Rsaer: </p>
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<div id="sponsormap"><img align="left" src="https://static.igem.org/mediawiki/2012/a/a0/Ubcigemnotebookmenu.jpg" usemap="#sponsormap" alt="UBC iGEM 2012 notebook"> </div> <br />
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</div><div id=note><br />
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'''Our new home at the Life Sciences Institute, UBC'''<br />
<br />
[[File:Ubclsi.jpg | center]]<br />
<br />
<br />
[[File:UBCiGEMatwork.jpg | center]]<br />
<br />
Our team is hosted by [http://www.cmde.science.ubc.ca/hallam/index.php Steven Hallam's laboratory].<br />
<br />
<br />
'''August 2''' <br />
<br />
Field trip to Chevron Burnaby site!<br />
<br />
[[file:Ubchev.jpg | center]]<br />
<br />
[[file:Ubcigemchevcollage.jpg | center]]<br />
<br />
[[file:Ubcchev.jpg | center]]<br />
<br />
<br />
<br />
<br />
'''Sept 15-16'''<br />
<br />
Traveling to Edmonton for aGEM!<br />
<br />
Our team attended the Alberta Genetically Engineered Machines (aGEM) competition alongside the 2012 University of Alberta, University of Calgary and Lethbridge University iGEM teams. aGEM consisted of a jamboree-style presentation by each team and feedback from a judging panel. We obtained a lot of constructive advice from the panel regarding our presentation. Overall, the judges found our project to be very exciting and applicable industrially, and thought that we needed to present our story more coherently and confidently! We are looking forward to meeting with the teams from Alberta again at the West Americas regionals and hopefully the Worlds Championship.<br />
<br />
[[file:Ubcagem1.jpg | center]]<br />
<br />
[[file:Ubcagem2.jpg | center]]<br />
<br />
[[file:Ubcagem3.jpg | center]]<br />
<br />
<h2>[https://2012.igem.org/Team:British_Columbia/LabNotebook Back to top]</h2></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PathwayTeam:British Columbia/Pathway2012-10-04T03:26:03Z<p>Rsaer: </p>
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<p align=center><font face=arial narrow size=5><b>Our Pathway Model<br />
</b></font></p><font face=arial narrow><br />
<br />
The study of environmental genomics attempts to capture the taxonomic and functional diversity of natural microbial communities. Our host at UBC, the Hallam lab, designs novel tools for analyzing the gene content in the context of distributed metabolism. Recently, a pipeline has been developed for the automated construction and visualizing of metabolic pathways from genomic data by integrating software such as Pathway Tools, Pathologic and Metacyc [1]. This provided us an opportunity to model pathway compartmentalization and distribution amongst microbes in the natural environment as it applies to our project. </br></br><br />
<br />
<h2><b>Summary of the pipeline for community-level metabolic analysis (Figure 1):</b></h2> First, open reading frames are predicted from sequence data with Prodigal, are then annotated by protein BLAST, and later summarized in a GenBank file. Pathway/genome databases (PGDBs) are generated from sequence data in a manner which does not constrain predictions within the scope of model organisms. This produces a community-based analysis that can be visualized using Pathway Tools. <br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/9c/UbcigemSlide1.jpg"></p><br />
<br />
<h2><b>Modeling Metabolism:</b></h2> <i>Rhodococcus erythropolis</i> and <i>Pseudomnas fluorescens</i> are often both prevalent in similar niches. With a high likelihood that these organisms encounter each other, studies have been conducted to assess their metabolic properties in co-culture. In a study by Kayser et al, it was shown that the 4S biodesulferization pathway in <i>R. erythropolis</i> demonstrated higher activity the presence of <i>P. fluorescens</i> [2]. As <i>P. fluorescens</i> does not biodesulferize DBT and sulfate is known to repress the 4S pathway, we looked to analyze the genomes of the two organisms in the context of sulfur metabolism. </br></br><br />
<br />
The 4S pathway releases sulfite, which is toxic to the cell, and therefore genomes were analyzed with the aforementioned pipeline for pathways involved in metabolizing sulfite. It was found that both organisms have annotated genes which convert sulfite into sulfate via a reductase, however the organisms differ in downstream metabolism of sulfate. While both organisms contain a pathway for assimilatory sulfate metabolism, only <i>P. fluorescens</i> has the capability for dissimilatory sulfate metabolism (Figure 2, 3). Based on these findings, we can hypothesize that <i>R. erythropolis</i> and <i>P. fluorescens</i> likely excrete and catabolize any excess sulfate. This provides an explanation for the improved desulfurization found in co-culture conditions. <i>P. fluorescens</i> potentially removes sulfate from the environment, allowing increased secretion by <i>R. erythropolis</i>, and thereby derepressing 4S pathway. The prediction of distributed sulfite metabolism to improve biodesulferization in the environment provides both a testable hypothesis, as well as a grounds for improving biodesulferization through synthetic pathway distribution. </br></br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/4/43/UbcigemerSlide2.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/d/d1/UbcigemerSlide3.jpg"></p><br />
<br />
<br />
We then scanned the literature for more evidence of shared metabolism between both <i>R. erythropolis</i> and <i>P. fluorescens</i> in order to further analyze gene content in the context of co-culture experiments. In a study by Goswami et al., the metabolism of chlorinated aromatic compounds and phenol was compared in monoculture versus co-culture using <i>R. erythropolis</i> and <i>P. fluorescens</i> [3]. This study showed that the growth rate of pure cultures of <i>R. erythropolis</i> was higher than <i>P. fluorescens</i> on chlorinated aromatics, however in mixed culture, <i>P. fluorescens</i> showed a higher growth rate. For the degradation of phenol, <i>R. erythropolis</i> showed higher growth rates in both pure and mixed culture. The authors of this study suggested that these results were likely a product of substrate competition. We attempted to analyze the genomes of both <i>R. erythropolis</i> and <i>P. fluorescens</i>, separately and together in an attempt to offer an alternate interpretation of the co-culture results. The first pathways assessed were those involved in chlorinated aromatic degradation. It was found that <i>P. fluorescens</i> contains a higher diversity of genes involved in catabolizing chlorinated aromatics; however, only <i>R. erythropolis</i> seems to be able to degrade phenol (Figure 4, 5). This suggests the possibility of the compartmentalization of different components of these metabolic processes, leading to the different growth kinetics observed in co-culture. For example, while <i>R. erythropolis</i> may be more efficient at degrading certain chlorinated aromatics, in co-culture, the diversity of catabolism of chlorinated aromatics allows <i>P. fluorescens</i> to grow more rapidly. Chlorinated aromatic degradation by <i>P. fluorescens</i>, however, would result in the accumulation of downstream products, such as phenol, that only <i>R. erythropolis</i> can catabolism. This provides a metabolic network which could both select and sustain both microbes in the presence of diverse chlorinated aromatics. </br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/b/bd/UbcigemSlide4.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/99/UbcigemSlide5.jpg"></p><br />
<br />
<br />
<br />
<br />
The sums of general aromatic degradation pathways were compared for the organisms genomes separately and together (Figure 6). This resulted in emergent predicted pathways in combination as well as combinatorial increases araomatic degradation potential. <br /><br />
<br /><br />
Ultimately, gene annotation-based models for distributed metabolism in the environment may help to engineer and optimize complex metabolism through synthetic consortia. </br><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/a/aa/UbcigemSlide6.jpg"></p></br></br><br />
<br />
<h2><b>References</b></h2><br /><br />
[1] Hanson, N.W; Page, A.P; Konwar, K.M; Howes, C.G; Hallam, S.J. Metabolic interaction networks for the whole community, 2012. Unpublished.</br></br><br />
<br />
[2] Kayser, K.J; Biolaga-Jones, B.A.; Jackowski, K; Odusan, O; Kildane, J.J. Utilzation of organosulfur compounds by anexic and mixed culture of <i>Rhodococcus rhodochrous</i> IGTS8, 1993. Journal of General Microbioology, 139: 3123-3129.</br></br><br />
<br />
[3] Goswami, M; Shivaraman, N; Singh, R.P. Microbial metabolism of 2-chlorophenol, phenol and p-cresol by <i>Rhodococcus erythropolis</i> M1 and co-culture with <i>Pseudomonas fluorescens</i> P1, 2005. Microbiological Research, 160: 101-109.</br></br></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PathwayTeam:British Columbia/Pathway2012-10-04T03:24:53Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<style><br />
#break {width:950px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}</style><br />
<div id=break></div><br />
<br />
<p align=center><font face=arial narrow size=5><b>Our Pathway Model<br />
</b></font></p><font face=arial narrow><br />
<br />
The study of environmental genomics attempts to capture the taxonomic and functional diversity of natural microbial communities. Our host at UBC, the Hallam lab, designs novel tools for analyzing the gene content in the context of distributed metabolism. Recently, a pipeline has been developed for the automated construction and visualizing of metabolic pathways from genomic data by integrating software such as Pathway Tools, Pathologic and Metacyc [1]. This provided us an opportunity to model pathway compartmentalization and distribution amongst microbes in the natural environment as it applies to our project. </br></br><br />
<br />
<h2><b>Summary of the pipeline for community-level metabolic analysis (Figure 1):</b></h2> First, open reading frames are predicted from sequence data with Prodigal, are then annotated by protein BLAST, and later summarized in a GenBank file. Pathway/genome databases (PGDBs) are generated from sequence data in a manner which does not constrain predictions within the scope of model organisms. This produces a community-based analysis that can be visualized using Pathway Tools. <br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/9c/UbcigemSlide1.jpg"></p><br />
<br />
<h2><b>Modeling Metabolism:</b></h2> <i>Rhodococcus erythropolis</i> and <i>Pseudomnas fluorescens</i> are often both prevalent in similar niches. With a high likelihood that these organisms encounter each other, studies have been conducted to assess their metabolic properties in co-culture. In a study by Kayser et al, it was shown that the 4S biodesulferization pathway in <i>R. erythropolis</i> demonstrated higher activity the presence of <i>P. fluorescens</i> [2]. As <i>P. fluorescens</i> does not biodesulferize DBT and sulfate is known to repress the 4S pathway, we looked to analyze the genomes of the two organisms in the context of sulfur metabolism. </br></br><br />
<br />
The 4S pathway releases sulfite, which is toxic to the cell, and therefore genomes were analyzed with the aforementioned pipeline for pathways involved in metabolizing sulfite. It was found that both organisms have annotated genes which convert sulfite into sulfate via a reductase, however the organisms differ in downstream metabolism of sulfate. While both organisms contain a pathway for assimilatory sulfate metabolism, only <i>P. fluorescens</i> has the capability for dissimilatory sulfate metabolism (Figure 2, 3). Based on these findings, we can hypothesize that <i>R. erythropolis</i> and <i>P. fluorescens</i> likely excrete and catabolize any excess sulfate. This provides an explanation for the improved desulfurization found in co-culture conditions. <i>P. fluorescens</i> potentially removes sulfate from the environment, allowing increased secretion by <i>R. erythropolis</i>, and thereby derepressing 4S pathway. The prediction of distributed sulfite metabolism to improve biodesulferization in the environment provides both a testable hypothesis, as well as a grounds for improving biodesulferization through synthetic pathway distribution. </br></br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/4/43/UbcigemerSlide2.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/d/d1/UbcigemerSlide3.jpg"></p><br />
<br />
<br />
We then scanned the literature for more evidence of shared metabolism between both <i>R. erythropolis</i> and <i>P. fluorescens</i> in order to further analyze gene content in the context of co-culture experiments. In a study by Goswami et al., the metabolism of chlorinated aromatic compounds and phenol was compared in monoculture versus co-culture using <i>R. erythropolis</i> and <i>P. fluorescens</i> [3]. This study showed that the growth rate of pure cultures of <i>R. erythropolis</i> was higher than <i>P. fluorescens</i> on chlorinated aromatics, however in mixed culture, <i>P. fluorescens</i> showed a higher growth rate. For the degradation of phenol, <i>R. erythropolis</i> showed higher growth rates in both pure and mixed culture. The authors of this study suggested that these results were likely a product of substrate competition. We attempted to analyze the genomes of both <i>R. erythropolis</i> and <i>P. fluorescens</i>, separately and together in an attempt to offer an alternate interpretation of the co-culture results. The first pathways assessed were those involved in chlorinated aromatic degradation. It was found that <i>P. fluorescens</i> contains a higher diversity of genes involved in catabolizing chlorinated aromatics; however, only <i>R. erythropolis</i> seems to be able to degrade phenol (Figure 4, 5). This suggests the possibility of the compartmentalization of different components of these metabolic processes, leading to the different growth kinetics observed in co-culture. For example, while <i>R. erythropolis</i> may be more efficient at degrading certain chlorinated aromatics, in co-culture, the diversity of catabolism of chlorinated aromatics allows <i>P. fluorescens</i> to grow more rapidly. Chlorinated aromatic degradation by <i>P. fluorescens</i>, however, would result in the accumulation of downstream products, such as phenol, that only <i>R. erythropolis</i> can catabolism. This provides a metabolic network which could both select and sustain both microbes in the presence of diverse chlorinated aromatics. </br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/b/bd/UbcigemSlide4.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/99/UbcigemSlide5.jpg"></p><br />
<br />
<br />
<br />
<br />
The sums of general aromatic degradation pathways were compared for the organisms genomes separately and together (Figure 6). This resulted in emergent predicted pathways in combination as well as combinatorial increases araomatic degradation potential. <br /><br />
<br /><br />
Ultimately, gene annotation-based models for distributed metabolism in the environment may help to engineer and optimize complex metabolism through synthetic consortia. </br><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/a/aa/UbcigemSlide6.jpg"></p></br></br><br />
<br />
<h2><b>References</b></h2><br /><br />
[1] Hanson, N.W; Page, A.P; Konwar, K.M; Howes, C.G; Hallam, S.J. Metabolic interaction networks for the whole community, 2012. Unpublished.</br></br><br />
<br />
[2] Kayser, K.J; Biolaga-Jones, B.A.; Jackowski, K; Odusan, O; Kildane, J.J. Utilzation of organosulfur compounds by anexic and mixed culture of ''Rhodococcus rhodochrous'' IGTS8, 1993. Journal of General Microbioology, 139: 3123-3129.</br></br><br />
<br />
[3] Goswami, M; Shivaraman, N; Singh, R.P. Microbial metabolism of 2-chlorophenol, phenol and p-cresol by ''Rhodococcus erythropolis'' M1 and co-culture with ''Pseudomonas fluorescens'' P1, 2005. Microbiological Research, 160: 101-109.</br></br></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PathwayTeam:British Columbia/Pathway2012-10-04T03:23:57Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<style><br />
#break {width:950px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}</style><br />
<div id=break></div><br />
<br />
<p align=center><font face=arial narrow size=5><b>Our Pathway Model<br />
</b></font></p><font face=arial narrow><br />
<br />
The study of environmental genomics attempts to capture the taxonomic and functional diversity of natural microbial communities. Our host at UBC, the Hallam lab, designs novel tools for analyzing the gene content in the context of distributed metabolism. Recently, a pipeline has been developed for the automated construction and visualizing of metabolic pathways from genomic data by integrating software such as Pathway Tools, Pathologic and Metacyc [1]. This provided us an opportunity to model pathway compartmentalization and distribution amongst microbes in the natural environment as it applies to our project. </br></br><br />
<br />
<h2><b>Summary of the pipeline for community-level metabolic analysis (Figure 1):</b></h2> First, open reading frames are predicted from sequence data with Prodigal, are then annotated by protein BLAST, and later summarized in a GenBank file. Pathway/genome databases (PGDBs) are generated from sequence data in a manner which does not constrain predictions within the scope of model organisms. This produces a community-based analysis that can be visualized using Pathway Tools. <br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/9c/UbcigemSlide1.jpg"></p><br />
<br />
<h2><b>Modeling Metabolism:</b></h2> <i>Rhodococcus erythropolis</i> and <i>Pseudomnas fluorescens</i> are often both prevalent in similar niches. With a high likelihood that these organisms encounter each other, studies have been conducted to assess their metabolic properties in co-culture. In a study by Kayser et al, it was shown that the 4S biodesulferization pathway in <i>R. erythropolis</i> demonstrated higher activity the presence of <i>P. fluorescens</i> [2]. As <i>P. fluorescens</i> does not biodesulferize DBT and sulfate is known to repress the 4S pathway, we looked to analyze the genomes of the two organisms in the context of sulfur metabolism. </br></br><br />
<br />
The 4S pathway releases sulfite, which is toxic to the cell, and therefore genomes were analyzed with the aforementioned pipeline for pathways involved in metabolizing sulfite. It was found that both organisms have annotated genes which convert sulfite into sulfate via a reductase, however the organisms differ in downstream metabolism of sulfate. While both organisms contain a pathway for assimilatory sulfate metabolism, only <i>P. fluorescens</i> has the capability for dissimilatory sulfate metabolism (Figure 2, 3). Based on these findings, we can hypothesize that <i>R. erythropolis</i> and <i>P. fluorescens</i> likely excrete and catabolize any excess sulfate. This provides an explanation for the improved desulfurization found in co-culture conditions. <i>P. fluorescens</i> potentially removes sulfate from the environment, allowing increased secretion by <i>R. erythropolis</i>, and thereby derepressing 4S pathway. The prediction of distributed sulfite metabolism to improve biodesulferization in the environment provides both a testable hypothesis, as well as a grounds for improving biodesulferization through synthetic pathway distribution. </br></br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/4/43/UbcigemerSlide2.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/d/d1/UbcigemerSlide3.jpg"></p><br />
<br />
<br />
We then scanned the literature for more evidence of shared metabolism between both <i>R. erythropolis</i> and <i>P. fluorescens</i> in order to further analyze gene content in the context of co-culture experiments. In a study by Goswami et al., the metabolism of chlorinated aromatic compounds and phenol was compared in monoculture versus co-culture using <i>R. erythropolis</i> and <i>P. fluorescens</i> [3]. This study showed that the growth rate of pure cultures of <i>R. erythropolis</i> was higher than <i>P. fluorescens</i> on chlorinated aromatics, however in mixed culture, <i>P. fluorescens</i> showed a higher growth rate. For the degradation of phenol, <i>R. erythropolis</i> showed higher growth rates in both pure and mixed culture. The authors of this study suggested that these results were likely a product of substrate competition. We attempted to analyze the genomes of both <i>R. erythropolis</i> and <i>P. fluorescens</i>, separately and together in an attempt to offer an alternate interpretation of the co-culture results. The first pathways assessed were those involved in chlorinated aromatic degradation. It was found that <i>P. fluorescens</i> contains a higher diversity of genes involved in catabolizing chlorinated aromatics; however, only <i>R. erythropolis</i> seems to be able to degrade phenol (Figure 4, 5). This suggests the possibility of the compartmentalization of different components of these metabolic processes, leading to the different growth kinetics observed in co-culture. For example, while <i>R. erythropolis</i> may be more efficient at degrading certain chlorinated aromatics, in co-culture, the diversity of catabolism of chlorinated aromatics allows <i>P. fluorescens</i> to grow more rapidly. Chlorinated aromatic degradation by <i>P. fluorescens</i>, however, would result in the accumulation of downstream products, such as phenol, that only <i>R. erythropolis</i> can catabolism. This provides a metabolic network which could both select and sustain both microbes in the presence of diverse chlorinated aromatics. </br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/b/bd/UbcigemSlide4.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/99/UbcigemSlide5.jpg"></p><br />
<br />
<br />
<br />
<br />
The sums of general aromatic degradation pathways were compared for the organisms genomes separately and together (Figure 6). This resulted in emergent predicted pathways in combination as well as combinatorial increases araomatic degradation potential. <br /><br />
<br /><br />
Ultimately, gene annotation-based models for distributed metabolism in the environment may help to engineer and optimize complex metabolism through synthetic consortia. </br><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/a/aa/UbcigemSlide6.jpg"></p></br></br><br />
<br />
<h2><b>References</b></h2><br /><br />
[1] Hanson, N.W; Page, A.P; Konwar, K.M; Howes, C.G; Hallam, S.J. Metabolic interaction networks for the whole community, 2012. Unpublished.</br></br><br />
<br />
[2] Kayser, K.J; Biolaga-Jones, B.A.; Jackowski, K; Odusan, O; Kildane, J.J. Utilzation of organosulfur compounds by anexic and mixed culture of Rhodococcus rhodochrous IGTS8, 1993. Journal of General Microbioology, 139: 3123-3129.</br></br><br />
<br />
[3] Goswami, M; Shivaraman, N; Singh, R.P. Microbial metabolism of 2-chlorophenol, phenol and p-cresol by Rhodococcus erythropolis M1 and co-culture with Pseudomonas fluorescens P1, 2005. Microbiological Research, 160: 101-109.</br></br></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PathwayTeam:British Columbia/Pathway2012-10-04T03:23:26Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<style><br />
#break {width:950px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}</style><br />
<div id=break></div><br />
<br />
<p align=center><font face=arial narrow size=5><b>Our Pathway Model<br />
</b></font></p><font face=arial narrow><br />
<br />
The study of environmental genomics attempts to capture the taxonomic and functional diversity of natural microbial communities. Our host at UBC, the Hallam lab, designs novel tools for analyzing the gene content in the context of distributed metabolism. Recently, a pipeline has been developed for the automated construction and visualizing of metabolic pathways from genomic data by integrating software such as Pathway Tools, Pathologic and Metacyc [1]. This provided us an opportunity to model pathway compartmentalization and distribution amongst microbes in the natural environment as it applies to our project. </br></br><br />
<br />
<h2><b>Summary of the pipeline for community-level metabolic analysis (Figure 1):</b></h2> First, open reading frames are predicted from sequence data with Prodigal, are then annotated by protein BLAST, and later summarized in a GenBank file. Pathway/genome databases (PGDBs) are generated from sequence data in a manner which does not constrain predictions within the scope of model organisms. This produces a community-based analysis that can be visualized using Pathway Tools. <br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/9c/UbcigemSlide1.jpg"></p><br />
<br />
<h2><b>Modeling Metabolism:</b></h2> <i>Rhodococcus erythropolis</i> and <i>Pseudomnas fluorescens</i> are often both prevalent in similar niches. With a high likelihood that these organisms encounter each other, studies have been conducted to assess their metabolic properties in co-culture. In a study by Kayser et al, it was shown that the 4S biodesulferization pathway in <i>R. erythropolis</i> demonstrated higher activity the presence of <i>P. fluorescens</i> [2]. As <i>P. fluorescens</i> does not biodesulferize DBT and sulfate is known to repress the 4S pathway, we looked to analyze the genomes of the two organisms in the context of sulfur metabolism. </br></br><br />
<br />
The 4S pathway releases sulfite, which is toxic to the cell, and therefore genomes were analyzed with the aforementioned pipeline for pathways involved in metabolizing sulfite. It was found that both organisms have annotated genes which convert sulfite into sulfate via a reductase, however the organisms differ in downstream metabolism of sulfate. While both organisms contain a pathway for assimilatory sulfate metabolism, only <i>P. fluorescens</i> has the capability for dissimilatory sulfate metabolism (Figure 2, 3). Based on these findings, we can hypothesize that <i>R. erythropolis</i> and <i>P. fluorescens</i> likely excrete and catabolize any excess sulfate. This provides an explanation for the improved desulfurization found in co-culture conditions. <i>P. fluorescens</i> potentially removes sulfate from the environment, allowing increased secretion by <i>R. erythropolis</i>, and thereby derepressing 4S pathway. The prediction of distributed sulfite metabolism to improve biodesulferization in the environment provides both a testable hypothesis, as well as a grounds for improving biodesulferization through synthetic pathway distribution. </br></br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/4/43/UbcigemerSlide2.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/d/d1/UbcigemerSlide3.jpg"></p><br />
<br />
<br />
We then scanned the literature for more evidence of shared metabolism between both <i>R. erythropolis</i> and <i>P. fluorescens</i> in order to further analyze gene content in the context of co-culture experiments. In a study by Goswami et al., the metabolism of chlorinated aromatic compounds and phenol was compared in monoculture versus co-culture using <i>R. erythropolis</i> and <i>P. fluorescens</i> [3]. This study showed that the growth rate of pure cultures of <i>R. erythropolis</i> was higher than <i>P. fluorescens</i> on chlorinated aromatics, however in mixed culture, <i>P. fluorescens</i> showed a higher growth rate. For the degradation of phenol, <i>R. erythropolis</i> showed higher growth rates in both pure and mixed culture. The authors of this study suggested that these results were likely a product of substrate competition. We attempted to analyze the genomes of both <i>R. erythropolis</i> and <i>P. fluorescens</i>, separately and together in an attempt to offer an alternate interpretation of the co-culture results. The first pathways assessed were those involved in chlorinated aromatic degradation. It was found that <i>P. fluorescens</i> contains a higher diversity of genes involved in catabolizing chlorinated aromatics; however, only <i>R. erythropolis</i> seems to be able to degrade phenol (Figure 4, 5). This suggests the possibility of the compartmentalization of different components of these metabolic processes, leading to the different growth kinetics observed in co-culture. For example, while <i>R. erythropolis</i> may be more efficient at degrading certain chlorinated aromatics, in co-culture, the diversity of catabolism of chlorinated aromatics allows <i>P. fluorescens</i> to grow more rapidly. Chlorinated aromatic degradation by <i>P. fluorescens</i>, however, would result in the accumulation of downstream products, such as phenol, that only <i>R. erythropolis</i> can catabolism. This provides a metabolic network which could both select and sustain both microbes in the presence of diverse chlorinated aromatics. </br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/b/bd/UbcigemSlide4.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/99/UbcigemSlide5.jpg"></p><br />
<br />
<br />
<br />
<br />
The sums of general aromatic degradation pathways were compared for the organisms genomes separately and together (Figure 6). This resulted in emergent predicted pathways in combination as well as combinatorial increases araomatic degradation potential. <br /><br />
<br /><br />
Ultimately, gene annotation-based models for distributed metabolism in the environment may help to engineer and optimize complex metabolism through synthetic consortia. </br><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/a/aa/UbcigemSlide6.jpg"></p></br></br><br />
<br />
<h2>References</h2></br><br />
[1] Hanson, N.W; Page, A.P; Konwar, K.M; Howes, C.G; Hallam, S.J. Metabolic interaction networks for the whole community, 2012. Unpublished.</br></br><br />
<br />
[2] Kayser, K.J; Biolaga-Jones, B.A.; Jackowski, K; Odusan, O; Kildane, J.J. Utilzation of organosulfur compounds by anexic and mixed culture of Rhodococcus rhodochrous IGTS8, 1993. Journal of General Microbioology, 139: 3123-3129.</br></br><br />
<br />
[3] Goswami, M; Shivaraman, N; Singh, R.P. Microbial metabolism of 2-chlorophenol, phenol and p-cresol by Rhodococcus erythropolis M1 and co-culture with Pseudomonas fluorescens P1, 2005. Microbiological Research, 160: 101-109.</br></br></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DataTeam:British Columbia/Data2012-10-04T03:21:26Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
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<font face=arial narrow size=5><b>Our System</b></font></br></br><font face=arial narrow><br />
<br />
<p>We have designed a tunable microbial consortium to distribute the 4S biodesulfurization pathway, responsible for the bio-desulfurization of DBT, as a metabolic network.</p><br />
<br />
<h2>There are two major genetic circuits contained within our system:</h2><br><br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/ProjectConsortia"> first</a> is responsible for tuning the relative populations of the bacteria within the consortium. It is composed of different fluorescence markers under constitutive promoters, used to differentiate member of the population. As well as an amino acid biosynthesis genes under a inducible promoter, used to regulate the bacterial populations within the consortium.</p><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/Desulfurization"> second</a> codes for the distributed 4S pathway, and was created by splitting the <i>dsz</i> operon into each member species of our tunable consortium.</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/3/3f/Data_Page_diagram_.png"></p><br />
<br />
<font face=arial narrow size=4><b>Data for our Favourite New Parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000"><b>Main Page</a> - Strong Constitutive Promoter-ECFP generator, BBa_K804000:</b> This is an Enhanced Cyan Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses ECFP (BBa_E0420). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, ECFP excites at 439nm and emits at 476nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001"><b>Main Page</a> - Strong constitutive promoter-EYFP generator, BBa_K804001:</b> This is an Enhanced Yellow Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses EYFP (BBa_E0430). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, EYFP excites at 514nm and emits at 527nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804012"><b>Main Page</a> - Rhamnose Inducible TrpA coding gene, BBa_K804012:</b> This part contains a rhamnose inducible (pRha) TrpA coding gene. Upon induction with rhamnose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<br />
<font face=arial narrow size=4><b>Data for Pre-Existing Parts</b></font></br></br><font face=arial narrow><br />
<b><a href="http://partsregistry.org/Part:BBa_K902065:Experience">Experience</a> - Rhamnose inducible, glucose repressible promoter (pRha), BBa_K902065 (Calgary, iGEM 2012):</b> We placed the Rhamnose promoter upstream of our TrpA and MetA coding genes. In the respective auxotrophs, induction by arabinose resulted in growth compared to a negative control as measured by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0030:Experience">Experience</a> - Enhanced yellow fluorescent protein, BBa_E0030 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001">BBa_K804001</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0020:Experience">Experience</a> - Engineered cyan fluorescent protein, BBa_E0020 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000">BBa_K804000</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<br />
<font face=arial narrow size=4><b>We've also characterized the following parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804007"><b>Main Page</a> - Constitutive ECFP generator - Arabinose inducible TrpA coding gene, BBa_K804007:</b> This part contains a constitutive ECFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804009"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible TrpA coding gene, BBa_K804009:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804011"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible MetA coding gene, BBa_K804011:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804002"><b>Main Page</a> - TrpA coding gene, BBa_K804002:</b> This is the TrpA coding gene. It codes for the alpha subunit of tryptophan synthase (TSase α), and functions as both a binding site for indole-3-glycerol-phosphate (InGP) and can catalyze the cleavage of InGP to indole and glyceraldehyde-3-phosphate. This is the same part as BBa_K187028, but is in the pSB1C3 plasmid.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804003"><b>Main Page</a> - TyrA coding gene, BBa_K804003:</b> This is a TyrA coding gene for both the tyrosine and phenylalanine bio-synthetic pathways. TyrA expresses a bifunctional chorismate mutase/prehenate dehydrogenase which catalyzes the conversion of chorismate into prephenate and NAD+-dependent oxidative decarboxylation of prephanate.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804004"><b>Main Page</a> - MetA coding gene, BBa_K804004:</b> This is a MetA coding gene for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><b>Main Page</a> - DszC coding gene, BBa_K804005:</b> This is a DszC coding gene from the DszABC operon in the 4S pathway of Rhodococcus erythropolis IGTS8. It encodes one of the three biodesulfurizing enzymes in the DszABC operon. DszC enzymes have been shown to catalyze the oxidation of dibenzothiophene (DBT)to dibenzothiophene-5-oxide (DBTO) in the first reaction and then from DBTO to DBT sulfoxide (DBTO2) in the second reaction, both in the presence of NADH, oxygen, FMN and flavin reductases.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804006"><b>Main Page</a> - DszD coding gene, BBa_K804006:</b> This is a DszD coding gene isolated from Rhodococcus erythropolis IGTS8, which encodes for a NADH:FMN oxidoreductase to enhance the activities of DszA and DszC in the DszABC operon.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804008"><b>Main Page</a> - Arabinose inducible TrpA coding gene, BBa_K804008:</b> This part contains an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804010"><b>Main Page</a> - Arabinose inducible MetA coding gene, BBa_K804010:</b> This part contains an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804013"><b>Main Page</a> - Rhamnose Inducible MetA coding gene, BBa_K804013:</b> This part contains a rhamnose inducible (pRha) MetA coding gene. Upon induction with rhamnose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
</html></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DataTeam:British Columbia/Data2012-10-04T03:21:16Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<br />
<font face=arial narrow size=5><b>Our System</b></font></br></br><font face=arial narrow><br />
<br />
<p>We have designed a tunable microbial consortium to distribute the 4S biodesulfurization pathway, responsible for the bio-desulfurization of DBT, as a metabolic network.</p><br />
<br />
<h2>There are two major genetic circuits contained within our system:</h2><br><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/ProjectConsortia"> first</a> is responsible for tuning the relative populations of the bacteria within the consortium. It is composed of different fluorescence markers under constitutive promoters, used to differentiate member of the population. As well as an amino acid biosynthesis genes under a inducible promoter, used to regulate the bacterial populations within the consortium.</p><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/Desulfurization"> second</a> codes for the distributed 4S pathway, and was created by splitting the <i>dsz</i> operon into each member species of our tunable consortium.</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/3/3f/Data_Page_diagram_.png"></p><br />
<br />
<font face=arial narrow size=4><b>Data for our Favourite New Parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000"><b>Main Page</a> - Strong Constitutive Promoter-ECFP generator, BBa_K804000:</b> This is an Enhanced Cyan Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses ECFP (BBa_E0420). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, ECFP excites at 439nm and emits at 476nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001"><b>Main Page</a> - Strong constitutive promoter-EYFP generator, BBa_K804001:</b> This is an Enhanced Yellow Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses EYFP (BBa_E0430). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, EYFP excites at 514nm and emits at 527nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804012"><b>Main Page</a> - Rhamnose Inducible TrpA coding gene, BBa_K804012:</b> This part contains a rhamnose inducible (pRha) TrpA coding gene. Upon induction with rhamnose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<br />
<font face=arial narrow size=4><b>Data for Pre-Existing Parts</b></font></br></br><font face=arial narrow><br />
<b><a href="http://partsregistry.org/Part:BBa_K902065:Experience">Experience</a> - Rhamnose inducible, glucose repressible promoter (pRha), BBa_K902065 (Calgary, iGEM 2012):</b> We placed the Rhamnose promoter upstream of our TrpA and MetA coding genes. In the respective auxotrophs, induction by arabinose resulted in growth compared to a negative control as measured by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0030:Experience">Experience</a> - Enhanced yellow fluorescent protein, BBa_E0030 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001">BBa_K804001</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0020:Experience">Experience</a> - Engineered cyan fluorescent protein, BBa_E0020 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000">BBa_K804000</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<br />
<font face=arial narrow size=4><b>We've also characterized the following parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804007"><b>Main Page</a> - Constitutive ECFP generator - Arabinose inducible TrpA coding gene, BBa_K804007:</b> This part contains a constitutive ECFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804009"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible TrpA coding gene, BBa_K804009:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804011"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible MetA coding gene, BBa_K804011:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804002"><b>Main Page</a> - TrpA coding gene, BBa_K804002:</b> This is the TrpA coding gene. It codes for the alpha subunit of tryptophan synthase (TSase α), and functions as both a binding site for indole-3-glycerol-phosphate (InGP) and can catalyze the cleavage of InGP to indole and glyceraldehyde-3-phosphate. This is the same part as BBa_K187028, but is in the pSB1C3 plasmid.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804003"><b>Main Page</a> - TyrA coding gene, BBa_K804003:</b> This is a TyrA coding gene for both the tyrosine and phenylalanine bio-synthetic pathways. TyrA expresses a bifunctional chorismate mutase/prehenate dehydrogenase which catalyzes the conversion of chorismate into prephenate and NAD+-dependent oxidative decarboxylation of prephanate.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804004"><b>Main Page</a> - MetA coding gene, BBa_K804004:</b> This is a MetA coding gene for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><b>Main Page</a> - DszC coding gene, BBa_K804005:</b> This is a DszC coding gene from the DszABC operon in the 4S pathway of Rhodococcus erythropolis IGTS8. It encodes one of the three biodesulfurizing enzymes in the DszABC operon. DszC enzymes have been shown to catalyze the oxidation of dibenzothiophene (DBT)to dibenzothiophene-5-oxide (DBTO) in the first reaction and then from DBTO to DBT sulfoxide (DBTO2) in the second reaction, both in the presence of NADH, oxygen, FMN and flavin reductases.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804006"><b>Main Page</a> - DszD coding gene, BBa_K804006:</b> This is a DszD coding gene isolated from Rhodococcus erythropolis IGTS8, which encodes for a NADH:FMN oxidoreductase to enhance the activities of DszA and DszC in the DszABC operon.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804008"><b>Main Page</a> - Arabinose inducible TrpA coding gene, BBa_K804008:</b> This part contains an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804010"><b>Main Page</a> - Arabinose inducible MetA coding gene, BBa_K804010:</b> This part contains an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804013"><b>Main Page</a> - Rhamnose Inducible MetA coding gene, BBa_K804013:</b> This part contains a rhamnose inducible (pRha) MetA coding gene. Upon induction with rhamnose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
</html></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DataTeam:British Columbia/Data2012-10-04T03:21:01Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<br />
<font face=arial narrow size=5><b>Our System</b></font></br></br><font face=arial narrow><br />
<br />
<p>We have designed a tunable microbial consortium to distribute the 4S biodesulfurization pathway, responsible for the bio-desulfurization of DBT, as a metabolic network.</p><br />
<br />
<h2><b>There are two major genetic circuits contained within our system:</b></h2><br><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/ProjectConsortia"> first</a> is responsible for tuning the relative populations of the bacteria within the consortium. It is composed of different fluorescence markers under constitutive promoters, used to differentiate member of the population. As well as an amino acid biosynthesis genes under a inducible promoter, used to regulate the bacterial populations within the consortium.</p><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/Desulfurization"> second</a> codes for the distributed 4S pathway, and was created by splitting the <i>dsz</i> operon into each member species of our tunable consortium.</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/3/3f/Data_Page_diagram_.png"></p><br />
<br />
<font face=arial narrow size=4><b>Data for our Favourite New Parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000"><b>Main Page</a> - Strong Constitutive Promoter-ECFP generator, BBa_K804000:</b> This is an Enhanced Cyan Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses ECFP (BBa_E0420). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, ECFP excites at 439nm and emits at 476nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001"><b>Main Page</a> - Strong constitutive promoter-EYFP generator, BBa_K804001:</b> This is an Enhanced Yellow Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses EYFP (BBa_E0430). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, EYFP excites at 514nm and emits at 527nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804012"><b>Main Page</a> - Rhamnose Inducible TrpA coding gene, BBa_K804012:</b> This part contains a rhamnose inducible (pRha) TrpA coding gene. Upon induction with rhamnose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<br />
<font face=arial narrow size=4><b>Data for Pre-Existing Parts</b></font></br></br><font face=arial narrow><br />
<b><a href="http://partsregistry.org/Part:BBa_K902065:Experience">Experience</a> - Rhamnose inducible, glucose repressible promoter (pRha), BBa_K902065 (Calgary, iGEM 2012):</b> We placed the Rhamnose promoter upstream of our TrpA and MetA coding genes. In the respective auxotrophs, induction by arabinose resulted in growth compared to a negative control as measured by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0030:Experience">Experience</a> - Enhanced yellow fluorescent protein, BBa_E0030 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001">BBa_K804001</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0020:Experience">Experience</a> - Engineered cyan fluorescent protein, BBa_E0020 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000">BBa_K804000</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<br />
<font face=arial narrow size=4><b>We've also characterized the following parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804007"><b>Main Page</a> - Constitutive ECFP generator - Arabinose inducible TrpA coding gene, BBa_K804007:</b> This part contains a constitutive ECFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804009"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible TrpA coding gene, BBa_K804009:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804011"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible MetA coding gene, BBa_K804011:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804002"><b>Main Page</a> - TrpA coding gene, BBa_K804002:</b> This is the TrpA coding gene. It codes for the alpha subunit of tryptophan synthase (TSase α), and functions as both a binding site for indole-3-glycerol-phosphate (InGP) and can catalyze the cleavage of InGP to indole and glyceraldehyde-3-phosphate. This is the same part as BBa_K187028, but is in the pSB1C3 plasmid.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804003"><b>Main Page</a> - TyrA coding gene, BBa_K804003:</b> This is a TyrA coding gene for both the tyrosine and phenylalanine bio-synthetic pathways. TyrA expresses a bifunctional chorismate mutase/prehenate dehydrogenase which catalyzes the conversion of chorismate into prephenate and NAD+-dependent oxidative decarboxylation of prephanate.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804004"><b>Main Page</a> - MetA coding gene, BBa_K804004:</b> This is a MetA coding gene for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><b>Main Page</a> - DszC coding gene, BBa_K804005:</b> This is a DszC coding gene from the DszABC operon in the 4S pathway of Rhodococcus erythropolis IGTS8. It encodes one of the three biodesulfurizing enzymes in the DszABC operon. DszC enzymes have been shown to catalyze the oxidation of dibenzothiophene (DBT)to dibenzothiophene-5-oxide (DBTO) in the first reaction and then from DBTO to DBT sulfoxide (DBTO2) in the second reaction, both in the presence of NADH, oxygen, FMN and flavin reductases.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804006"><b>Main Page</a> - DszD coding gene, BBa_K804006:</b> This is a DszD coding gene isolated from Rhodococcus erythropolis IGTS8, which encodes for a NADH:FMN oxidoreductase to enhance the activities of DszA and DszC in the DszABC operon.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804008"><b>Main Page</a> - Arabinose inducible TrpA coding gene, BBa_K804008:</b> This part contains an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804010"><b>Main Page</a> - Arabinose inducible MetA coding gene, BBa_K804010:</b> This part contains an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804013"><b>Main Page</a> - Rhamnose Inducible MetA coding gene, BBa_K804013:</b> This part contains a rhamnose inducible (pRha) MetA coding gene. Upon induction with rhamnose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
</html></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DataTeam:British Columbia/Data2012-10-04T03:19:54Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<br />
<font face=arial narrow size=5><b>Our System</b></font></br></br><font face=arial narrow><br />
<br />
<p>We have designed a tunable microbial consortium to distribute the 4S biodesulfurization pathway, responsible for the bio-desulfurization of DBT, as a metabolic network.</p><br />
<br />
<b>There are two major genetic circuits contained within our system:</b><br><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/ProjectConsortia"> first</a> is responsible for tuning the relative populations of the bacteria within the consortium. It is composed of different fluorescence markers under constitutive promoters, used to differentiate member of the population. As well as an amino acid biosynthesis genes under a inducible promoter, used to regulate the bacterial populations within the consortium.</p><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/Desulfurization"> second</a> codes for the distributed 4S pathway, and was created by splitting the <i>dsz</i> operon into each member species of our tunable consortium.</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/3/3f/Data_Page_diagram_.png"></p><br />
<br />
<font face=arial narrow size=4><b>Data for our Favourite New Parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000"><b>Main Page</a> - Strong Constitutive Promoter-ECFP generator, BBa_K804000:</b> This is an Enhanced Cyan Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses ECFP (BBa_E0420). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, ECFP excites at 439nm and emits at 476nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001"><b>Main Page</a> - Strong constitutive promoter-EYFP generator, BBa_K804001:</b> This is an Enhanced Yellow Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses EYFP (BBa_E0430). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, EYFP excites at 514nm and emits at 527nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804012"><b>Main Page</a> - Rhamnose Inducible TrpA coding gene, BBa_K804012:</b> This part contains a rhamnose inducible (pRha) TrpA coding gene. Upon induction with rhamnose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<br />
<font face=arial narrow size=4><b>Data for Pre-Existing Parts</b></font></br></br><font face=arial narrow><br />
<b><a href="http://partsregistry.org/Part:BBa_K902065:Experience">Experience</a> - Rhamnose inducible, glucose repressible promoter (pRha), BBa_K902065 (Calgary, iGEM 2012):</b> We placed the Rhamnose promoter upstream of our TrpA and MetA coding genes. In the respective auxotrophs, induction by arabinose resulted in growth compared to a negative control as measured by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0030:Experience">Experience</a> - Enhanced yellow fluorescent protein, BBa_E0030 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001">BBa_K804001</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0020:Experience">Experience</a> - Engineered cyan fluorescent protein, BBa_E0020 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000">BBa_K804000</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<br />
<font face=arial narrow size=4><b>We've also characterized the following parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804007"><b>Main Page</a> - Constitutive ECFP generator - Arabinose inducible TrpA coding gene, BBa_K804007:</b> This part contains a constitutive ECFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804009"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible TrpA coding gene, BBa_K804009:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804011"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible MetA coding gene, BBa_K804011:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804002"><b>Main Page</a> - TrpA coding gene, BBa_K804002:</b> This is the TrpA coding gene. It codes for the alpha subunit of tryptophan synthase (TSase α), and functions as both a binding site for indole-3-glycerol-phosphate (InGP) and can catalyze the cleavage of InGP to indole and glyceraldehyde-3-phosphate. This is the same part as BBa_K187028, but is in the pSB1C3 plasmid.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804003"><b>Main Page</a> - TyrA coding gene, BBa_K804003:</b> This is a TyrA coding gene for both the tyrosine and phenylalanine bio-synthetic pathways. TyrA expresses a bifunctional chorismate mutase/prehenate dehydrogenase which catalyzes the conversion of chorismate into prephenate and NAD+-dependent oxidative decarboxylation of prephanate.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804004"><b>Main Page</a> - MetA coding gene, BBa_K804004:</b> This is a MetA coding gene for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><b>Main Page</a> - DszC coding gene, BBa_K804005:</b> This is a DszC coding gene from the DszABC operon in the 4S pathway of Rhodococcus erythropolis IGTS8. It encodes one of the three biodesulfurizing enzymes in the DszABC operon. DszC enzymes have been shown to catalyze the oxidation of dibenzothiophene (DBT)to dibenzothiophene-5-oxide (DBTO) in the first reaction and then from DBTO to DBT sulfoxide (DBTO2) in the second reaction, both in the presence of NADH, oxygen, FMN and flavin reductases.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804006"><b>Main Page</a> - DszD coding gene, BBa_K804006:</b> This is a DszD coding gene isolated from Rhodococcus erythropolis IGTS8, which encodes for a NADH:FMN oxidoreductase to enhance the activities of DszA and DszC in the DszABC operon.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804008"><b>Main Page</a> - Arabinose inducible TrpA coding gene, BBa_K804008:</b> This part contains an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804010"><b>Main Page</a> - Arabinose inducible MetA coding gene, BBa_K804010:</b> This part contains an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804013"><b>Main Page</a> - Rhamnose Inducible MetA coding gene, BBa_K804013:</b> This part contains a rhamnose inducible (pRha) MetA coding gene. Upon induction with rhamnose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
</html></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DataTeam:British Columbia/Data2012-10-04T03:19:27Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<br />
<font face=arial narrow size=5><b>Our System</b></font></br></br><font face=arial narrow><br />
<br />
<p>We have designed a tunable microbial consortium to distribute the 4S biodesulfurization pathway, responsible for the bio-desulfurization of DBT, as a metabolic network.</p><br />
<br />
<b>There are two major genetic circuits contained within our system:</b><br><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/ProjectConsortia"> first</a> is responsible for tuning the relative populations of the bacteria within the consortium. It is composed of different fluorescence markers under constitutive promoters, used to differentiate member of the population. As well as an amino acid biosynthesis genes under a inducible promoter, used to regulate the bacterial populations within the consortium.</p><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/Desulfurization"> second</a> codes for the distributed 4S pathway, and was created by splitting the <i>Dsz</i> operon into each member species of our tunable consortium.</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/3/3f/Data_Page_diagram_.png"></p><br />
<br />
<font face=arial narrow size=4><b>Data for our Favourite New Parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000"><b>Main Page</a> - Strong Constitutive Promoter-ECFP generator, BBa_K804000:</b> This is an Enhanced Cyan Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses ECFP (BBa_E0420). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, ECFP excites at 439nm and emits at 476nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001"><b>Main Page</a> - Strong constitutive promoter-EYFP generator, BBa_K804001:</b> This is an Enhanced Yellow Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses EYFP (BBa_E0430). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, EYFP excites at 514nm and emits at 527nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804012"><b>Main Page</a> - Rhamnose Inducible TrpA coding gene, BBa_K804012:</b> This part contains a rhamnose inducible (pRha) TrpA coding gene. Upon induction with rhamnose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<br />
<font face=arial narrow size=4><b>Data for Pre-Existing Parts</b></font></br></br><font face=arial narrow><br />
<b><a href="http://partsregistry.org/Part:BBa_K902065:Experience">Experience</a> - Rhamnose inducible, glucose repressible promoter (pRha), BBa_K902065 (Calgary, iGEM 2012):</b> We placed the Rhamnose promoter upstream of our TrpA and MetA coding genes. In the respective auxotrophs, induction by arabinose resulted in growth compared to a negative control as measured by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0030:Experience">Experience</a> - Enhanced yellow fluorescent protein, BBa_E0030 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001">BBa_K804001</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0020:Experience">Experience</a> - Engineered cyan fluorescent protein, BBa_E0020 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000">BBa_K804000</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<br />
<font face=arial narrow size=4><b>We've also characterized the following parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804007"><b>Main Page</a> - Constitutive ECFP generator - Arabinose inducible TrpA coding gene, BBa_K804007:</b> This part contains a constitutive ECFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804009"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible TrpA coding gene, BBa_K804009:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804011"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible MetA coding gene, BBa_K804011:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804002"><b>Main Page</a> - TrpA coding gene, BBa_K804002:</b> This is the TrpA coding gene. It codes for the alpha subunit of tryptophan synthase (TSase α), and functions as both a binding site for indole-3-glycerol-phosphate (InGP) and can catalyze the cleavage of InGP to indole and glyceraldehyde-3-phosphate. This is the same part as BBa_K187028, but is in the pSB1C3 plasmid.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804003"><b>Main Page</a> - TyrA coding gene, BBa_K804003:</b> This is a TyrA coding gene for both the tyrosine and phenylalanine bio-synthetic pathways. TyrA expresses a bifunctional chorismate mutase/prehenate dehydrogenase which catalyzes the conversion of chorismate into prephenate and NAD+-dependent oxidative decarboxylation of prephanate.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804004"><b>Main Page</a> - MetA coding gene, BBa_K804004:</b> This is a MetA coding gene for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><b>Main Page</a> - DszC coding gene, BBa_K804005:</b> This is a DszC coding gene from the DszABC operon in the 4S pathway of Rhodococcus erythropolis IGTS8. It encodes one of the three biodesulfurizing enzymes in the DszABC operon. DszC enzymes have been shown to catalyze the oxidation of dibenzothiophene (DBT)to dibenzothiophene-5-oxide (DBTO) in the first reaction and then from DBTO to DBT sulfoxide (DBTO2) in the second reaction, both in the presence of NADH, oxygen, FMN and flavin reductases.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804006"><b>Main Page</a> - DszD coding gene, BBa_K804006:</b> This is a DszD coding gene isolated from Rhodococcus erythropolis IGTS8, which encodes for a NADH:FMN oxidoreductase to enhance the activities of DszA and DszC in the DszABC operon.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804008"><b>Main Page</a> - Arabinose inducible TrpA coding gene, BBa_K804008:</b> This part contains an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804010"><b>Main Page</a> - Arabinose inducible MetA coding gene, BBa_K804010:</b> This part contains an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804013"><b>Main Page</a> - Rhamnose Inducible MetA coding gene, BBa_K804013:</b> This part contains a rhamnose inducible (pRha) MetA coding gene. Upon induction with rhamnose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
</html></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DataTeam:British Columbia/Data2012-10-04T03:18:50Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<br />
<font face=arial narrow size=5><b>Our System</b></font></br></br><font face=arial narrow><br />
<br />
<p>We have designed a tunable microbial consortium to distribute the 4S pathway, responsible for the bio-desulfurization of DBT, as a metabolic network.</p><br />
<br />
<b>There are two major genetic circuits contained within our system:</b><br><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/ProjectConsortia"> first</a> is responsible for tuning the relative populations of the bacteria within the consortium. It is composed of different fluorescence markers under constitutive promoters, used to differentiate member of the population. As well as an amino acid biosynthesis genes under a inducible promoter, used to regulate the bacterial populations within the consortium.</p><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/Desulfurization"> second</a> codes for the distributed 4S desulfurization pathway, and was created by splitting the <i>Dsz</i> operon into each member species of our tunable consortium.</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/3/3f/Data_Page_diagram_.png"></p><br />
<br />
<font face=arial narrow size=4><b>Data for our Favourite New Parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000"><b>Main Page</a> - Strong Constitutive Promoter-ECFP generator, BBa_K804000:</b> This is an Enhanced Cyan Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses ECFP (BBa_E0420). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, ECFP excites at 439nm and emits at 476nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001"><b>Main Page</a> - Strong constitutive promoter-EYFP generator, BBa_K804001:</b> This is an Enhanced Yellow Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses EYFP (BBa_E0430). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, EYFP excites at 514nm and emits at 527nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804012"><b>Main Page</a> - Rhamnose Inducible TrpA coding gene, BBa_K804012:</b> This part contains a rhamnose inducible (pRha) TrpA coding gene. Upon induction with rhamnose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<br />
<font face=arial narrow size=4><b>Data for Pre-Existing Parts</b></font></br></br><font face=arial narrow><br />
<b><a href="http://partsregistry.org/Part:BBa_K902065:Experience">Experience</a> - Rhamnose inducible, glucose repressible promoter (pRha), BBa_K902065 (Calgary, iGEM 2012):</b> We placed the Rhamnose promoter upstream of our TrpA and MetA coding genes. In the respective auxotrophs, induction by arabinose resulted in growth compared to a negative control as measured by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0030:Experience">Experience</a> - Enhanced yellow fluorescent protein, BBa_E0030 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001">BBa_K804001</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0020:Experience">Experience</a> - Engineered cyan fluorescent protein, BBa_E0020 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000">BBa_K804000</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<br />
<font face=arial narrow size=4><b>We've also characterized the following parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804007"><b>Main Page</a> - Constitutive ECFP generator - Arabinose inducible TrpA coding gene, BBa_K804007:</b> This part contains a constitutive ECFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804009"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible TrpA coding gene, BBa_K804009:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804011"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible MetA coding gene, BBa_K804011:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804002"><b>Main Page</a> - TrpA coding gene, BBa_K804002:</b> This is the TrpA coding gene. It codes for the alpha subunit of tryptophan synthase (TSase α), and functions as both a binding site for indole-3-glycerol-phosphate (InGP) and can catalyze the cleavage of InGP to indole and glyceraldehyde-3-phosphate. This is the same part as BBa_K187028, but is in the pSB1C3 plasmid.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804003"><b>Main Page</a> - TyrA coding gene, BBa_K804003:</b> This is a TyrA coding gene for both the tyrosine and phenylalanine bio-synthetic pathways. TyrA expresses a bifunctional chorismate mutase/prehenate dehydrogenase which catalyzes the conversion of chorismate into prephenate and NAD+-dependent oxidative decarboxylation of prephanate.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804004"><b>Main Page</a> - MetA coding gene, BBa_K804004:</b> This is a MetA coding gene for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><b>Main Page</a> - DszC coding gene, BBa_K804005:</b> This is a DszC coding gene from the DszABC operon in the 4S pathway of Rhodococcus erythropolis IGTS8. It encodes one of the three biodesulfurizing enzymes in the DszABC operon. DszC enzymes have been shown to catalyze the oxidation of dibenzothiophene (DBT)to dibenzothiophene-5-oxide (DBTO) in the first reaction and then from DBTO to DBT sulfoxide (DBTO2) in the second reaction, both in the presence of NADH, oxygen, FMN and flavin reductases.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804006"><b>Main Page</a> - DszD coding gene, BBa_K804006:</b> This is a DszD coding gene isolated from Rhodococcus erythropolis IGTS8, which encodes for a NADH:FMN oxidoreductase to enhance the activities of DszA and DszC in the DszABC operon.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804008"><b>Main Page</a> - Arabinose inducible TrpA coding gene, BBa_K804008:</b> This part contains an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804010"><b>Main Page</a> - Arabinose inducible MetA coding gene, BBa_K804010:</b> This part contains an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804013"><b>Main Page</a> - Rhamnose Inducible MetA coding gene, BBa_K804013:</b> This part contains a rhamnose inducible (pRha) MetA coding gene. Upon induction with rhamnose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
</html></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DataTeam:British Columbia/Data2012-10-04T03:16:48Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<br />
<font face=arial narrow size=5><b>Our System</b></font></br></br><font face=arial narrow><br />
<br />
<p>We have designed a tunable microbial consortium to distribute the 4S pathway, responsible for the bio-desulfurization of DBT, as a metabolic network.</p><br />
<br />
<b>There are two major genetic circuits contained within our system:</b><br><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/ProjectConsortia"> first</a> is responsible for tuning the relative populations of the bacteria within the consortium. It is composed of different fluorescence markers under constitutive promoters, used to differentiate member of the population. As well as an amino acid biosynthesis genes under a inducible promoter, used to regulate the bacterial populations within the consortium.</p><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/Desulfurization"> second</a> is responsible for the 4S desulfurization-distributed metabolic network and was created by splitting the Dsz operon into each member species of our tunable consortium.</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/3/3f/Data_Page_diagram_.png"></p><br />
<br />
<font face=arial narrow size=4><b>Data for our Favourite New Parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000"><b>Main Page</a> - Strong Constitutive Promoter-ECFP generator, BBa_K804000:</b> This is an Enhanced Cyan Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses ECFP (BBa_E0420). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, ECFP excites at 439nm and emits at 476nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001"><b>Main Page</a> - Strong constitutive promoter-EYFP generator, BBa_K804001:</b> This is an Enhanced Yellow Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses EYFP (BBa_E0430). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, EYFP excites at 514nm and emits at 527nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804012"><b>Main Page</a> - Rhamnose Inducible TrpA coding gene, BBa_K804012:</b> This part contains a rhamnose inducible (pRha) TrpA coding gene. Upon induction with rhamnose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<br />
<font face=arial narrow size=4><b>Data for Pre-Existing Parts</b></font></br></br><font face=arial narrow><br />
<b><a href="http://partsregistry.org/Part:BBa_K902065:Experience">Experience</a> - Rhamnose inducible, glucose repressible promoter (pRha), BBa_K902065 (Calgary, iGEM 2012):</b> We placed the Rhamnose promoter upstream of our TrpA and MetA coding genes. In the respective auxotrophs, induction by arabinose resulted in growth compared to a negative control as measured by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0030:Experience">Experience</a> - Enhanced yellow fluorescent protein, BBa_E0030 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001">BBa_K804001</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0020:Experience">Experience</a> - Engineered cyan fluorescent protein, BBa_E0020 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000">BBa_K804000</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<br />
<font face=arial narrow size=4><b>We've also characterized the following parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804007"><b>Main Page</a> - Constitutive ECFP generator - Arabinose inducible TrpA coding gene, BBa_K804007:</b> This part contains a constitutive ECFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804009"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible TrpA coding gene, BBa_K804009:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804011"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible MetA coding gene, BBa_K804011:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804002"><b>Main Page</a> - TrpA coding gene, BBa_K804002:</b> This is the TrpA coding gene. It codes for the alpha subunit of tryptophan synthase (TSase α), and functions as both a binding site for indole-3-glycerol-phosphate (InGP) and can catalyze the cleavage of InGP to indole and glyceraldehyde-3-phosphate. This is the same part as BBa_K187028, but is in the pSB1C3 plasmid.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804003"><b>Main Page</a> - TyrA coding gene, BBa_K804003:</b> This is a TyrA coding gene for both the tyrosine and phenylalanine bio-synthetic pathways. TyrA expresses a bifunctional chorismate mutase/prehenate dehydrogenase which catalyzes the conversion of chorismate into prephenate and NAD+-dependent oxidative decarboxylation of prephanate.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804004"><b>Main Page</a> - MetA coding gene, BBa_K804004:</b> This is a MetA coding gene for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><b>Main Page</a> - DszC coding gene, BBa_K804005:</b> This is a DszC coding gene from the DszABC operon in the 4S pathway of Rhodococcus erythropolis IGTS8. It encodes one of the three biodesulfurizing enzymes in the DszABC operon. DszC enzymes have been shown to catalyze the oxidation of dibenzothiophene (DBT)to dibenzothiophene-5-oxide (DBTO) in the first reaction and then from DBTO to DBT sulfoxide (DBTO2) in the second reaction, both in the presence of NADH, oxygen, FMN and flavin reductases.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804006"><b>Main Page</a> - DszD coding gene, BBa_K804006:</b> This is a DszD coding gene isolated from Rhodococcus erythropolis IGTS8, which encodes for a NADH:FMN oxidoreductase to enhance the activities of DszA and DszC in the DszABC operon.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804008"><b>Main Page</a> - Arabinose inducible TrpA coding gene, BBa_K804008:</b> This part contains an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804010"><b>Main Page</a> - Arabinose inducible MetA coding gene, BBa_K804010:</b> This part contains an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804013"><b>Main Page</a> - Rhamnose Inducible MetA coding gene, BBa_K804013:</b> This part contains a rhamnose inducible (pRha) MetA coding gene. Upon induction with rhamnose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
</html></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DataTeam:British Columbia/Data2012-10-04T03:16:31Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<br />
<font face=arial narrow size=5><b>Our System</b></font></br></br><font face=arial narrow><br />
<br />
<p>We have designed a tunable microbial consortium to distribute the 4S pathway, responsible for the bio-desulfurization of DBT, as a metabolic network.</p><br />
<br />
<b>There are two major genetic circuits contained within our system:</b><br><br />
<br />
*<p>The <a href="https://2012.igem.org/Team:British_Columbia/ProjectConsortia"> first</a> is responsible for tuning the relative populations of the bacteria within the consortium. It is composed of different fluorescence markers under constitutive promoters, used to differentiate member of the population. As well as an amino acid biosynthesis genes under a inducible promoter, used to regulate the bacterial populations within the consortium.</p><br />
<br />
*<p>The <a href="https://2012.igem.org/Team:British_Columbia/Desulfurization"> second</a> is responsible for the 4S desulfurization-distributed metabolic network and was created by splitting the Dsz operon into each member species of our tunable consortium.</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/3/3f/Data_Page_diagram_.png"></p><br />
<br />
<font face=arial narrow size=4><b>Data for our Favourite New Parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000"><b>Main Page</a> - Strong Constitutive Promoter-ECFP generator, BBa_K804000:</b> This is an Enhanced Cyan Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses ECFP (BBa_E0420). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, ECFP excites at 439nm and emits at 476nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001"><b>Main Page</a> - Strong constitutive promoter-EYFP generator, BBa_K804001:</b> This is an Enhanced Yellow Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses EYFP (BBa_E0430). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, EYFP excites at 514nm and emits at 527nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804012"><b>Main Page</a> - Rhamnose Inducible TrpA coding gene, BBa_K804012:</b> This part contains a rhamnose inducible (pRha) TrpA coding gene. Upon induction with rhamnose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<br />
<font face=arial narrow size=4><b>Data for Pre-Existing Parts</b></font></br></br><font face=arial narrow><br />
<b><a href="http://partsregistry.org/Part:BBa_K902065:Experience">Experience</a> - Rhamnose inducible, glucose repressible promoter (pRha), BBa_K902065 (Calgary, iGEM 2012):</b> We placed the Rhamnose promoter upstream of our TrpA and MetA coding genes. In the respective auxotrophs, induction by arabinose resulted in growth compared to a negative control as measured by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0030:Experience">Experience</a> - Enhanced yellow fluorescent protein, BBa_E0030 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001">BBa_K804001</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0020:Experience">Experience</a> - Engineered cyan fluorescent protein, BBa_E0020 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000">BBa_K804000</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<br />
<font face=arial narrow size=4><b>We've also characterized the following parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804007"><b>Main Page</a> - Constitutive ECFP generator - Arabinose inducible TrpA coding gene, BBa_K804007:</b> This part contains a constitutive ECFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804009"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible TrpA coding gene, BBa_K804009:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804011"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible MetA coding gene, BBa_K804011:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804002"><b>Main Page</a> - TrpA coding gene, BBa_K804002:</b> This is the TrpA coding gene. It codes for the alpha subunit of tryptophan synthase (TSase α), and functions as both a binding site for indole-3-glycerol-phosphate (InGP) and can catalyze the cleavage of InGP to indole and glyceraldehyde-3-phosphate. This is the same part as BBa_K187028, but is in the pSB1C3 plasmid.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804003"><b>Main Page</a> - TyrA coding gene, BBa_K804003:</b> This is a TyrA coding gene for both the tyrosine and phenylalanine bio-synthetic pathways. TyrA expresses a bifunctional chorismate mutase/prehenate dehydrogenase which catalyzes the conversion of chorismate into prephenate and NAD+-dependent oxidative decarboxylation of prephanate.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804004"><b>Main Page</a> - MetA coding gene, BBa_K804004:</b> This is a MetA coding gene for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><b>Main Page</a> - DszC coding gene, BBa_K804005:</b> This is a DszC coding gene from the DszABC operon in the 4S pathway of Rhodococcus erythropolis IGTS8. It encodes one of the three biodesulfurizing enzymes in the DszABC operon. DszC enzymes have been shown to catalyze the oxidation of dibenzothiophene (DBT)to dibenzothiophene-5-oxide (DBTO) in the first reaction and then from DBTO to DBT sulfoxide (DBTO2) in the second reaction, both in the presence of NADH, oxygen, FMN and flavin reductases.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804006"><b>Main Page</a> - DszD coding gene, BBa_K804006:</b> This is a DszD coding gene isolated from Rhodococcus erythropolis IGTS8, which encodes for a NADH:FMN oxidoreductase to enhance the activities of DszA and DszC in the DszABC operon.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804008"><b>Main Page</a> - Arabinose inducible TrpA coding gene, BBa_K804008:</b> This part contains an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804010"><b>Main Page</a> - Arabinose inducible MetA coding gene, BBa_K804010:</b> This part contains an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804013"><b>Main Page</a> - Rhamnose Inducible MetA coding gene, BBa_K804013:</b> This part contains a rhamnose inducible (pRha) MetA coding gene. Upon induction with rhamnose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
</html></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DataTeam:British Columbia/Data2012-10-04T03:16:06Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<br />
<font face=arial narrow size=5><b>Our System</b></font></br></br><font face=arial narrow><br />
<br />
<p>We have designed a tunable microbial consortium to distribute the 4S pathway, responsible for the bio-desulfurization of DBT, as a metabolic network.</p><br />
<br />
<b>There are two major genetic circuits contained within our system:</b><br><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/ProjectConsortia"> first</a> is responsible for tuning the relative populations of the bacteria within the consortium. It is composed of different fluorescence markers under constitutive promoters, used to differentiate member of the population. As well as an amino acid biosynthesis genes under a inducible promoter, used to regulate the bacterial populations within the consortium.</p><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/Desulfurization"> second</a> is responsible for the 4S desulfurization-distributed metabolic network and was created by splitting the Dsz operon into each member species of our tunable consortium.</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/3/3f/Data_Page_diagram_.png"></p><br />
<br />
<font face=arial narrow size=4><b>Data for our Favourite New Parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000"><b>Main Page</a> - Strong Constitutive Promoter-ECFP generator, BBa_K804000:</b> This is an Enhanced Cyan Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses ECFP (BBa_E0420). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, ECFP excites at 439nm and emits at 476nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001"><b>Main Page</a> - Strong constitutive promoter-EYFP generator, BBa_K804001:</b> This is an Enhanced Yellow Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses EYFP (BBa_E0430). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, EYFP excites at 514nm and emits at 527nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804012"><b>Main Page</a> - Rhamnose Inducible TrpA coding gene, BBa_K804012:</b> This part contains a rhamnose inducible (pRha) TrpA coding gene. Upon induction with rhamnose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<br />
<font face=arial narrow size=4><b>Data for Pre-Existing Parts</b></font></br></br><font face=arial narrow><br />
<b><a href="http://partsregistry.org/Part:BBa_K902065:Experience">Experience</a> - Rhamnose inducible, glucose repressible promoter (pRha), BBa_K902065 (Calgary, iGEM 2012):</b> We placed the Rhamnose promoter upstream of our TrpA and MetA coding genes. In the respective auxotrophs, induction by arabinose resulted in growth compared to a negative control as measured by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0030:Experience">Experience</a> - Enhanced yellow fluorescent protein, BBa_E0030 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001">BBa_K804001</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0020:Experience">Experience</a> - Engineered cyan fluorescent protein, BBa_E0020 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000">BBa_K804000</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<br />
<font face=arial narrow size=4><b>We've also characterized the following parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804007"><b>Main Page</a> - Constitutive ECFP generator - Arabinose inducible TrpA coding gene, BBa_K804007:</b> This part contains a constitutive ECFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804009"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible TrpA coding gene, BBa_K804009:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804011"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible MetA coding gene, BBa_K804011:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804002"><b>Main Page</a> - TrpA coding gene, BBa_K804002:</b> This is the TrpA coding gene. It codes for the alpha subunit of tryptophan synthase (TSase α), and functions as both a binding site for indole-3-glycerol-phosphate (InGP) and can catalyze the cleavage of InGP to indole and glyceraldehyde-3-phosphate. This is the same part as BBa_K187028, but is in the pSB1C3 plasmid.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804003"><b>Main Page</a> - TyrA coding gene, BBa_K804003:</b> This is a TyrA coding gene for both the tyrosine and phenylalanine bio-synthetic pathways. TyrA expresses a bifunctional chorismate mutase/prehenate dehydrogenase which catalyzes the conversion of chorismate into prephenate and NAD+-dependent oxidative decarboxylation of prephanate.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804004"><b>Main Page</a> - MetA coding gene, BBa_K804004:</b> This is a MetA coding gene for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><b>Main Page</a> - DszC coding gene, BBa_K804005:</b> This is a DszC coding gene from the DszABC operon in the 4S pathway of Rhodococcus erythropolis IGTS8. It encodes one of the three biodesulfurizing enzymes in the DszABC operon. DszC enzymes have been shown to catalyze the oxidation of dibenzothiophene (DBT)to dibenzothiophene-5-oxide (DBTO) in the first reaction and then from DBTO to DBT sulfoxide (DBTO2) in the second reaction, both in the presence of NADH, oxygen, FMN and flavin reductases.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804006"><b>Main Page</a> - DszD coding gene, BBa_K804006:</b> This is a DszD coding gene isolated from Rhodococcus erythropolis IGTS8, which encodes for a NADH:FMN oxidoreductase to enhance the activities of DszA and DszC in the DszABC operon.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804008"><b>Main Page</a> - Arabinose inducible TrpA coding gene, BBa_K804008:</b> This part contains an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804010"><b>Main Page</a> - Arabinose inducible MetA coding gene, BBa_K804010:</b> This part contains an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804013"><b>Main Page</a> - Rhamnose Inducible MetA coding gene, BBa_K804013:</b> This part contains a rhamnose inducible (pRha) MetA coding gene. Upon induction with rhamnose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
</html></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DataTeam:British Columbia/Data2012-10-04T03:14:05Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<br />
<font face=arial narrow size=5><b>Our System</b></font></br></br><font face=arial narrow><br />
<br />
<p>We have designed a tunable microbial consortium that distributes the 4S pathway, responsible for the bio-desulfurization of DBT, as a metabolic network.</p><br />
<br />
<b>There are two major genetic circuits contained within our system:</b><br><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/ProjectConsortia"> first</a> is responsible for tuning the relative populations of the bacteria within the consortium. It is composed of different fluorescence markers under constitutive promoters, used to differentiate member of the population. As well as an amino acid biosynthesis genes under a inducible promoter, used to regulate the bacterial populations within the consortium.</p><br />
<br />
<p>The <a href="https://2012.igem.org/Team:British_Columbia/Desulfurization"> second</a> is responsible for the 4S-desulfurization distributed metabolic network and was created by splitting the Dsz operon into each member species of our tunable consortium.</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/3/3f/Data_Page_diagram_.png"></p><br />
<br />
<font face=arial narrow size=4><b>Data for our Favourite New Parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000"><b>Main Page</a> - Strong Constitutive Promoter-ECFP generator, BBa_K804000:</b> This is an Enhanced Cyan Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses ECFP (BBa_E0420). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, ECFP excites at 439nm and emits at 476nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001"><b>Main Page</a> - Strong constitutive promoter-EYFP generator, BBa_K804001:</b> This is an Enhanced Yellow Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses EYFP (BBa_E0430). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, EYFP excites at 514nm and emits at 527nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804012"><b>Main Page</a> - Rhamnose Inducible TrpA coding gene, BBa_K804012:</b> This part contains a rhamnose inducible (pRha) TrpA coding gene. Upon induction with rhamnose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<br />
<font face=arial narrow size=4><b>Data for Pre-Existing Parts</b></font></br></br><font face=arial narrow><br />
<b><a href="http://partsregistry.org/Part:BBa_K902065:Experience">Experience</a> - Rhamnose inducible, glucose repressible promoter (pRha), BBa_K902065 (Calgary, iGEM 2012):</b> We placed the Rhamnose promoter upstream of our TrpA and MetA coding genes. In the respective auxotrophs, induction by arabinose resulted in growth compared to a negative control as measured by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0030:Experience">Experience</a> - Enhanced yellow fluorescent protein, BBa_E0030 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001">BBa_K804001</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<b><a href="http://partsregistry.org/Part:BBa_E0020:Experience">Experience</a> - Engineered cyan fluorescent protein, BBa_E0020 (Registry, iGEM 2004):</b> We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000">BBa_K804000</a>) and measured fluorescence and OD by a plate reader.</br><br />
</br><br />
<br />
<font face=arial narrow size=4><b>We've also characterized the following parts</b></font></br></br><font face=arial narrow><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804007"><b>Main Page</a> - Constitutive ECFP generator - Arabinose inducible TrpA coding gene, BBa_K804007:</b> This part contains a constitutive ECFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804009"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible TrpA coding gene, BBa_K804009:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804011"><b>Main Page</a> - Constitutive EYFP generator - Arabinose inducible MetA coding gene, BBa_K804011:</b> This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs. </br></br><br />
<br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804002"><b>Main Page</a> - TrpA coding gene, BBa_K804002:</b> This is the TrpA coding gene. It codes for the alpha subunit of tryptophan synthase (TSase α), and functions as both a binding site for indole-3-glycerol-phosphate (InGP) and can catalyze the cleavage of InGP to indole and glyceraldehyde-3-phosphate. This is the same part as BBa_K187028, but is in the pSB1C3 plasmid.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804003"><b>Main Page</a> - TyrA coding gene, BBa_K804003:</b> This is a TyrA coding gene for both the tyrosine and phenylalanine bio-synthetic pathways. TyrA expresses a bifunctional chorismate mutase/prehenate dehydrogenase which catalyzes the conversion of chorismate into prephenate and NAD+-dependent oxidative decarboxylation of prephanate.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804004"><b>Main Page</a> - MetA coding gene, BBa_K804004:</b> This is a MetA coding gene for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><b>Main Page</a> - DszC coding gene, BBa_K804005:</b> This is a DszC coding gene from the DszABC operon in the 4S pathway of Rhodococcus erythropolis IGTS8. It encodes one of the three biodesulfurizing enzymes in the DszABC operon. DszC enzymes have been shown to catalyze the oxidation of dibenzothiophene (DBT)to dibenzothiophene-5-oxide (DBTO) in the first reaction and then from DBTO to DBT sulfoxide (DBTO2) in the second reaction, both in the presence of NADH, oxygen, FMN and flavin reductases.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804006"><b>Main Page</a> - DszD coding gene, BBa_K804006:</b> This is a DszD coding gene isolated from Rhodococcus erythropolis IGTS8, which encodes for a NADH:FMN oxidoreductase to enhance the activities of DszA and DszC in the DszABC operon.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804008"><b>Main Page</a> - Arabinose inducible TrpA coding gene, BBa_K804008:</b> This part contains an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.</br></br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804010"><b>Main Page</a> - Arabinose inducible MetA coding gene, BBa_K804010:</b> This part contains an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804013"><b>Main Page</a> - Rhamnose Inducible MetA coding gene, BBa_K804013:</b> This part contains a rhamnose inducible (pRha) MetA coding gene. Upon induction with rhamnose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.</br></br><br />
</html></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/Human_Practices/IPTeam:British Columbia/Human Practices/IP2012-10-04T03:10:20Z<p>Rsaer: </p>
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<p align=center><font face=arial narrow size=5><b>Investigating Intellectual Property in iGEM</b></font></p><font face=arial narrow><br />
<br />
Working with novel concepts such as tunable consortia and distribution of the Dsz pathway, the UBC team became curious of whether iGEM projects would be owned as intellectual property (IP). We also realize that almost all research projects require the use of some sort of IP-related component. Even if iGEM teams are not planning to patent their own work, they will likely come across something (e.g., reagent, strain, etc.) that is already patented by someone else. To use these materials properly, we need to understand how to navigate around the legal aspect of patents.</br></br><br />
<br />
The purpose of this part of our Human Practices project is to convey IP knowledge to the community in an iGEM-relevant fashion. We surveyed various iGEM teams about their interest in such an IP primer and asked them about what they already knew, what the would like to know, and how they would like the information presented to them. In constructing our guide, we have met up with professions in the field of biotechnological patenting and incorporated the results of these meetings here. In this respect, our project will serve as a connection between iGEM and experts outside our community.</br></br><br />
<br />
We do not intend to make an argument for or against the idea of intellectual property. Our aim is to create a user-friendly platform for all iGEM teams to learn about relevant IP issues. It will present information in a manner that will allow users to make an informed decision about what stance to take when confronted with IP-related decisions. In addition, the guide will help teams navigate through the processes of obtaining or using patents and build a foundation upon which they can then proceed with their projects.</br></br><br />
<br />
<p align=center><font face=arial narrow size=4><b>Our Survey</b></font></p><font face=arial narrow><br />
<br />
Our survey methodology was relatively simple. We wanted to confirm our suspicions that other iGEM teams knew as little about "patents and stuff" as we did. We then came up with a set of questions for other teams, trying to evaluate their existing knowledge, their self-assessment of their knowledge, and their desire, if any, to increase that knowledge. Some of us wondered if we'd see evidence of a <a href="http://en.wikipedia.org/wiki/Dunning–Kruger_effect">Dunning–Kruger effect</a>.</br></br><br />
<br />
After implementing our survey using Google Drive, we sent it to team <a href="https://2012.igem.org/Team:Calgary">Calgary</a> for testing. They <b>all</b> took it, and verified it worked. Thanks, Calgary! We then sent an email to <a href="https://igem.org/Team_List?year=2012">every team's primary contact</a>, asking them to take our survey and pass it along to their team. We had great turnout, garnering 328 individual responses.</br></br></br><br />
<br />
<p align=center><font face=arial narrow size=4><b>Best Respondents</b></font></p><font face=arial narrow><br />
Big thanks to teams <a href="https://2012.igem.org/Team:Bielefeld-Germany">Bielefeld</a>, <a href="https://2012.igem.org/Team:Bonn">Bonn</a>, <a href="https://2012.igem.org/Team:Calgary">Calgary</a>, <a href="https://2012.igem.org/Team:UC_Davis">UC Davis</a>, and <a href="https://2012.igem.org/Team:Warsaw">Warsaw</a> for having more than 80% of their team members, advisors, and instructors answer our survey! They represent the best parts of the iGEM community, helping others create knowledge without asking for much in return.</br></br></br></br><br />
<br />
<p align=center><font face=arial narrow size=4><b>Demographics</b></font></p><font face=arial narrow><br />
<div id=graph1><br />
<img src="https://static.igem.org/mediawiki/2012/a/ae/UBCLocation.png"><img src="https://static.igem.org/mediawiki/2012/f/f4/UBCTeamRole.png"></br><br />
</div><br />
<div id=note1></br></br></br>These questions were relatively straightforward. We just wanted to know who was answering our survey! Happily, it looks like we got a reasonably representative sample of respondents.</br></div><br />
<br />
<div id=break><p align=center><br />
<font face=arial narrow size=4><b></br></br>Experience With IP</b></font></p><font face=arial narrow></div><br />
<div id=break><div id=graph><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/0/0c/UBCExperience.png"></p></br><br />
</div><br />
<div id=note></br></br>Unsurprisingly, the size of the "Yes" pie slice is almost the same size as the sum of "Advisors" and "Instructors" pie slices above, but let's look into that further...</div></div><br />
<div id=break><br />
<div id=graph><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/1/14/UBCRoleExperience.png"></p></br><br />
</div><br />
<div id=note></br></br>While the bulk of respondents are still members with no patent experience, this breakdown reveals that approximately half of the responding advisors and instructors have patent experience and half don't.</div></div><br />
<div id=break><br />
<div id=graph><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/8/8c/UBCExperienceContext.png"></p></br><br />
</div><br />
<div id=note></br></br>Having a larger number of responses to this question than "Yes" answers to the previous one was perplexing. We expected those who indicated having no experience with patents to skip this question. A more sophisticated survey might have only revealed this question upon receiving a yes answer to the previous one.</div></div><br />
<div id=break><br />
<div id=graph><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/7/7a/UBCPastExperienceNature.png"></p></br><br />
</div><br />
<div id=note></br></br>This was a check-box poll, where respondents could select more than one box, and could also fill in a text field for custom "Other" responses.<br />
<br />
Some of the "Other" responses included involvement with software copyrights & patents, research in patent databases, and being in the process of obtaining a patent. </div></div><br />
<br />
<br />
<div id=break><p align=center><br />
<font face=arial narrow size=4><b>Need a for Guide</b></font></p><font face=arial narrow><br />
<img src="https://static.igem.org/mediawiki/2012/9/9f/UBCDepthGainWish.png"></br><br />
<br />
The above graph displays responses from three questions. Note that respondent's median self-assessed level of knowledge is 3, while the median desired level of knowledge is 8 and the median level thought to be obtainable from a brief guide is 6. Respondents thought that a brief guide can get them a good amount of the way to their goal, and this reinforced our motivation to produce such a guide.</br></br><br />
<br />
It is interesting to note how many respondents want to be patent lawyers.</br></br></div><br />
<br />
<div id=graph><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/1/16/UBCFormat.png"></br></p><br />
</div><br />
<div id=note></br></br>This check-box poll confirmed our suspicions that a FAQ-style guide was the way to go. We likely lead the respondents to that conclusion. We didn't claim to be perfect social scientists. What's a little push-polling between friends?</div><br />
<br />
<div id=graph><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/4/41/UBCLearningInterest.png"></br></p><br />
</div><br />
<div id=note></br></br>Reinforcing that we aren't perfect social scientists, this question should have forced the respondent to make a choice. "Both" is an easy no-thought choice that damages what we intended to learn from the question. Regardless, we still learned that respondents are generally enthusiastic to learn about both sides of patents.</br></br></div><br />
<br />
<br />
<div id=break><p align=center><br />
<font face=arial narrow size=4><b></br></br>Patent Knowledge</b></font></p><font face=arial narrow><br />
<p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/0/09/UBCPatentCost.png"><img src="https://static.igem.org/mediawiki/2012/8/85/UBCPatentWorldCost.png"></p><br />
</div><br />
<div id=break>Our <a href="https://2012.igem.org/Team:British_Columbia/Human_Practices/IP_FAQ#How_much_does_a_patent_cost.3F">research for the FAQ</a> turned up a ballpark figure $US30,000 for a biotech patent in the United States. Accordingly, it seems like the median respondent is underestimating the cost of a patent by a decimal place or two. </br></br></div><br />
<div id=break><br />
<div id=graph><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/UBCPatentTime.png"></br></p><br />
</div><br />
<div id=note></br></br>We <a href="https://2012.igem.org/Team:British_Columbia/Human_Practices/IP_FAQ#How_long_does_it_take_to_get_a_patent.3F">turned up a ballpark number</a>for this too. Three to six years is a typical length of time for the patent process to go from initial application, through prosecution, to issuance.</br></br></div></div><br />
<div id=break><br />
<br />
<div id=graph><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/6/62/UBCPatentExtend.png"></br></p><br />
</div><br />
<div id=note></br></br>We suspect you'll notice a theme when we tell you we <a href="https://2012.igem.org/Team:British_Columbia/Human_Practices/IP_FAQ#What_regions_is_my_patent_effective_in.3F">found out about this</a> too. The Patent Cooperation Treaty gives an applicant 18 months after their first application in a participating country to apply for patents in other participating countries. These applications in the other participating countries will have an effective filing date of the first application.<br />
<br />
</br></br></div></div><br />
<br />
<div id=break><p align=center></br></br><br />
<font face=arial narrow size=4><b>Intellectual Property & Current Projects</b></font></p><font face=arial narrow><br />
<div id=graph><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/f/fd/UBCApplyingForIP.png"></br></p><br />
</div><br />
<div id=note></br></br>This check-box poll illustrates that iGEM participants are reasonably resourceful when they start thinking about applying for IP protection. The majority of people have chosen to use the internet to gather information. This was what we had expected. This may explain why learning about IP for iGEM is such a big concern. The world of IP is vast, ever-changing, and often ambiguous. The internet, as a result, contains a sea of information that tends to overwhelm people new to IP. Like us, many might like for someone to pick and choose the relevant information instead of sifting through everything themselves.</br></br></div><br />
<div id=break><br />
<div id=graph><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/5/59/UBCConsideringApplying.png"></br></p><br />
</div><br />
<div id=note></br></br>As you can see, a large part of the survey-takers don't know if they want to apply for a patent. This is interest because we can start to ask why. Have they just never given IP much thought, or do they not know enough about it to even consider applying? Either way, it shows that iGEMers need to pay more attention to IP issues. As for the other results, like expected, patents looks to be the most prevalent type of IP that iGEMers applying for IP protection is concerned about. Now, if we look at the people who answered that they didn't want to consider getting protection, the majority of the people gave moral reasons and lack of effort as the explanation. We're hoping that our IP guide can help address both of these groups. For the iGEMers who think tackling IP problems are too much work, a centralized collection of information (aka Team UBC's IP guide) may help them out. As for the ones who don't want to seek IP protection, our guide can provide information for them to make informed decisions. This aim really applies to everyone, because even people who don't want to get a patent/copyright/trademark themselves, they will still need to work in a world where these things exist.</br></br></div></div><br />
<div id=break><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/e/ec/UBCPatentConcerns.png"><img src="https://static.igem.org/mediawiki/2012/1/1e/UBCOtherNegative.png"><img src="https://static.igem.org/mediawiki/2012/8/81/UBCPatentDepend.png"></p><br />
So, apparently most of us have not been affected negatively by IP or even been too bothered by it in general. A lot of us also don't know if we have been affected. These two groups might not actually be mutually exclusive.<br />
</div><br />
<br />
<div id=break><p align=center><br />
<font face=arial narrow size=4><b></br></br>Opinions</b></font></p><font face=arial narrow><br />
<div id=graph><p align=center><img src="https://static.igem.org/mediawiki/2012/1/17/UBCPersonalInterest.png"></br></p></div><br />
<div id=note></br></br>Although 13% of respondents said they had been negatively affected by patents and 9% by other forms of IP, only 7% of respondents indicated they didn't want to protect their own IP. The majority replied that they wanted IP protection, which lets us know that an IP guide is something that would benefit a big part of the iGEM community.</br></br></div></div><br />
<br />
<div id=break><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/e/ea/UBCBioBrickCan.png"><img src="https://static.igem.org/mediawiki/2012/e/ef/UBCBioBrickShould.png"><img src="https://static.igem.org/mediawiki/2012/3/3f/UBCPatentableProjects.png"><br />
</p></br><br />
There is a pretty even spread of answers as to whether BioBricks can be patented, but more than half think that we ''shouldn't'' patent them. Despite all of that, 75% of the survey-takers agree that iGEM teams are capable of producing projects that can be patented. Seeing as how BioBricks play such a big role, to what extent should or can we patent iGEM projects, if half of us think BioBricks shouldn't be IP protected?<br />
<br />
<div id=break><p align=center><br />
<div id=break><p align=center><br />
<p align=center><font face=arial narrow size=4><b>Conclusions</b></font></p><font face=arial narrow><br />
With a considerable sample size and responses from both students and advisors, our survey results are fairly representative of the iGEM community. We've learned from the results that the majority of iGEMers want to learn more about intellectual property and think they will benefit from a custom-tailored guide, especially one on patents. There were a lot of undecided responses, which indicate that many are probably lacking the background needed to take a stance. Overall, the survey results show that the iGEM community is interested in IP, and that there exists a large audience for our guide.</div>Rsaerhttp://2012.igem.org/Team:British_Columbia/Human_Practices/IndustryTeam:British Columbia/Human Practices/Industry2012-10-04T03:08:21Z<p>Rsaer: </p>
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<br />
<font face=arial narrow size=5><b>The Applications and Applicability of Engineered Microbial Consortia</b></font></br></br><font face=arial narrow><br />
Our project sets a foundational advance by engineering microbial consortia with the purpose of distributing metabolic pathways to increase their efficiency, whereby they become more desirable from a biotech standpoint. We thus set out to find the following information from our industrial partners:</br><br />
<ol><br />
<li>What are some potential industrial applications of engineered microbial consortia? <br />
<li>What advantages are there to employing biological methods versus current chemical methods?<br />
<li>How feasible is it to implement biological methods?<br />
<li>Are Research & Development sectors of current organizations/companies interested in pursuing synthetic biology options?<br />
</ol><br />
<br/><br />
<font face=arial narrow size=4><b>Our Approach</b></font></br></br><font face=arial narrow><br />
Our team contacted Chevron and arranged a visit to the Chevron refinery in Burnaby, BC, Canada. We communicated with Chevron representatives to find out more about the existing methods of desulfurization and costs of refining crude oil.</br></br><br />
<br />
We connected with Alberta Innovates – Technology Futures (AITF) representative Karen Budwill and Oil Sands Leadership Initiative (OSLI) representatives John Vidmar and Nicolas Choquette-Levy to discuss the progress of our project and obtain some industrial insights.</br></br><br />
<br />
<font face=arial narrow size=4><b>Industrial Insights</b></font></br><font face=arial narrow><br />
</br><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/5/5d/Ubchev.jpg"></br></p><br />
<br />
<div id=break><b>From our Chevron visit, we learned:</b></br><br />
<ol><br />
<li>How refinery desulfurization works:</li><br />
<ul><br />
<li>Sulfur-rich fuels are catalytically hydrogenated (hydrotreated) at high pressure (700 psi) and temperature (800 °F), creating H<sub>2</sub>S gas.</li><br />
<li>The H<sub>2</sub>S gas is absorbed from the fuel stream by being contacted with amines at high pressure.</li><br />
<li>The amines are then heated to release the H<sub>2</sub>S gas to the two-step Claus process.</li><br />
<li>In the first step of the Claus process, the H<sub>2</sub>S gas is partially combusted, creating water, elemental sulfur and sulfur oxides.</li><br />
<li>The Claus process's second step catalytically reacts the combustion products with more H<sub>2</sub>S, creating water and elemental sulfur with very high yields.</li><br />
</ul><br />
<li>Industrial-scale desulfurization is massive. The diesel hydrotreater at Chevron Burnaby treats 18000 barrels of diesel fuel every day, removing 99.5% of the sulfur and taking it from around 500 ppm to less than 15 ppm sulfur at a cost of about $2 per barrel.</li><br />
<li>Sulfur content in fuel is regulated by governments because sulfur-containing fuels lead to acid rain. As time has gone on, the permitted sulfur content has decreased. The kinetics of conventional hydrotreatment cause the cost of treating 100 ppm sulfur fuel down to 15 ppm to require more extreme process conditions and thus be substantially more expensive than treating 500 ppm sulfur fuel to 100 ppm.</li><br />
</ol><br />
<br />
<div id=break><b>From our correspondence with AITF-OSLI, we learned:</b></br><br />
<ol><br />
<li>In Alberta, upgrading and refining processes aim to reduce viscosity and desulfurize crude oil to facilitate transport by pipeline.</li><br />
<br />
<li>Presently, the industry does not possess infrastructure for the utilization of biological systems such as bioreactors or emulsifiers. However, this is an area of interest for them and could be implemented in a time span of approximately 5 years. There is also an interest in screening tailings ponds for new organisms or genes encoding parts capable of refining crude oil.</li><br />
<li>Our AITF and OSLI collaborators are currently looking into the economic and environmental costs of refining oil, as well as our project's potential impact on industry and applications other than desulfurization. They will get in touch with us within a few weeks once they have this information.</li><br />
</ol></br></br><br />
<br />
<br />
<font face=arial narrow size=4><b>Biocatalytic desulfurization of dibenzothiophene: Hypothetical Bioreactor Design</b></br></font></br><font face=arial narrow><br />
<br />
After talking with our industrial collaborators we had a good idea about what the industry was looking for in terms of bio-desulfurization. The next step was to devise a hypothetical design for a small scale bio-desulfurization plant. The general schematic of this plant can be found below (Diagram 1): <br />
<br />
<p align=center><b></br></br><b>Diagram 1.</b> General layout and flow of a bio-desulfurization unit.</p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/6/64/Bio_reactor.png"></p></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/MedalTeam:British Columbia/Medal2012-10-04T03:06:54Z<p>Rsaer: </p>
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<div id=note></br><font face=arial narrow size=5><b>Bronze Medal Requirements</b></br></br></font><font face=arial narrow><br />
Registered the team, had a great summer, and plan to have fun at the Regional Jamboree.</br><br />
Successfully completed and submitted the iGEM 2012 Judging form.</br><br />
Created and shared a <a href="https://2012.igem.org/Team:British_Columbia">Description</a> of the team's project using the iGEM wiki.</br><br />
Plan to present a Poster and Talk at the iGEM Jamboree.</br><br />
Submitted DNA for and entered information detailing <a href="https://2012.igem.org/Team:British_Columbia/Parts">15 new standard BioBrick Parts</a> in the Registry of Standard Biological Parts. <br />
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<div id=note></br><font face=arial narrow size=5><b>Silver Medal Requirements</b></br></br></font><font face=arial narrow><br />
Demonstrated that 14 new BioBrick Parts of our own design and construction works as expected and entered this information on the part's 'Main Page' section of the Registry</br><br />
Part Number(s): <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000">BBa_K804000</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001">BBa_K804001</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804002">BBa_K804002</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804003">BBa_K804003</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804004">BBa_K804004</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005">BBa_K804005</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804006">BBa_K804006</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804007">BBa_K804007</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804008">BBa_K804008</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804009">BBa_K804009</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804010">BBa_K804010</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804011">BBa_K804011</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804012">BBa_K804012</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804013">BBa_K804013</a><br />
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<br />
<div id=note></br><font face=arial narrow size=5><b>Gold Medal Requirements</b></br></br></font><font face=arial narrow><br />
<b>Criteria 1: Improve an existing BioBrick Part or Device and enter this information back on the Experience Page of the Registry.</b></br></br><br />
We standardized ECFP and EYFP fluorescent protein BioBricks by putting them under the same constitutive promoters and plasmid. Thereby, we have improved the fluorescent markers by making them into three more usable parts that can be used as a new tool kit for future iGEM teams. </br><br />
Part Number(s): <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804000">BBa_K804000</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804001">BBa_K804001</a></br></br><br />
<br />
<b>Criteria 2: Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system.</b></br></br><br />
<br />
We helped the University of Calgary team troubleshoot the rhamnose promoter that they were having trouble with and proved that it was not functional through careful experiments. Our two teams also distributed the work of making BioBricks of the DSZ genes. Read more about our <a href="https://2012.igem.org/Team:British_Columbia/Attributions">collaboration on our attributions page.</a></br></br><br />
<br />
<b>Criteria 3: Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.</b></br></br><br />
<br />
Through a survey of over 380 iGEM community members, we identified intellectual property as an issue that impacts the work of the majority of the iGEM community. To address this, we engaged with patent agents and IP experts to create an <a href="https://2012.igem.org/Team:British_Columbia/Human_Practices/IP_FAQ">iGEM-specific patent guide</a> to act as a primer that can be used to help iGEMers navigate intellectual property issues.</br><br />
</div><br />
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</br><br />
<font face=arial narrow size=5><b>Eligibility for Special Prizes</b></br></br></font><br />
<font face=arial narrow size=3><b>Best Human Practice Advance:</b></font></br><font face=arial narrow><br />
</br><br />
<b>IP survey and guide:</b> We designed and administered an <a href="https://2012.igem.org/Team:British_Columbia/Patent">intellectual property survey</a> to gather high impact data from more than 300 members of the iGEM community. The information was useful to other iGEMers, such as Team INSA Lyon who cited the majority of our data for their human practice. Our team also engaged people outside of the iGEM community, such as a patent agent, patent lawyer, University of British Columbia University-Industry Liaison Office and technical experts. The end result, with use of feedback from experts and other iGEM teams, was an <a href="https://2012.igem.org/Team:British_Columbia/Human_Practices/IP_FAQ">intellectual property guide</a> geared specifically towards the iGEM competition.</br><br />
</br><br />
<b>Assessing the industrial relevance of our research:</b> We went to the Chevron refinery in Burnaby, British Columbia and talked to an industry professional about the processes and costs pertinent to our project. We also communicated with Alberta Innovates – Technology Futures (AITF) and Oil Sands Leadership Initiative (OSLI) to discuss the progress of our project and obtain some <a href="https://2012.igem.org/Team:British_Columbia/Industry">industrial insights</a>.</br></br><br />
<br />
<font face=arial narrow size=3><b>Best Model:</b></font></br><font face=arial narrow></br><br />
<b>Consortia Model:</b> To model the population dynamics of our engineered consortia, we used a lot primary wet lab data to improve the accuracy. Our <a href="https://2012.igem.org/Team:British_Columbia/Consortia">model</a> improves on the two-organism consortia model found in literature by simulating the significantly more complex synthetic three-organism community, which has not yet been done to our knowledge. Furthermore, our model was designed to be easily adaptable to predict any limiting-substrate interdependent consortia population dynamics in general.</br><br />
</br><br />
<b>Pathway Model:</b> Our <a href="https://2012.igem.org/Team:British_Columbia/Pathway">distributed pathway model</a> streamlines and informs future consortia project planning. Our model explained experimental observations found in literature of synergistic pathways in consortia and demonstrated that distributed metabolisms, like the one we are engineering, occur in nature. This model helps investigators probe for possible new metabolisms derived from combining different organisms with different metabolic capacities.<br />
</br></br><br />
<font face=arial narrow size=3><b>Best Foundational Advance:</b></font></br></br><br />
BioBrick standard biological parts are DNA sequences of defined structure and function designed to be incorporated into living cells to construct new biological systems. Synthetic biologists have been building BioRooms by engineering single microbes that contain purposeful compositions of BioBricks to perform tasks such as bio-sensing, bio-degradation, bio-transformation or bio-synthesis. However, most of these BioRooms are designed to be self-containing, self-sufficient systems in stark contrast to how microbes normally exist in community in natural environments. </br></br><br />
<br />
The UBC iGEM 2012 team sets a foundational advance by engineering microbial BioRooms that are compatible with each other and can be regulated based on their interdependencies. In the future, it may be possible to mix and match BioRooms to create BioFactories with novel synergistic metabolisms.<br />
</br><br />
</br></br><br />
</br><br />
<font face=arial narrow size=4><b>Inter-Team Collaborations</b></br></br></font><font face=arial narrow><br />
<br />
<div id=collab2><br />
<b>Calgary</b>:</br>Calgary submitted and characterized DszA and B, and UBC submitted and characterized DszC and D of the same operon. <a href="https://2012.igem.org/Team:British_Columbia/Attributions">More details</a> on our attributions page.</div><br />
<br />
<div id=collab2><br />
<b>TU Munich</b>:</br>Our team members participated in <a href="https://2012.igem.org/Team:TU_Munich/Results/RFC">TU Munich's survey</a> on the improvement of part descriptions.</br></br><br />
<br />
<br />
</div><div id=collab2><b>INSA Lyon</b>:</br><br />
<a href="https://2012.igem.org/Team:Lyon-INSA/humanPractice">INSA Lyon</a> cited our Intellectual Property survey results and provided feedback on our patent guide. <br />
</html></div>Rsaerhttp://2012.igem.org/Team:British_ColumbiaTeam:British Columbia2012-10-04T03:02:03Z<p>Rsaer: </p>
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<p align=center><font face=arial narrow size=5><b>Synthetic Syntrophy</b></font></p><font face=arial narrow><br />
The field of synthetic biology has seen the development of many biological monocultures capable of performing a wide range of novel functions. In contrast to this current paradigm, microbes have naturally evolved to survive as members of dynamic communities with distributed metabolism. This “divide and conquer” strategy allows the community to perform more complicated metabolic processing than would be possible in single microorganisms while being resilient to environmental changes. Despite very recent proof of concepts in developing model microbial consortia, or synthetic ecology, questions remain as to whether complex metabolic pathways can be engineered in context of microbial populations. The 2012 University of British Columbia iGEM team sets a precedent by engineering a tunable consortium with a distributed 4S desulfurization pathway for increased efficiency in the removal of organosulfurs in heavy oils and bitumen resources.</br></br><br />
<br />
<p align=center><font face=arial narrow size=4><b>Foundational Advance: BioBricks to BioRooms to BioFactories</b></font></p><font face=arial narrow><br />
BioBrick standard biological parts are DNA sequences of defined structure and function designed to be incorporated into living cells to construct new biological systems. Synthetic biologists have been building BioRooms by engineering single microbes that contain purposeful compositions of BioBricks to perform tasks such as bio-sensing, bio-degradation, bio-transformation or bio-synthesis. However, most of these BioRooms are designed to be self-containing, self-sufficient systems in stark contrast to how microbes normally exist in community in natural environments. </br></br><b>The UBC iGEM 2012 team sets a foundational advance by engineering microbial BioRooms that are compatible with each other and can be regulated based on their interdependencies. In the future, it may be possible to mix and match BioRooms to create BioFactories with novel synergistic metabolisms.</br><br />
<div id=slide><p align=center><img src="https://static.igem.org/mediawiki/2012/6/66/Ubcigemslide1.jpg" width=350px></p></div><br />
<div id=caption><b></br></br>Synthetic biologists have created systems for bio-sensing, bio-degradation, bio-transformation and bio-synthesis.</div><br />
<div id=break2></div><br />
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<div id=slide><p align=center><img src="https://static.igem.org/mediawiki/2012/1/11/Ubcigemslide2.jpg" width=300px></p></div><br />
<div id=caption></br>In nature, microbes normally exist within communities (consortia), where metabolic pathways are distributed across different species.</div><div id=break2></div><br />
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<div id=slide><p align=center><img src="https://static.igem.org/mediawiki/2012/f/f2/Ubcigemslide3.jpg" width=300px></p></div><br />
<div id=caption></br>We hypothesize that distributing metabolic pathways in the field of synthetic biology may proffer separate advantages compared to engineering a single microbe containing an entire independent metabolism. For instance, distributing pathways can (i) reduce the metabolic burden on any one microbe and (ii) increase compartmentalization so that there is reduced cross-talk regulation/feedback inhibition and each reaction is sequestered within a more conducive cellular environment.<br />
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<div id=slide><p align=center><img src="https://static.igem.org/mediawiki/2012/d/d8/Ubcigemslide4.jpg"></p></div><br />
<div id=caption></br>Microbial consortia have also been engineered to be tunable so that addition of certain inducers can easily modulate population dynamics within the community. This is tenable in the context of up- or down-regulating certain steps of the metabolic pathway to optimize efficiency and alleviate bottle-necks.</br></br><br />
Our team utilizes three different strains of <i>E. coli</i> with complementary prototrophies and auxotrophies: each strain expresses a specific amino acid synthetase under a particular inducible promoter, which is required for the growth of another strain. <br />
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<font face=arial narrow size=5><b>The Applications and Applicability of Engineered Microbial Consortia</b></font></br></br><font face=arial narrow><br />
Our project sets a foundational advance by engineering microbial consortia with the purpose of distributing metabolic pathways to increase their efficiency, whereby they become more desirable from a biotech standpoint. We thus set out to find the following information from our industrial partners:</br><br />
<ol><br />
<li>What are some potential industrial applications of engineered microbial consortia? <br />
<li>What advantages are there to employing biological methods versus current chemical methods?<br />
<li>How feasible is it to implement biological methods?<br />
<li>Are Research & Development sectors of current organizations/companies interested in pursuing synthetic biology options?<br />
</ol><br />
<br/><br />
<font face=arial narrow size=5><b>Our Approach</b></font></br></br><font face=arial narrow><br />
Our team contacted Chevron and arranged a visit to the Chevron refinery in Burnaby, BC, Canada. We communicated with Chevron representatives to find out more about the existing methods of desulfurization and costs of refining crude oil.</br></br><br />
<br />
We connected with Alberta Innovates – Technology Futures (AITF) representative Karen Budwill and Oil Sands Leadership Initiative (OSLI) representatives John Vidmar and Nicolas Choquette-Levy to discuss the progress of our project and obtain some industrial insights.</br></br><br />
<br />
<font face=arial narrow size=5><b>Industrial Insights</b></font></br><font face=arial narrow><br />
</br><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/5/5d/Ubchev.jpg"></br></p><br />
<br />
<div id=break><b>From our Chevron visit, we learned:</b></br><br />
<ol><br />
<li>How refinery desulfurization works:</li><br />
<ul><br />
<li>Sulfur-rich fuels are catalytically hydrogenated (hydrotreated) at high pressure (700 psi) and temperature (800 °F), creating H<sub>2</sub>S gas.</li><br />
<li>The H<sub>2</sub>S gas is absorbed from the fuel stream by being contacted with amines at high pressure.</li><br />
<li>The amines are then heated to release the H<sub>2</sub>S gas to the two-step Claus process.</li><br />
<li>In the first step of the Claus process, the H<sub>2</sub>S gas is partially combusted, creating water, elemental sulfur and sulfur oxides.</li><br />
<li>The Claus process's second step catalytically reacts the combustion products with more H<sub>2</sub>S, creating water and elemental sulfur with very high yields.</li><br />
</ul><br />
<li>Industrial-scale desulfurization is massive. The diesel hydrotreater at Chevron Burnaby treats 18000 barrels of diesel fuel every day, removing 99.5% of the sulfur and taking it from around 500 ppm to less than 15 ppm sulfur at a cost of about $2 per barrel.</li><br />
<li>Sulfur content in fuel is regulated by governments because sulfur-containing fuels lead to acid rain. As time has gone on, the permitted sulfur content has decreased. The kinetics of conventional hydrotreatment cause the cost of treating 100 ppm sulfur fuel down to 15 ppm to require more extreme process conditions and thus be substantially more expensive than treating 500 ppm sulfur fuel to 100 ppm.</li><br />
</ol><br />
<br />
<div id=break><b>From our correspondence with AITF-OSLI, we learned:</b></br><br />
<ol><br />
<li>In Alberta, upgrading and refining processes aim to reduce viscosity and desulfurize crude oil to facilitate transport by pipeline.</li><br />
<br />
<li>Presently, the industry does not possess infrastructure for the utilization of biological systems such as bioreactors or emulsifiers. However, this is an area of interest for them and could be implemented in a time span of approximately 5 years. There is also an interest in screening tailings ponds for new organisms or genes encoding parts capable of refining crude oil.</li><br />
<li>Our AITF and OSLI collaborators are currently looking into the economic and environmental costs of refining oil, as well as our project's potential impact on industry and applications other than desulfurization. They will get in touch with us within a few weeks once they have this information.</li><br />
</ol></br></br><br />
<br />
<br />
<font face=arial narrow size=5><b>Biocatalytic desulfurization of dibenzothiophene: Hypothetical Bioreactor Design</b></br></font></br><font face=arial narrow><br />
<br />
After talking with our industrial collaborators we had a good idea about what the industry was looking for in terms of bio-desulfurization. The next step was to devise a hypothetical design for a small scale bio-desulfurization plant. The general schematic of this plant can be found below (Diagram 1): <br />
<br />
<p align=center><b></br></br><b>Diagram 1.</b> General layout and flow of a bio-desulfurization unit.</p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/6/64/Bio_reactor.png"></p></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/Human_Practices/IndustryTeam:British Columbia/Human Practices/Industry2012-10-04T02:59:40Z<p>Rsaer: </p>
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<font face=arial narrow size=5><b>The Applications and Applicability of Engineered Microbial Consortia</b></font></br></br><font face=arial narrow><br />
Our project sets a foundational advance by engineering microbial consortia with the purpose of distributing metabolic pathways to increase their efficiency, whereby they become more desirable from a biotech standpoint. We thus set out to find the following information from our industrial partners:</br><br />
<ol><br />
<li>What are some potential industrial applications of engineered microbial consortia? <br />
<li>What advantages are there to employing biological methods versus current chemical methods?<br />
<li>How feasible is it to implement biological methods?<br />
<li>Are Research & Development sectors of current organizations/companies interested in pursuing synthetic biology options?<br />
</ol><br />
<br/><br />
<font face=arial narrow size=5><b>Our Approach</b></font></br></br><font face=arial narrow><br />
Our team contacted Chevron and arranged a visit to the Chevron refinery in Burnaby, BC, Canada. We communicated with Chevron representatives to find out more about the existing methods of desulfurization and costs of refining crude oil.</br></br><br />
<br />
We connected with Alberta Innovates – Technology Futures (AITF) representative Karen Budwill and Oil Sands Leadership Initiative (OSLI) representatives John Vidmar and Nicolas Choquette-Levy to discuss the progress of our project and obtain some industrial insights.</br></br><br />
<br />
<font face=arial narrow size=5><b>Industrial Insights</b></font></br><font face=arial narrow><br />
</br><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/5/5d/Ubchev.jpg"></br></p><br />
<br />
<div id=break><b>From our Chevron visit, we learned:</b></br><br />
<ol><br />
<li>How refinery desulfurization works:</li><br />
<ul><br />
<li>Sulfur-rich fuels are catalytically hydrogenated (hydrotreated) at high pressure (700 psi) and temperature (800 °F), creating H<sub>2</sub>S gas.</li><br />
<li>The H<sub>2</sub>S gas is absorbed from the fuel stream by being contacted with amines at high pressure.</li><br />
<li>The amines are then heated to release the H<sub>2</sub>S gas to the two-step Claus process.</li><br />
<li>In the first step of the Claus process, the H<sub>2</sub>S gas is partially combusted, creating water, elemental sulfur and sulfur oxides.</li><br />
<li>The Claus process's second step catalytically reacts the combustion products with more H<sub>2</sub>S, creating water and elemental sulfur with very high yields.</li><br />
</ul><br />
<li>Industrial-scale desulfurization is massive. The diesel hydrotreater at Chevron Burnaby treats 18000 barrels of diesel fuel every day, removing 99.5% of the sulfur and taking it from around 500 ppm to less than 15 ppm sulfur at a cost of about $2 per barrel.</li><br />
<li>Sulfur content in fuel is regulated by governments because sulfur-containing fuels lead to acid rain. As time has gone on, the permitted sulfur content has decreased. The kinetics of conventional hydrotreatment cause the cost of treating 100 ppm sulfur fuel down to 15 ppm to require more extreme process conditions and thus be substantially more expensive than treating 500 ppm sulfur fuel to 100 ppm.</li><br />
</ol><br />
<br />
<div id=break><b>From our correspondence with AITF-OSLI, we learned:</b></br><br />
<ol><br />
<li>In Alberta, upgrading and refining processes aim to reduce viscosity and desulfurize crude oil to facilitate transport by pipeline.</li><br />
<br />
<li>Presently, the industry does not possess infrastructure for the utilization of biological systems such as bioreactors or emulsifiers. However, this is an area of interest for them and could be implemented in a time span of approximately 5 years. There is also an interest in screening tailings ponds for new organisms or genes encoding parts capable of refining crude oil.</li><br />
<li>Our AITF and OSLI collaborators are currently looking into the economic and environmental costs of refining oil, as well as our project's potential impact on industry and applications other than desulfurization. They will get in touch with us within a few weeks once they have this information.</li><br />
</ol></br></br><br />
<br />
<br />
<font face=arial narrow size=5><b>Biocatalytic desulfurization of dibenzothiophene: Hypothetical Bioreactor Design</b></br></font></br><font face=arial narrow><br />
<br />
After talking with our industrial collaborators we had a good idea about what the industry was looking for in terms of bio-desulfurization. The next step was to layout a hypothetical design for a small scale bio-desulfurization plant. The general schematic of this plant can be found below (Diagram 1): <br />
<br />
<p align=center><b></br></br><b>Diagram 1.</b> General layout and flow of a bio-desulfurization unit.</p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/6/64/Bio_reactor.png"></p></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/Human_Practices/IndustryTeam:British Columbia/Human Practices/Industry2012-10-04T02:58:17Z<p>Rsaer: </p>
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<font face=arial narrow size=5><b>The Applications and Applicability of Engineered Microbial Consortia</b></font></br></br><font face=arial narrow><br />
Our project sets a foundational advance by engineering microbial consortia with the purpose of distributing metabolic pathways to increase their efficiency, whereby they become more desirable from a biotech standpoint. We thus set out to find the following information from our industrial partners:</br><br />
<ol><br />
<li>What are some potential industrial applications of engineered microbial consortia? <br />
<li>What advantages are there to employing biological methods versus current chemical methods?<br />
<li>How feasible is it to implement biological methods?<br />
<li>Are Research & Development sectors of current organizations/companies interested in pursuing synthetic biology options?<br />
</ol><br />
<br/><br />
<font face=arial narrow size=5><b>Our Approach</b></font></br></br><font face=arial narrow><br />
Our team contacted Chevron and arranged a visit to the Chevron refinery in Burnaby, BC, Canada. We communicated with Chevron representatives to find out more about the existing methods of desulfurization and costs of refining crude oil.</br></br><br />
<br />
We connected with Alberta Innovates – Technology Futures (AITF) representative Karen Budwill and Oil Sands Leadership Initiative (OSLI) representatives John Vidmar and Nicolas Choquette-Levy to discuss the progress of our project and obtain some industrial insights.</br></br><br />
<br />
<font face=arial narrow size=5><b>Industrial Insights</b></font></br><font face=arial narrow><br />
</br><p align=center><br />
<img src="https://static.igem.org/mediawiki/2012/5/5d/Ubchev.jpg"></br></p><br />
<br />
<div id=break><b>From our Chevron visit, we learned:</b></br><br />
<ol><br />
<li>How refinery desulfurization works:</li><br />
<ul><br />
<li>Sulfur-rich fuels are catalytically hydrogenated (hydrotreated) at high pressure (700 psi) and temperature (800 °F), creating H<sub>2</sub>S gas.</li><br />
<li>The H<sub>2</sub>S gas is absorbed from the fuel stream by being contacted with amines at high pressure.</li><br />
<li>The amines are then heated to release the H<sub>2</sub>S gas to the two-step Claus process.</li><br />
<li>In the first step of the Claus process, the H<sub>2</sub>S gas is partially combusted, creating water, elemental sulfur and sulfur oxides.</li><br />
<li>The Claus process's second step catalytically reacts the combustion products with more H<sub>2</sub>S, creating water and elemental sulfur with very high yields.</li><br />
</ul><br />
<li>Industrial-scale desulfurization is massive. The diesel hydrotreater at Chevron Burnaby treats 18000 barrels of diesel fuel every day, removing 99.5% of the sulfur and taking it from around 500 ppm to less than 15 ppm sulfur at a cost of about $2 per barrel.</li><br />
<li>Sulfur content in fuel is regulated by governments because sulfur-containing fuels lead to acid rain. As time has gone on, the permitted sulfur content has decreased. The kinetics of conventional hydrotreatment cause the cost of treating 100 ppm sulfur fuel down to 15 ppm to require more extreme process conditions and thus be substantially more expensive than treating 500 ppm sulfur fuel to 100 ppm.</li><br />
</ol><br />
<br />
<div id=break><b>From our correspondence with AITF-OSLI, we learned:</b></br><br />
<ol><br />
<li>In Alberta, upgrading and refining processes aim to reduce viscosity and desulfurize crude oil to facilitate transport by pipeline.</li><br />
<br />
<li>Presently, the industry does not possess infrastructure for the utilization of biological systems such as bioreactors or emulsifiers. However, this is an area of interest for them and could be implemented in a time span of approximately 5 years. There is also an interest in screening tailings ponds for new organisms or genes encoding parts capable of refining crude oil.</li><br />
<li>Our AITF and OSLI collaborators are currently looking into the economic and environmental costs of refining oil, as well as our project's potential impact on industry and applications other than desulfurization. They will get in touch with us within a few weeks once they have this information.</li><br />
</ol></br></br><br />
<br />
<br />
<font face=arial narrow size=5><b>Biocatalytic desulfurization of dibenzothiophene: Hypothetical Bioreactor Design</b></br></font></br><font face=arial narrow><br />
<br />
After talking with our industrial collaborators we had a good idea about what the industry was looking for in terms of bio-desulfurization. Therefore we set out to layout a hypothetical design for a small scale bio-desulfurization plant. The general schematic of this plant can be found below (Diagram 1). <br />
<br />
<p align=center><b></br></br><b>Diagram 1.</b> General layout and flow of a bio-desulfurization unit.</p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/6/64/Bio_reactor.png"></p></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PathwayTeam:British Columbia/Pathway2012-10-04T02:46:49Z<p>Rsaer: </p>
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<p align=center><font face=arial narrow size=5><b>Our Pathway Model<br />
</b></font></p><font face=arial narrow><br />
<br />
The study of environmental genomics attempts to capture the taxonomic and functional diversity of natural microbial communities. Our host at UBC, the Hallam lab, designs novel tools for analyzing the gene content in the context of distributed metabolism. Recently, a pipeline has been developed for the automated construction and visualizing of metabolic pathways from genomic data by integrating software such as Pathway Tools, Pathologic and Metacyc [1]. This provided us an opportunity to model pathway compartmentalization and distribution amongst microbes in the natural environment as it applies to our project. </br></br><br />
<br />
<h2>Summary of the pipeline for community-level metabolic analysis (Figure 1):</h2> First, open reading frames are predicted from sequence data with Prodigal, are then annotated by protein BLAST, and later summarized in a GenBank file. Pathway/genome databases (PGDBs) are generated from sequence data in a manner which does not constrain predictions within the scope of model organisms. This produces a community-based analysis that can be visualized using Pathway Tools. <br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/9c/UbcigemSlide1.jpg"></p><br />
<br />
<h2>Modeling Metabolism:</h2> <i>Rhodococcus erythropolis</i> and <i>Pseudomnas fluorescens</i> are often both prevalent in similar niches. With a high likelihood that these organisms encounter each other, studies have been conducted to assess their metabolic properties in co-culture. In a study by Kayser et al, it was shown that the 4S biodesulferization pathway in <i>R. erythropolis</i> demonstrated higher activity the presence of <i>P. fluorescens</i> [2]. As <i>P. fluorescens</i> does not biodesulferize DBT and sulfate is known to repress the 4S pathway, we looked to analyze the genomes of the two organisms in the context of sulfur metabolism. </br></br><br />
<br />
The 4S pathway releases sulfite, which is toxic to the cell, and therefore genomes were analyzed with the aforementioned pipeline for pathways involved in metabolizing sulfite. It was found that both organisms have annotated genes which convert sulfite into sulfate via a reductase, however the organisms differ in downstream metabolism of sulfate. While both organisms contain a pathway for assimilatory sulfate metabolism, only <i>P. fluorescens</i> has the capability for dissimilatory sulfate metabolism (Figure 2, 3). Based on these findings, we can hypothesize that <i>R. erythropolis</i> and <i>P. fluorescens</i> likely excrete and catabolize any excess sulfate. This provides an explanation for the improved desulfurization found in co-culture conditions. <i>P. fluorescens</i> potentially removes sulfate from the environment, allowing increased secretion by <i>R. erythropolis</i>, and thereby derepressing 4S pathway. The prediction of distributed sulfite metabolism to improve biodesulferization in the environment provides both a testable hypothesis, as well as a grounds for improving biodesulferization through synthetic pathway distribution. </br></br><br />
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<p align=center><img src="https://static.igem.org/mediawiki/2012/4/43/UbcigemerSlide2.jpg"></p><br />
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<br />
<br />
We then scanned the literature for more evidence of shared metabolism between both <i>R. erythropolis</i> and <i>P. fluorescens</i> in order to further analyze gene content in the context of co-culture experiments. In a study by Goswami et al., the metabolism of chlorinated aromatic compounds and phenol was compared in monoculture versus co-culture using <i>R. erythropolis</i> and <i>P. fluorescens</i> [3]. This study showed that the growth rate of pure cultures of <i>R. erythropolis</i> was higher than <i>P. fluorescens</i> on chlorinated aromatics, however in mixed culture, <i>P. fluorescens</i> showed a higher growth rate. For the degradation of phenol, <i>R. erythropolis</i> showed higher growth rates in both pure and mixed culture. The authors of this study suggested that these results were likely a product of substrate competition. We attempted to analyze the genomes of both <i>R. erythropolis</i> and <i>P. fluorescens</i>, separately and together in an attempt to offer an alternate interpretation of the co-culture results. The first pathways assessed were those involved in chlorinated aromatic degradation. It was found that <i>P. fluorescens</i> contains a higher diversity of genes involved in catabolizing chlorinated aromatics; however, only <i>R. erythropolis</i> seems to be able to degrade phenol (Figure 4, 5). This suggests the possibility of the compartmentalization of different components of these metabolic processes, leading to the different growth kinetics observed in co-culture. For example, while <i>R. erythropolis</i> may be more efficient at degrading certain chlorinated aromatics, in co-culture, the diversity of catabolism of chlorinated aromatics allows <i>P. fluorescens</i> to grow more rapidly. Chlorinated aromatic degradation by <i>P. fluorescens</i>, however, would result in the accumulation of downstream products, such as phenol, that only <i>R. erythropolis</i> can catabolism. This provides a metabolic network which could both select and sustain both microbes in the presence of diverse chlorinated aromatics. </br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/b/bd/UbcigemSlide4.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/99/UbcigemSlide5.jpg"></p><br />
<br />
<br />
<br />
<br />
The sums of general aromatic degradation pathways were compared for the organisms genomes separately and together (Figure 6). This resulted in emergent predicted pathways in combination as well as combinatorial increases araomatic degradation potential. <br /><br />
<br /><br />
Ultimately, gene annotation-based models for distributed metabolism in the environment may help to engineer and optimize complex metabolism through synthetic consortia. </br><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/a/aa/UbcigemSlide6.jpg"></p></br></br><br />
<br />
[1] Hanson, N.W; Page, A.P; Konwar, K.M; Howes, C.G; Hallam, S.J. Metabolic interaction networks for the whole community, 2012. Unpublished.</br></br><br />
<br />
[2] Kayser, K.J; Biolaga-Jones, B.A.; Jackowski, K; Odusan, O; Kildane, J.J. Utilzation of organosulfur compounds by anexic and mixed culture of Rhodococcus rhodochrous IGTS8, 1993. Journal of General Microbioology, 139: 3123-3129.</br></br><br />
<br />
[3] Goswami, M; Shivaraman, N; Singh, R.P. Microbial metabolism of 2-chlorophenol, phenol and p-cresol by Rhodococcus erythropolis M1 and co-culture with Pseudomonas fluorescens P1, 2005. Microbiological Research, 160: 101-109.</br></br></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PathwayTeam:British Columbia/Pathway2012-10-04T02:46:17Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<style><br />
#break {width:950px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}</style><br />
<div id=break></div><br />
<br />
<p align=center><font face=arial narrow size=5><b>Our Pathway Model<br />
</b></font></p><font face=arial narrow><br />
<br />
The study of environmental genomics attempts to capture the taxonomic and functional diversity of natural microbial communities. Our host at UBC, the Hallam lab, designs novel tools for analyzing the gene content in the context of distributed metabolism. Recently, a pipeline has been developed for the automated construction and visualizing of metabolic pathways from genomic data by integrating software such as Pathway Tools, Pathologic and Metacyc [1]. This provided us an opportunity to model pathway compartmentalization and distribution amongst microbes in the natural environment as it applies to our project. </br></br><br />
<br />
<h2>Summary of the pipeline for community-level metabolic analysis (Figure 1):</h2> First, open reading frames are predicted from sequence data with Prodigal, are then annotated by protein BLAST, and later summarized in a GenBank file. Pathway/genome databases (PGDBs) are generated from sequence data in a manner which does not constrain predictions within the scope of model organisms. This produces a community-based analysis that can be visualized using Pathway Tools. <br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/9c/UbcigemSlide1.jpg"></p><br />
<br />
<h2>Modeling Metabolism:</h2> <i>Rhodococcus erythropolis</i> and <i>Pseudomnas fluorescens</i> are often both prevalent in similar niches. With a high likelihood that these organisms encounter each other, studies have been conducted to assess their metabolic properties in co-culture. In a study by Kayser et al, it was shown that the 4S biodesulferization pathway in <i>R. erythropolis</i> demonstrated higher activity the presence of <i>P. fluorescens</i> [2]. As <i>P. fluorescens</i> does not biodesulferize DBT and sulfate is known to repress the 4S pathway, we looked to analyze the genomes of the two organisms in the context of sulfur metabolism. </br></br><br />
<br />
The 4S pathway releases sulfite, which is toxic to the cell, and therefore genomes were analyzed with the aforementioned pipeline for pathways involved in metabolizing sulfite. It was found that both organisms have annotated genes which convert sulfite into sulfate via a reductase, however the organisms differ in downstream metabolism of sulfate. While both organisms contain a pathway for assimilatory sulfate metabolism, only <i>P. fluorescens</i> has the capability for dissimilatory sulfate metabolism (Figure 2, 3). Based on these findings, we can hypothesize that <i>R. erythropolis</i> and <i>P. fluorescens</i> likely excrete and catabolize any excess sulfate. This provides an explanation for the improved desulfurization found in co-culture conditions. <i>P. fluorescens</i> potentially removes sulfate from the environment, allowing increased secretion by <i>R. erythropolis</i>, and thereby derepressing 4S pathway. The prediction of distributed sulfite metabolism to improve biodesulferization in the environment provides both a testable hypothesis, as well as a grounds for improving biodesulferization through synthetic pathway distribution. </br></br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/4/43/UbcigemerSlide2.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/d/d1/UbcigemerSlide3.jpg"></p><br />
<br />
<br />
We then scanned the literature for more evidence of shared metabolism between both <i>R. erythropolis</i> and <i>P. fluorescens</i> in order to further analyze gene content in the context of co-culture experiments. In a study by Goswami et al., the metabolism of chlorinated aromatic compounds and phenol was compared in monoculture versus co-culture using <i>R. erythropolis</i> and <i>P. fluorescens</i> [3]. This study showed that the growth rate of pure cultures of <i>R. erythropolis</i> was higher than <i>P. fluorescens</i> on chlorinated aromatics, however in mixed culture, <i>P. fluorescens</i> showed a higher growth rate. For the degradation of phenol, <i>R. erythropolis</i> showed higher growth rates in both pure and mixed culture. The authors of this study suggested that these results were likely a product of substrate competition. We attempted to analyze the genomes of both <i>R. erythropolis</i> and <i>P. fluorescens</i>, separately and together in an attempt to offer an alternate interpretation of the co-culture results. The first pathways assessed were those involved in chlorinated aromatic degradation. It was found that <i>P. fluorescens</i> contains a higher diversity of genes involved in catabolizing chlorinated aromatics; however, only <i>R. erythropolis</i> seems to be able to degrade phenol (Figure 4, 5). This suggests the possibility of the compartmentalization of different components of these metabolic processes, leading to the different growth kinetics observed in co-culture. For example, while <i>R. erythropolis</i> may be more efficient at degrading certain chlorinated aromatics, in co-culture, the diversity of catabolism of chlorinated aromatics allows <i>P. fluorescens</i> to grow more rapidly. Chlorinated aromatic degradation by <i>P. fluorescens</i>, however, would result in the accumulation of downstream products, such as phenol, that only <i>R. erythropolis</i> can catabolism. This provides a metabolic network which could both select and sustain both microbes in the presence of diverse chlorinated aromatics. </br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/b/bd/UbcigemSlide4.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/99/UbcigemSlide5.jpg"></p><br />
<br />
<br />
<br />
<br />
The sums of general aromatic degradation pathways were compared for the organisms genomes separately and together (Figure 6). This resulted in emergent predicted pathways in combination as well as combinatorial increases araomatic degradation potential. <br /><br />
<br />
Ultimately, gene annotation-based models for distributed metabolism in the environment may help to engineer and optimize complex metabolism through synthetic consortia. </br><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/a/aa/UbcigemSlide6.jpg"></p></br></br><br />
<br />
[1] Hanson, N.W; Page, A.P; Konwar, K.M; Howes, C.G; Hallam, S.J. Metabolic interaction networks for the whole community, 2012. Unpublished.</br></br><br />
<br />
[2] Kayser, K.J; Biolaga-Jones, B.A.; Jackowski, K; Odusan, O; Kildane, J.J. Utilzation of organosulfur compounds by anexic and mixed culture of Rhodococcus rhodochrous IGTS8, 1993. Journal of General Microbioology, 139: 3123-3129.</br></br><br />
<br />
[3] Goswami, M; Shivaraman, N; Singh, R.P. Microbial metabolism of 2-chlorophenol, phenol and p-cresol by Rhodococcus erythropolis M1 and co-culture with Pseudomonas fluorescens P1, 2005. Microbiological Research, 160: 101-109.</br></br></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PathwayTeam:British Columbia/Pathway2012-10-04T02:44:30Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<style><br />
#break {width:950px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}</style><br />
<div id=break></div><br />
<br />
<p align=center><font face=arial narrow size=5><b>Our Pathway Model<br />
</b></font></p><font face=arial narrow><br />
<br />
The study of environmental genomics attempts to capture the taxonomic and functional diversity of natural microbial communities. Our host at UBC, the Hallam lab, designs novel tools for analyzing the gene content in the context of distributed metabolism. Recently, a pipeline has been developed for the automated construction and visualizing of metabolic pathways from genomic data by integrating software such as Pathway Tools, Pathologic and Metacyc [1]. This provided us an opportunity to model pathway compartmentalization and distribution amongst microbes in the natural environment as it applies to our project. </br></br><br />
<br />
<h2>Summary of the pipeline for community-level metabolic analysis (Figure 1):</h2> First, open reading frames are predicted from sequence data with Prodigal, are then annotated by protein BLAST, and later summarized in a GenBank file. Pathway/genome databases (PGDBs) are generated from sequence data in a manner which does not constrain predictions within the scope of model organisms. This produces a community-based analysis that can be visualized using Pathway Tools. <br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/9c/UbcigemSlide1.jpg"></p><br />
<br />
<h2>Modeling Metabolism:</h2> <i>Rhodococcus erythropolis</i> and <i>Pseudomnas fluorescens</i> are often both prevalent in similar niches. With a high likelihood that these organisms encounter each other, studies have been conducted to assess their metabolic properties in co-culture. In a study by Kayser et al, it was shown that the 4S biodesulferization pathway in <i>R. erythropolis</i> demonstrated higher activity the presence of <i>P. fluorescens</i> [2]. As <i>P. fluorescens</i> does not biodesulferize DBT and sulfate is known to repress the 4S pathway, we looked to analyze the genomes of the two organisms in the context of sulfur metabolism. </br></br><br />
<br />
The 4S pathway releases sulfite, which is toxic to the cell, and therefore genomes were analyzed with the aforementioned pipeline for pathways involved in metabolizing sulfite. It was found that both organisms have annotated genes which convert sulfite into sulfate via a reductase, however the organisms differ in downstream metabolism of sulfate. While both organisms contain a pathway for assimilatory sulfate metabolism, only <i>P. fluorescens</i> has the capability for dissimilatory sulfate metabolism (Figure 2, 3). Based on these findings, we can hypothesize that <i>R. erythropolis</i> and <i>P. fluorescens</i> likely excrete and catabolize any excess sulfate. This provides an explanation for the improved desulfurization found in co-culture conditions. <i>P. fluorescens</i> potentially removes sulfate from the environment, allowing increased secretion by <i>R. erythropolis</i>, and thereby derepressing 4S pathway. The prediction of distributed sulfite metabolism to improve biodesulferization in the environment provides both a testable hypothesis, as well as a grounds for improving biodesulferization through synthetic pathway distribution. </br></br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/4/43/UbcigemerSlide2.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/d/d1/UbcigemerSlide3.jpg"></p><br />
<br />
<br />
We then scanned the literature for more evidence of shared metabolism between both <i>R. erythropolis</i> and <i>P. fluorescens</i> in order to further analyze gene content in the context of co-culture experiments. In a study by Goswami et al., the metabolism of chlorinated aromatic compounds and phenol was compared in monoculture versus co-culture using <i>R. erythropolis</i> and <i>P. fluorescens</i> [3]. This study showed that the growth rate of pure cultures of <i>R. erythropolis</i> was higher than <i>P. fluorescens</i> on chlorinated aromatics, however in mixed culture, <i>P. fluorescens</i> showed a higher growth rate. For the degradation of phenol, <i>R. erythropolis</i> showed higher growth rates in both pure and mixed culture. The authors of this study suggested that these results were likely a product of substrate competition. We attempted to analyze the genomes of both <i>R. erythropolis</i> and <i>P. fluorescens</i>, separately and together in an attempt to offer an alternate interpretation of the co-culture results. The first pathways assessed were those involved in chlorinated aromatic degradation. It was found that <i>P. fluorescens</i> contains a higher diversity of genes involved in catabolizing chlorinated aromatics; however, only <i>R. erythropolis</i> seems to be able to degrade phenol (Figure 4, 5). This suggests the possibility of the compartmentalization of different components of these metabolic processes, leading to the different growth kinetics observed in co-culture. For example, while <i>R. erythropolis</i> may be more efficient at degrading certain chlorinated aromatics, in co-culture, the diversity of catabolism of chlorinated aromatics allows <i>P. fluorescens</i> to grow more rapidly. Chlorinated aromatic degradation by <i>P. fluorescens</i>, however, would result in the accumulation of downstream products, such as phenol, that only <i>R. erythropolis</i> can catabolism. This provides a metabolic network which could both select and sustain both microbes in the presence of diverse chlorinated aromatics. </br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/b/bd/UbcigemSlide4.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/99/UbcigemSlide5.jpg"></p><br />
<br />
<br />
<br />
<br />
Finally, the sums of general aromatic degradation pathways were compared for the organisms genomes separately and together (Figure 6). This resulted in emergent predicted pathways in combination as well as combinatorial increases araomatic degradation potential. Ultimately, a gene annotation-based models for distributed metabolism in the environment may help to engineer and optimize complex metabolism through synthetic consortia. </br><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/a/aa/UbcigemSlide6.jpg"></p></br></br><br />
<br />
[1] Hanson, N.W; Page, A.P; Konwar, K.M; Howes, C.G; Hallam, S.J. Metabolic interaction networks for the whole community, 2012. Unpublished.</br></br><br />
<br />
[2] Kayser, K.J; Biolaga-Jones, B.A.; Jackowski, K; Odusan, O; Kildane, J.J. Utilzation of organosulfur compounds by anexic and mixed culture of Rhodococcus rhodochrous IGTS8, 1993. Journal of General Microbioology, 139: 3123-3129.</br></br><br />
<br />
[3] Goswami, M; Shivaraman, N; Singh, R.P. Microbial metabolism of 2-chlorophenol, phenol and p-cresol by Rhodococcus erythropolis M1 and co-culture with Pseudomonas fluorescens P1, 2005. Microbiological Research, 160: 101-109.</br></br></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PathwayTeam:British Columbia/Pathway2012-10-04T02:34:34Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<style><br />
#break {width:950px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}</style><br />
<div id=break></div><br />
<br />
<p align=center><font face=arial narrow size=5><b>Our Pathway Model<br />
</b></font></p><font face=arial narrow><br />
<br />
The study of environmental genomics attempts to capture the taxonomic and functional diversity of natural microbial communities. Our host at UBC, the Hallam lab, designs novel tools for analyzing the gene content in the context of distributed metabolism. Recently, a pipeline has been developed for the automated construction and visualizing of metabolic pathways from genomic data by integrating software such as Pathway Tools, Pathologic and Metacyc [1]. This provided us an opportunity to model pathway compartmentalization and distribution amongst microbes in the natural environment as it applies to our project. </br></br><br />
<br />
<h2>Summary of the pipeline for community-level metabolic analysis (Figure 1):</h2> First, open reading frames are predicted from sequence data with Prodigal, are then annotated by protein BLAST, and later summarized in a GenBank file. Pathway/genome databases (PGDBs) are generated from sequence data in a manner which does not constrain predictions within the scope of model organisms. This produces a community-based analysis that can be visualized using Pathway Tools. <br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/9c/UbcigemSlide1.jpg"></p><br />
<br />
<h2>Modeling Metabolism:</h2> <i>Rhodococcus erythropolis</i> and <i>Pseudomnas fluorescens</i> are often both prevalent in similar niches. With a high likelihood that these organisms encounter each other, studies have been conducted to assess their metabolic properties in co-culture. In a study by Kayser et al, it was shown that the 4S biodesulferization pathway in <i>R. erythropolis</i> demonstrated higher activity the presence of <i>P. fluorescens</i> [2]. As <i>P. fluorescens</i> does not biodesulferize DBT and sulfate is known to repress the 4S pathway, we looked to analyze the genomes of the two organisms in the context of sulfur metabolism. </br></br><br />
<br />
The 4S pathway releases sulfite, which is toxic to the cell, and therefore genomes were analyzed with the aforementioned pipeline for pathways involved in metabolizing sulfite. It was found that both organisms have annotated genes which convert sulfite into sulfate via a reductase, however the organisms differ in downstream metabolism of sulfate. While both organisms contain a pathway for assimilatory sulfate metabolism, only <i>P. fluorescens</i> has the capability for dissimilatory sulfate metabolism (Figure 2, 3). Based on these findings, we can hypothesize that <i>R. erythropolis</i> and <i>P. fluorescens</i> likely excrete and catabolize any excess sulfate. This provides an explanation for the improved desulfurization found in co-culture conditions. <i>P. fluorescens</i> potentially removes sulfate from the environment, allowing increased secretion by <i>R. erythropolis</i>, and thereby derepressing 4S pathway. The prediction of distributed sulfite metabolism to improve biodesulferization in the environment provides both a testable hypothesis, as well as a grounds for improving biodesulferization through synthetic pathway distribution. </br></br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/4/43/UbcigemerSlide2.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/d/d1/UbcigemerSlide3.jpg"></p><br />
<br />
<br />
We then looked through the literature for more evidence of shared metabolism between both <i>R. erythropolis</i> and <i>P. fluorescens</i> to further analyze gene content in the context of co-culture experiments. In a study by Goswami et al. the metabolism of chlorinated aromatic compounds and phenol was compared in monoculture and co-culture with both <i>R. erythropolis</i> and <i>P. fluorescens</i> [3]. This study showed the growth rate of pure culture <i>R. erythropolis</i> was higher than <i>P. fluorescens</i> on chlorinated aromatics; however, in mixed culture, <i>P. fluorescens</i> showed a higher growth rate. For the degradation of phenol, <i>R. erythropolis</i> showed higher growth rates in both pure and mixed culture. The authors of this study suggested that these results were likely a product of substrate competition. We attempted to analyze the genomes of both <i>R. erythropolis</i> and <i>P. fluorescens</i>, separately and together in an attempt to offer an alternate interpretation of the co-culture results. The first pathways assessed were those involved in chlorinated aromatic degradation. It was found that <i>P. fluorescens</i> contains a higher diversity of genes involved in catabolizing chlorinated aromatics; however, only <i>R. erythropolis</i> seems to be able to degrade phenol (Figure 4, 5). This suggests the possibility of the compartmentalization of different components of these metabolic processes lead to different growth kinetics seen in co-culture. For example, while <i>R. erythropolis</i> may be more efficient at degrading certain chlorinated aromatics, in co-culture, the diversity of catabolism of chlorinated aromatics allows <i>P. fluorescens</i> to grow more rapidly. However, chlorinated aromatic degradation by <i>P. fluorescens</i> would result in the accumulation of some downstream products, such as phenol, that only <i>R. erythropolis</i> can catabolism. This provides a metabolic network which could both select and sustain both microbes in the presence of diverse chlorinated aromatics. </br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/b/bd/UbcigemSlide4.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/99/UbcigemSlide5.jpg"></p><br />
<br />
<br />
<br />
<br />
Finally, the sums of general aromatic degradation pathways were compared for the organisms genomes separately and together (Figure 6). This resulted in emergent predicted pathways in combination as well as combinatorial increases araomatic degradation potential. Ultimately, a gene annotation based models for distributed metabolism in the environment may help to engineer and optimize complex metabolism through synthetic consortia. </br><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/a/aa/UbcigemSlide6.jpg"></p></br></br><br />
<br />
[1] Hanson, N.W; Page, A.P; Konwar, K.M; Howes, C.G; Hallam, S.J. Metabolic interaction networks for the whole community, 2012. Unpublished.</br></br><br />
<br />
[2] Kayser, K.J; Biolaga-Jones, B.A.; Jackowski, K; Odusan, O; Kildane, J.J. Utilzation of organosulfur compounds by anexic and mixed culture of Rhodococcus rhodochrous IGTS8, 1993. Journal of General Microbioology, 139: 3123-3129.</br></br><br />
<br />
[3] Goswami, M; Shivaraman, N; Singh, R.P. Microbial metabolism of 2-chlorophenol, phenol and p-cresol by Rhodococcus erythropolis M1 and co-culture with Pseudomonas fluorescens P1, 2005. Microbiological Research, 160: 101-109.</br></br></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PathwayTeam:British Columbia/Pathway2012-10-04T02:24:06Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<style><br />
#break {width:950px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}</style><br />
<div id=break></div><br />
<br />
<p align=center><font face=arial narrow size=5><b>Our Pathway Model<br />
</b></font></p><font face=arial narrow><br />
<br />
The study of environmental genomics attempts to capture the taxonomic and functional diversity of natural microbial communities. Our host at UBC, the Hallam lab, designs novel tools for analyzing the gene content in the context of distributed metabolism. Recently, a pipeline has been developed for the automated construction and visualizing of metabolic pathways from genomic data by integrating software such as Pathway Tools, Pathologic and Metacyc [1]. This provided us an opportunity to model pathway compartmentalization and distribution amongst microbes in the natural environment as it applies to our project. </br></br><br />
<br />
<h2>Summary of the pipeline for community-level metabolic analysis (Figure 1):</h2> First, open reading frames predicted from sequence data using Prodigal are annotated by protein BLAST and summarized in a GenBank file. Pathway/genome databases (PGDBs) are generated from sequence data in a manner that does not constrain predictions within the scope of model organisms. This allows for a community-based analysis that can be visualized using Pathway Tools. <br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/9c/UbcigemSlide1.jpg"></p><br />
<br />
<h2>Modeling Metabolism:</h2> <i>Rhodococcus erythropolis</i> and <i>Pseudomnas fluorescens</i> are often both prevalent in similar niches. With a high likelihood that these organisms encounter each other, studies have been conducted to assess their metabolic properties in co-culture. In a study by Kayser et al, it was shown that the 4-S biodesulferization pathway in <i>R. erythropolis</i> was more active in the presence of <i>P. fluorescens</i> [2]. As <i>P. fluorescens</i> does not biodesulferize DBT and sulfate is known to repress the 4-S pathway, we looked to analyze the genomes of the two organisms in the context of sulfur metabolism. </br></br><br />
<br />
The 4-S pathway releases sulfite, which is toxic to the cell, therefore genomes were analyzed with the aforementioned pipeline for pathways involved in metabolizing sulfite. It was found that both organisms have annotated genes converting sulfite into sulfate via a reductase; however, the organisms differ on the downstream metabolism of the sulfate. Where both organisms have an assimilatory sulfate metabolism, only <i>P. fluorescens</i> has a dissimilatory sulfate metabolism (Figure 2, 3). Based on these findings, we can hypothesize that <i>R. erythropolis</i> and <i>P. fluorescens</i> likely excrete and catabolize any excess sulfate, respectively. This provides an explanation for the improved desulfurization found in co-culture conditions. <i>P. fluorescens</i> potentially removes sulfate from the environment allowing increased secretion by <i>R. erythropolis</i> and thereby removing suppression of the 4-S pathway. The prediction of distributed sulfite metabolism to improve biodesulferization in the environment provides both a testable hypothesis and direction in improving biodesulferization through synthetic pathway distribution. </br></br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/4/43/UbcigemerSlide2.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/d/d1/UbcigemerSlide3.jpg"></p><br />
<br />
<br />
We then looked through the literature for more evidence of shared metabolism between both <i>R. erythropolis</i> and <i>P. fluorescens</i> to further analyze gene content in the context of co-culture experiments. In a study by Goswami et al. the metabolism of chlorinated aromatic compounds and phenol was compared in monoculture and co-culture with both <i>R. erythropolis</i> and <i>P. fluorescens</i> [3]. This study showed the growth rate of pure culture <i>R. erythropolis</i> was higher than <i>P. fluorescens</i> on chlorinated aromatics; however, in mixed culture, <i>P. fluorescens</i> showed a higher growth rate. For the degradation of phenol, <i>R. erythropolis</i> showed higher growth rates in both pure and mixed culture. The authors of this study suggested that these results were likely a product of substrate competition. We attempted to analyze the genomes of both <i>R. erythropolis</i> and <i>P. fluorescens</i>, separately and together in an attempt to offer an alternate interpretation of the co-culture results. The first pathways assessed were those involved in chlorinated aromatic degradation. It was found that <i>P. fluorescens</i> contains a higher diversity of genes involved in catabolizing chlorinated aromatics; however, only <i>R. erythropolis</i> seems to be able to degrade phenol (Figure 4, 5). This suggests the possibility of the compartmentalization of different components of these metabolic processes lead to different growth kinetics seen in co-culture. For example, while <i>R. erythropolis</i> may be more efficient at degrading certain chlorinated aromatics, in co-culture, the diversity of catabolism of chlorinated aromatics allows <i>P. fluorescens</i> to grow more rapidly. However, chlorinated aromatic degradation by <i>P. fluorescens</i> would result in the accumulation of some downstream products, such as phenol, that only <i>R. erythropolis</i> can catabolism. This provides a metabolic network which could both select and sustain both microbes in the presence of diverse chlorinated aromatics. </br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/b/bd/UbcigemSlide4.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/99/UbcigemSlide5.jpg"></p><br />
<br />
<br />
<br />
<br />
Finally, the sums of general aromatic degradation pathways were compared for the organisms genomes separately and together (Figure 6). This resulted in emergent predicted pathways in combination as well as combinatorial increases araomatic degradation potential. Ultimately, a gene annotation based models for distributed metabolism in the environment may help to engineer and optimize complex metabolism through synthetic consortia. </br><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/a/aa/UbcigemSlide6.jpg"></p></br></br><br />
<br />
[1] Hanson, N.W; Page, A.P; Konwar, K.M; Howes, C.G; Hallam, S.J. Metabolic interaction networks for the whole community, 2012. Unpublished.</br></br><br />
<br />
[2] Kayser, K.J; Biolaga-Jones, B.A.; Jackowski, K; Odusan, O; Kildane, J.J. Utilzation of organosulfur compounds by anexic and mixed culture of Rhodococcus rhodochrous IGTS8, 1993. Journal of General Microbioology, 139: 3123-3129.</br></br><br />
<br />
[3] Goswami, M; Shivaraman, N; Singh, R.P. Microbial metabolism of 2-chlorophenol, phenol and p-cresol by Rhodococcus erythropolis M1 and co-culture with Pseudomonas fluorescens P1, 2005. Microbiological Research, 160: 101-109.</br></br></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PathwayTeam:British Columbia/Pathway2012-10-04T02:23:45Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
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<div id=break></div><br />
<br />
<p align=center><font face=arial narrow size=5><b>Our Pathway Model<br />
</b></font></p><font face=arial narrow><br />
<br />
The study of environmental genomics attempts to capture the taxonomic and functional diversity of natural microbial communities. Our host at UBC, the Hallam lab, designs novel tools for analyzing the gene content in the context of distributed metabolism. Recently, a pipeline has been developed for the automated construction and visualizing of metabolic pathways from genomic data by integrating software such as Pathway Tools, Pathologic and Metacyc [1]. This provided us an opportunity to model pathway compartmentalization and distribution amongst microbes in the natural environment as it applies to our project. </br></br><br />
<br />
<h2>Summary of the pipeline for community-level metabolic analysis (Figure 1):</h2> First, open reading frames predicted from sequence data using Prodigal are annotated by protein BLAST and summarized in a GenBank file. Pathway/genome databases (PGDBs) are generated from sequence data in a manner that does not constrain predictions within the scope of model organisms. This allows for a community-based analysis that can be visualized using Pathway Tools. <br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/9c/UbcigemSlide1.jpg"></p><br />
<br />
<b>Modeling Metabolism:</b> <i>Rhodococcus erythropolis</i> and <i>Pseudomnas fluorescens</i> are often both prevalent in similar niches. With a high likelihood that these organisms encounter each other, studies have been conducted to assess their metabolic properties in co-culture. In a study by Kayser et al, it was shown that the 4-S biodesulferization pathway in <i>R. erythropolis</i> was more active in the presence of <i>P. fluorescens</i> [2]. As <i>P. fluorescens</i> does not biodesulferize DBT and sulfate is known to repress the 4-S pathway, we looked to analyze the genomes of the two organisms in the context of sulfur metabolism. </br></br><br />
<br />
The 4-S pathway releases sulfite, which is toxic to the cell, therefore genomes were analyzed with the aforementioned pipeline for pathways involved in metabolizing sulfite. It was found that both organisms have annotated genes converting sulfite into sulfate via a reductase; however, the organisms differ on the downstream metabolism of the sulfate. Where both organisms have an assimilatory sulfate metabolism, only <i>P. fluorescens</i> has a dissimilatory sulfate metabolism (Figure 2, 3). Based on these findings, we can hypothesize that <i>R. erythropolis</i> and <i>P. fluorescens</i> likely excrete and catabolize any excess sulfate, respectively. This provides an explanation for the improved desulfurization found in co-culture conditions. <i>P. fluorescens</i> potentially removes sulfate from the environment allowing increased secretion by <i>R. erythropolis</i> and thereby removing suppression of the 4-S pathway. The prediction of distributed sulfite metabolism to improve biodesulferization in the environment provides both a testable hypothesis and direction in improving biodesulferization through synthetic pathway distribution. </br></br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/4/43/UbcigemerSlide2.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/d/d1/UbcigemerSlide3.jpg"></p><br />
<br />
<br />
We then looked through the literature for more evidence of shared metabolism between both <i>R. erythropolis</i> and <i>P. fluorescens</i> to further analyze gene content in the context of co-culture experiments. In a study by Goswami et al. the metabolism of chlorinated aromatic compounds and phenol was compared in monoculture and co-culture with both <i>R. erythropolis</i> and <i>P. fluorescens</i> [3]. This study showed the growth rate of pure culture <i>R. erythropolis</i> was higher than <i>P. fluorescens</i> on chlorinated aromatics; however, in mixed culture, <i>P. fluorescens</i> showed a higher growth rate. For the degradation of phenol, <i>R. erythropolis</i> showed higher growth rates in both pure and mixed culture. The authors of this study suggested that these results were likely a product of substrate competition. We attempted to analyze the genomes of both <i>R. erythropolis</i> and <i>P. fluorescens</i>, separately and together in an attempt to offer an alternate interpretation of the co-culture results. The first pathways assessed were those involved in chlorinated aromatic degradation. It was found that <i>P. fluorescens</i> contains a higher diversity of genes involved in catabolizing chlorinated aromatics; however, only <i>R. erythropolis</i> seems to be able to degrade phenol (Figure 4, 5). This suggests the possibility of the compartmentalization of different components of these metabolic processes lead to different growth kinetics seen in co-culture. For example, while <i>R. erythropolis</i> may be more efficient at degrading certain chlorinated aromatics, in co-culture, the diversity of catabolism of chlorinated aromatics allows <i>P. fluorescens</i> to grow more rapidly. However, chlorinated aromatic degradation by <i>P. fluorescens</i> would result in the accumulation of some downstream products, such as phenol, that only <i>R. erythropolis</i> can catabolism. This provides a metabolic network which could both select and sustain both microbes in the presence of diverse chlorinated aromatics. </br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/b/bd/UbcigemSlide4.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/99/UbcigemSlide5.jpg"></p><br />
<br />
<br />
<br />
<br />
Finally, the sums of general aromatic degradation pathways were compared for the organisms genomes separately and together (Figure 6). This resulted in emergent predicted pathways in combination as well as combinatorial increases araomatic degradation potential. Ultimately, a gene annotation based models for distributed metabolism in the environment may help to engineer and optimize complex metabolism through synthetic consortia. </br><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/a/aa/UbcigemSlide6.jpg"></p></br></br><br />
<br />
[1] Hanson, N.W; Page, A.P; Konwar, K.M; Howes, C.G; Hallam, S.J. Metabolic interaction networks for the whole community, 2012. Unpublished.</br></br><br />
<br />
[2] Kayser, K.J; Biolaga-Jones, B.A.; Jackowski, K; Odusan, O; Kildane, J.J. Utilzation of organosulfur compounds by anexic and mixed culture of Rhodococcus rhodochrous IGTS8, 1993. Journal of General Microbioology, 139: 3123-3129.</br></br><br />
<br />
[3] Goswami, M; Shivaraman, N; Singh, R.P. Microbial metabolism of 2-chlorophenol, phenol and p-cresol by Rhodococcus erythropolis M1 and co-culture with Pseudomonas fluorescens P1, 2005. Microbiological Research, 160: 101-109.</br></br></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/PathwayTeam:British Columbia/Pathway2012-10-04T02:23:08Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<style><br />
#break {width:950px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}</style><br />
<div id=break></div><br />
<br />
<p align=center><font face=arial narrow size=5><b>Our Pathway Model<br />
</b></font></p><font face=arial narrow><br />
<br />
The study of environmental genomics attempts to capture the taxonomic and functional diversity of natural microbial communities. Our host at UBC, the Hallam lab, designs novel tools for analyzing the gene content in the context of distributed metabolism. Recently, a pipeline has been developed for the automated construction and visualizing of metabolic pathways from genomic data by integrating software such as Pathway Tools, Pathologic and Metacyc [1]. This provided us an opportunity to model pathway compartmentalization and distribution amongst microbes in the natural environment as it applies to our project. </br></br><br />
<br />
<b>Summary of the pipeline for community-level metabolic analysis (Figure 1):</b> First, open reading frames predicted from sequence data using Prodigal are annotated by protein BLAST and summarized in a GenBank file. Pathway/genome databases (PGDBs) are generated from sequence data in a manner that does not constrain predictions within the scope of model organisms. This allows for a community-based analysis that can be visualized using Pathway Tools. <br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/9c/UbcigemSlide1.jpg"></p><br />
<br />
<b>Modeling Metabolism:</b> <i>Rhodococcus erythropolis</i> and <i>Pseudomnas fluorescens</i> are often both prevalent in similar niches. With a high likelihood that these organisms encounter each other, studies have been conducted to assess their metabolic properties in co-culture. In a study by Kayser et al, it was shown that the 4-S biodesulferization pathway in <i>R. erythropolis</i> was more active in the presence of <i>P. fluorescens</i> [2]. As <i>P. fluorescens</i> does not biodesulferize DBT and sulfate is known to repress the 4-S pathway, we looked to analyze the genomes of the two organisms in the context of sulfur metabolism. </br></br><br />
<br />
The 4-S pathway releases sulfite, which is toxic to the cell, therefore genomes were analyzed with the aforementioned pipeline for pathways involved in metabolizing sulfite. It was found that both organisms have annotated genes converting sulfite into sulfate via a reductase; however, the organisms differ on the downstream metabolism of the sulfate. Where both organisms have an assimilatory sulfate metabolism, only <i>P. fluorescens</i> has a dissimilatory sulfate metabolism (Figure 2, 3). Based on these findings, we can hypothesize that <i>R. erythropolis</i> and <i>P. fluorescens</i> likely excrete and catabolize any excess sulfate, respectively. This provides an explanation for the improved desulfurization found in co-culture conditions. <i>P. fluorescens</i> potentially removes sulfate from the environment allowing increased secretion by <i>R. erythropolis</i> and thereby removing suppression of the 4-S pathway. The prediction of distributed sulfite metabolism to improve biodesulferization in the environment provides both a testable hypothesis and direction in improving biodesulferization through synthetic pathway distribution. </br></br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/4/43/UbcigemerSlide2.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/d/d1/UbcigemerSlide3.jpg"></p><br />
<br />
<br />
We then looked through the literature for more evidence of shared metabolism between both <i>R. erythropolis</i> and <i>P. fluorescens</i> to further analyze gene content in the context of co-culture experiments. In a study by Goswami et al. the metabolism of chlorinated aromatic compounds and phenol was compared in monoculture and co-culture with both <i>R. erythropolis</i> and <i>P. fluorescens</i> [3]. This study showed the growth rate of pure culture <i>R. erythropolis</i> was higher than <i>P. fluorescens</i> on chlorinated aromatics; however, in mixed culture, <i>P. fluorescens</i> showed a higher growth rate. For the degradation of phenol, <i>R. erythropolis</i> showed higher growth rates in both pure and mixed culture. The authors of this study suggested that these results were likely a product of substrate competition. We attempted to analyze the genomes of both <i>R. erythropolis</i> and <i>P. fluorescens</i>, separately and together in an attempt to offer an alternate interpretation of the co-culture results. The first pathways assessed were those involved in chlorinated aromatic degradation. It was found that <i>P. fluorescens</i> contains a higher diversity of genes involved in catabolizing chlorinated aromatics; however, only <i>R. erythropolis</i> seems to be able to degrade phenol (Figure 4, 5). This suggests the possibility of the compartmentalization of different components of these metabolic processes lead to different growth kinetics seen in co-culture. For example, while <i>R. erythropolis</i> may be more efficient at degrading certain chlorinated aromatics, in co-culture, the diversity of catabolism of chlorinated aromatics allows <i>P. fluorescens</i> to grow more rapidly. However, chlorinated aromatic degradation by <i>P. fluorescens</i> would result in the accumulation of some downstream products, such as phenol, that only <i>R. erythropolis</i> can catabolism. This provides a metabolic network which could both select and sustain both microbes in the presence of diverse chlorinated aromatics. </br><br />
<br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/b/bd/UbcigemSlide4.jpg"></p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/9/99/UbcigemSlide5.jpg"></p><br />
<br />
<br />
<br />
<br />
Finally, the sums of general aromatic degradation pathways were compared for the organisms genomes separately and together (Figure 6). This resulted in emergent predicted pathways in combination as well as combinatorial increases araomatic degradation potential. Ultimately, a gene annotation based models for distributed metabolism in the environment may help to engineer and optimize complex metabolism through synthetic consortia. </br><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2012/a/aa/UbcigemSlide6.jpg"></p></br></br><br />
<br />
[1] Hanson, N.W; Page, A.P; Konwar, K.M; Howes, C.G; Hallam, S.J. Metabolic interaction networks for the whole community, 2012. Unpublished.</br></br><br />
<br />
[2] Kayser, K.J; Biolaga-Jones, B.A.; Jackowski, K; Odusan, O; Kildane, J.J. Utilzation of organosulfur compounds by anexic and mixed culture of Rhodococcus rhodochrous IGTS8, 1993. Journal of General Microbioology, 139: 3123-3129.</br></br><br />
<br />
[3] Goswami, M; Shivaraman, N; Singh, R.P. Microbial metabolism of 2-chlorophenol, phenol and p-cresol by Rhodococcus erythropolis M1 and co-culture with Pseudomonas fluorescens P1, 2005. Microbiological Research, 160: 101-109.</br></br></div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DesulfurizationTeam:British Columbia/Desulfurization2012-10-04T02:12:33Z<p>Rsaer: </p>
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<br />
<div id=image><p align=center><img src="https://static.igem.org/mediawiki/2012/4/4d/Dsz_operon.png" width=400px></p></div><br />
<br />
<div id=note></br><br />
<p align=center><font face=arial narrow size=5><b>Bio-desulfurization: A Real-world Consortial Case Study</b></font></p><br />
<br />
Once we are able to tune our biological consortium it was time to test it on a system of economic relevance: the desulfurization of dibenzothiophene (DBT). DBT is a polyaromatic sulfur compound present in oil crudes whose combustion by oil-powered machines, such as automobiles, causes the release of sulfur dioxide (SO<sub>2</sub>) into the atmosphere. Sulfur dioxide is a principle source of acid rain and air pollution, and thus the removal of this compound from oil crudes provides benefits to the air quality and general environmental health of developed areas. DBT is highly resistant to hydrodesulfurization, the current process of removing sulphur compounds from crude oil, and thus novel methods of removing this compound are desired. Biodesulfurization of DBT is one means of removing such a compound. Specifically, the 4S pathway from Rhodococcus erythropolis IGTS8, which utilizes the gene products of the Dsz operon, is ideally suited for such a task because it removes DBT from crude oil with minimal impact on the energy density of the crude (i.e. the desirable compounds in the crude that actually serve as fuel, are preserved). As a result of the 4S pathway, DBT is converted to hydroxybiphenyl (HPB, Figure 1).</br></br><br />
</div><div id=break></br><br />
<div align="left"><font face=arial narrow size=4><b>To create a distributed metabolic network, the three catalytic genes of the Dsz operon were separated into three <i>Escherichia coli</i> strains developed for our tunable consortium. This is crucial to optimizing the metabolic potential of the consortium.</b></br></br></font></div></div><br />
<br />
<div id=note><br />
</br></br></br><p align=center><br />
<font face=arial narrow size=5><b>Why the Dsz operon?</b></font></p><font face=arial narrow><br />
<br />
We choose the Dsz operon for a number of reasons. As stated above, bio-desulfurization has the potential to help relieve serious environmental problem associated with DBT content of fossil fuels. However, the Dsz operon also has a number of advantages directly related to our consortium. First, the pathway has previously been expressed functionally in <i>E. coli</i>, allowing us to obtain a working copy of the pathway expressed in an <i>E. coli</i> monoculture [1]. This will let us directly compare the efficiency of the single cell pathway with our distributed metabolic network. Also, the availability of a working pathway in <i>E. coli</i> indicates that all of the required enzymes will be active when recombinantly expressed in our <i>E. coli</i> consortium strains. Finally, the desulfurization activity of the 4S pathway can be easily monitored and quantitated by HPLC analysis [2].</br></div><br />
<br />
<div id=image><img src="https://static.igem.org/mediawiki/2012/f/ff/Dsz_Consortium.png" width=500></div><br />
<br />
<b><h2>References</h2></b><br />
<br />
<b>[1]</b> Reichmuth DM <i>et al</i>. Biodesulfurization of Dibenzothiophene in <i>Escherichia coli</i> Is Enhanced by Expression of a <i>Vibrio harveyi</i> Oxidoreductase Gene, 2000. Biotechnol Bioeng. 67(1):72-9.<br /><br />
<br />
<br />
<b>[2]</b> Bhatia S and Sharma DK. Thermophilic desulfurization of dibenzothiophene<br />
and different petroleum oils by <i>Klebsiella</i> sp. 13T, 2011. Environ Sci Pollut Res. 19:3491–3497</div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DesulfurizationTeam:British Columbia/Desulfurization2012-10-04T02:11:20Z<p>Rsaer: </p>
<hr />
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#note {width:380px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}<br />
</style><br />
<br />
<div id=image><p align=center><img src="https://static.igem.org/mediawiki/2012/4/4d/Dsz_operon.png" width=400px></p></div><br />
<br />
<div id=note></br><br />
<p align=center><font face=arial narrow size=5><b>Bio-desulfurization: A Real-world Consortial Case Study</b></font></p><br />
<br />
Once we are able to tune our biological consortium it was time to test it on a system of economic relevance: the desulfurization of dibenzothiophene (DBT). DBT is a polyaromatic sulfur compound present in oil crudes whose combustion by oil-powered machines, such as automobiles, causes the release of sulfur dioxide (SO<sub>2</sub>) into the atmosphere. Sulfur dioxide is a principle source of acid rain and air pollution, and thus the removal of this compound from oil crudes provides benefits to the air quality and general environmental health of developed areas. DBT is highly resistant to hydrodesulfurization, the current process of removing sulphur compounds from crude oil, and thus novel methods of removing this compound are desired. Biodesulfurization of DBT is one means of removing such a compound. Specifically, the 4S pathway from Rhodococcus erythropolis IGTS8, which utilizes the gene products of the Dsz operon, is ideally suited for such a task because it removes DBT from crude oil with minimal impact on the energy density of the crude (i.e. the desirable compounds in the crude that actually serve as fuel, are preserved). As a result of the 4S pathway, DBT is converted to hydroxybiphenyl (HPB, Figure 1).</br></br><br />
</div><div id=break></br><br />
<div align="left"><font face=arial narrow size=4><b>To create a distributed metabolic network, the three catalytic genes of the Dsz operon were separated into three <i>Escherichia coli</i> strains developed for our tunable consortium. This is crucial to optimizing the metabolic potential of the consortium.</b></br></br></font></div></div><br />
<br />
<div id=note><br />
</br></br></br><p align=center><br />
<font face=arial narrow size=5><b>Why the Dsz operon?</b></font></p><font face=arial narrow><br />
<br />
We choose the Dsz operon for a number of reasons. As stated above, bio-desulfurization has the potential to help relieve serious environmental problem associated with DBT content of fossil fuels. However, the Dsz operon also has a number of advantages directly related to our consortium. First, the pathway has previously been expressed functionally in <i>E. coli</i>, allowing us to obtain a working copy of the pathway expressed in an <i>E. coli</i> monoculture [1]. This will let us directly compare the efficiency of the single cell pathway with our distributed metabolic network. Also, the availability of a working pathway in <i>E. coli</i> indicates that all of the required enzymes will be active when recombinantly expressed in our <i>E. coli</i> consortium strains. Finally, the desulfurization activity of the 4S pathway can be easily monitored and quantitated by HPLC analysis [2].</br></div><br />
<br />
<div id=image><img src="https://static.igem.org/mediawiki/2012/f/ff/Dsz_Consortium.png" width=500></div><br />
<br />
<h2>References</h2><br />
<br />
<b>[1]</b> Reichmuth DM <i>et al</i>. Biodesulfurization of Dibenzothiophene in <i>Escherichia coli</i> Is Enhanced by Expression of a <i>Vibrio harveyi</i> Oxidoreductase Gene, 2000. Biotechnol Bioeng. 67(1):72-9.<br /><br />
<br />
<br />
<b>[2]</b> Bhatia S and Sharma DK. Thermophilic desulfurization of dibenzothiophene<br />
and different petroleum oils by <i>Klebsiella</i> sp. 13T, 2011. Environ Sci Pollut Res. 19:3491–3497</div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DesulfurizationTeam:British Columbia/Desulfurization2012-10-04T02:10:40Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
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#break {width:950px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}<br />
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<br />
<div id=image><p align=center><img src="https://static.igem.org/mediawiki/2012/4/4d/Dsz_operon.png" width=400px></p></div><br />
<br />
<div id=note></br><br />
<p align=center><font face=arial narrow size=5><b>Bio-desulfurization: A Real-world Consortial Case Study</b></font></p><br />
<br />
Once we are able to tune our biological consortium it was time to test it on a system of economic relevance: the desulfurization of dibenzothiophene (DBT). DBT is a polyaromatic sulfur compound present in oil crudes whose combustion by oil-powered machines, such as automobiles, causes the release of sulfur dioxide (SO<sub>2</sub>) into the atmosphere. Sulfur dioxide is a principle source of acid rain and air pollution, and thus the removal of this compound from oil crudes provides benefits to the air quality and general environmental health of developed areas. DBT is highly resistant to hydrodesulfurization, the current process of removing sulphur compounds from crude oil, and thus novel methods of removing this compound are desired. Biodesulfurization of DBT is one means of removing such a compound. Specifically, the 4S pathway from Rhodococcus erythropolis IGTS8, which utilizes the gene products of the Dsz operon, is ideally suited for such a task because it removes DBT from crude oil with minimal impact on the energy density of the crude (i.e. the desirable compounds in the crude that actually serve as fuel, are preserved). As a result of the 4S pathway, DBT is converted to hydroxybiphenyl (HPB, Figure 1).</br></br><br />
</div><div id=break></br><br />
<div align="left"><font face=arial narrow size=4><b>To create a distributed metabolic network, the three catalytic genes of the Dsz operon were separated into three <i>Escherichia coli</i> strains developed for our tunable consortium. This is crucial to optimizing the metabolic potential of the consortium.</b></br></br></font></div></div><br />
<br />
<div id=note><br />
</br></br></br><p align=center><br />
<font face=arial narrow size=5><b>Why the Dsz operon?</b></font></p><font face=arial narrow><br />
<br />
We choose the Dsz operon for a number of reasons. As stated above, bio-desulfurization has the potential to help relieve serious environmental problem associated with DBT content of fossil fuels. However, the Dsz operon also has a number of advantages directly related to our consortium. First, the pathway has previously been expressed functionally in <i>E. coli</i>, allowing us to obtain a working copy of the pathway expressed in an <i>E. coli</i> monoculture [1]. This will let us directly compare the efficiency of the single cell pathway with our distributed metabolic network. Also, the availability of a working pathway in <i>E. coli</i> indicates that all of the required enzymes will be active when recombinantly expressed in our <i>E. coli</i> consortium strains. Finally, the desulfurization activity of the 4S pathway can be easily monitored and quantitated by HPLC analysis [2].</br></div><br />
<br />
<div id=image><img src="https://static.igem.org/mediawiki/2012/f/ff/Dsz_Consortium.png" width=500></div><br />
<br />
<h2>References</h2><br />
<br />
<b>[1]</b> Reichmuth DM <i>et al</i>. Biodesulfurization of Dibenzothiophene in <i>Escherichia coli</i> Is Enhanced by Expression of a <i>Vibrio harveyi</i> Oxidoreductase Gene, 2000. Biotechnol Bioeng. 67(1):72-9.<br /><br />
<br />
<br />
<b>[2]</b> Bhatia S and Sharma DK. Thermophilic desulfurization of dibenzothiophene<br />
and different petroleum oils by Klebsiellasp. 13T, 2011. Environ Sci Pollut Res. 19:3491–3497</div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DesulfurizationTeam:British Columbia/Desulfurization2012-10-04T02:09:39Z<p>Rsaer: </p>
<hr />
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<div id=image><p align=center><img src="https://static.igem.org/mediawiki/2012/4/4d/Dsz_operon.png" width=400px></p></div><br />
<br />
<div id=note></br><br />
<p align=center><font face=arial narrow size=5><b>Bio-desulfurization: A Real-world Consortial Case Study</b></font></p><br />
<br />
Once we are able to tune our biological consortium it was time to test it on a system of economic relevance: the desulfurization of dibenzothiophene (DBT). DBT is a polyaromatic sulfur compound present in oil crudes whose combustion by oil-powered machines, such as automobiles, causes the release of sulfur dioxide (SO<sub>2</sub>) into the atmosphere. Sulfur dioxide is a principle source of acid rain and air pollution, and thus the removal of this compound from oil crudes provides benefits to the air quality and general environmental health of developed areas. DBT is highly resistant to hydrodesulfurization, the current process of removing sulphur compounds from crude oil, and thus novel methods of removing this compound are desired. Biodesulfurization of DBT is one means of removing such a compound. Specifically, the 4S pathway from Rhodococcus erythropolis IGTS8, which utilizes the gene products of the Dsz operon, is ideally suited for such a task because it removes DBT from crude oil with minimal impact on the energy density of the crude (i.e. the desirable compounds in the crude that actually serve as fuel, are preserved). As a result of the 4S pathway, DBT is converted to hydroxybiphenyl (HPB, Figure 1).</br></br><br />
</div><div id=break></br><br />
<div align="left"><font face=arial narrow size=4><b>To create a distributed metabolic network, the three catalytic genes of the Dsz operon were separated into three <i>Escherichia coli</i> strains developed for our tunable consortium. This is crucial to optimizing the metabolic potential of the consortium.</b></br></br></font></div></div><br />
<br />
<div id=note><br />
</br></br></br><p align=center><br />
<font face=arial narrow size=5><b>Why the Dsz operon?</b></font></p><font face=arial narrow><br />
<br />
We choose the Dsz operon for a number of reasons. As stated above, bio-desulfurization has the potential to help relieve serious environmental problem associated with DBT content of fossil fuels. However, the Dsz operon also has a number of advantages directly related to our consortium. First, the pathway has previously been expressed functionally in <i>E. coli</i>, allowing us to obtain a working copy of the pathway expressed in an <i>E. coli</i> monoculture [1]. This will let us directly compare the efficiency of the single cell pathway with our distributed metabolic network. Also, the availability of a working pathway in <i>E. coli</i> indicates that all of the required enzymes will be active when recombinantly expressed in our <i>E. coli</i> consortium strains. Finally, the desulfurization activity of the 4S pathway can be easily monitored and quantitated by HPLC analysis [2].</br></div><br />
<br />
<div id=image><img src="https://static.igem.org/mediawiki/2012/f/ff/Dsz_Consortium.png" width=500></div><br />
<br />
<h2>References</h2><br />
<br />
<b>[1]</b> Reichmuth DM <i>et al</i>. Biodesulfurization of Dibenzothiophene in <i>Escherichia coli</i> Is Enhanced by Expression of a <i>Vibrio harveyi</i> Oxidoreductase Gene, 2000. Biotechnol Bioeng. 67(1):72-9.<br />
<br />
<br />
<b>[2]</b> Bhatia S and Sharma DK. Thermophilic desulfurization of dibenzothiophene<br />
and different petroleum oils by Klebsiellasp. 13T, 2011. Environ Sci Pollut Res. 19:3491–3497</div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DesulfurizationTeam:British Columbia/Desulfurization2012-10-04T02:09:36Z<p>Rsaer: </p>
<hr />
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#break {width:950px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}<br />
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#note {width:380px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}<br />
</style><br />
<br />
<div id=image><p align=center><img src="https://static.igem.org/mediawiki/2012/4/4d/Dsz_operon.png" width=400px></p></div><br />
<br />
<div id=note></br><br />
<p align=center><font face=arial narrow size=5><b>Bio-desulfurization: A Real-world Consortial Case Study</b></font></p><br />
<br />
Once we are able to tune our biological consortium it was time to test it on a system of economic relevance: the desulfurization of dibenzothiophene (DBT). DBT is a polyaromatic sulfur compound present in oil crudes whose combustion by oil-powered machines, such as automobiles, causes the release of sulfur dioxide (SO<sub>2</sub>) into the atmosphere. Sulfur dioxide is a principle source of acid rain and air pollution, and thus the removal of this compound from oil crudes provides benefits to the air quality and general environmental health of developed areas. DBT is highly resistant to hydrodesulfurization, the current process of removing sulphur compounds from crude oil, and thus novel methods of removing this compound are desired. Biodesulfurization of DBT is one means of removing such a compound. Specifically, the 4S pathway from Rhodococcus erythropolis IGTS8, which utilizes the gene products of the Dsz operon, is ideally suited for such a task because it removes DBT from crude oil with minimal impact on the energy density of the crude (i.e. the desirable compounds in the crude that actually serve as fuel, are preserved). As a result of the 4S pathway, DBT is converted to hydroxybiphenyl (HPB, Figure 1).</br></br><br />
</div><div id=break></br><br />
<div align="left"><font face=arial narrow size=4><b>To create a distributed metabolic network, the three catalytic genes of the Dsz operon were separated into three <i>Escherichia coli</i> strains developed for our tunable consortium. This is crucial to optimizing the metabolic potential of the consortium.</b></br></br></font></div></div><br />
<br />
<div id=note><br />
</br></br></br><p align=center><br />
<font face=arial narrow size=5><b>Why the Dsz operon?</b></font></p><font face=arial narrow><br />
<br />
We choose the Dsz operon for a number of reasons. As stated above, bio-desulfurization has the potential to help relieve serious environmental problem associated with DBT content of fossil fuels. However, the Dsz operon also has a number of advantages directly related to our consortium. First, the pathway has previously been expressed functionally in <i>E. coli</i>, allowing us to obtain a working copy of the pathway expressed in an <i>E. coli</i> monoculture [1]. This will let us directly compare the efficiency of the single cell pathway with our distributed metabolic network. Also, the availability of a working pathway in <i>E. coli</i> indicates that all of the required enzymes will be active when recombinantly expressed in our <i>E. coli</i> consortium strains. Finally, the desulfurization activity of the 4S pathway can be easily monitored and quantitated by HPLC analysis [2].</br></div><br />
<br />
<div id=image><img src="https://static.igem.org/mediawiki/2012/f/ff/Dsz_Consortium.png" width=500></div><br />
<br />
<h2>References</h2><br />
<br />
<b>[1]</b> Reichmuth DM <i>et al</i>. Biodesulfurization of Dibenzothiophene in <i>Escherichia coli</i> Is Enhanced by Expression of a <i>Vibrio harveyi</i> Oxidoreductase Gene, 2000. Biotechnol Bioeng. 67(1):72-9.<br />
<br />
<br />
<b>[2]</b> Bhatia S and Sharma DK. Thermophilic desulfurization of dibenzothiophene<br />
and different petroleum oils by <i>Klebsiella</i> sp. 13T, 2011. Environ Sci Pollut Res. 19:3491–3497</div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DesulfurizationTeam:British Columbia/Desulfurization2012-10-04T02:04:08Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
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#break {width:950px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}<br />
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#note {width:380px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}<br />
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<br />
<div id=image><p align=center><img src="https://static.igem.org/mediawiki/2012/4/4d/Dsz_operon.png" width=400px></p></div><br />
<br />
<div id=note></br><br />
<p align=center><font face=arial narrow size=5><b>Bio-desulfurization: A Real-world Consortial Case Study</b></font></p><br />
<br />
Once we are able to tune our biological consortium it was time to test it on a system of economic relevance: the desulfurization of dibenzothiophene (DBT). DBT is a polyaromatic sulfur compound present in oil crudes whose combustion by oil-powered machines, such as automobiles, causes the release of sulfur dioxide (SO<sub>2</sub>) into the atmosphere. Sulfur dioxide is a principle source of acid rain and air pollution, and thus the removal of this compound from oil crudes provides benefits to the air quality and general environmental health of developed areas. DBT is highly resistant to hydrodesulfurization, the current process of removing sulphur compounds from crude oil, and thus novel methods of removing this compound are desired. Biodesulfurization of DBT is one means of removing such a compound. Specifically, the 4S pathway from Rhodococcus erythropolis IGTS8, which utilizes the gene products of the Dsz operon, is ideally suited for such a task because it removes DBT from crude oil with minimal impact on the energy density of the crude (i.e. the desirable compounds in the crude that actually serve as fuel, are preserved). As a result of the 4S pathway, DBT is converted to hydroxybiphenyl (HPB, Figure 1).</br></br><br />
</div><div id=break></br><br />
<div align="left"><font face=arial narrow size=4><b>To create a distributed metabolic network, the three catalytic genes of the Dsz operon were separated into three <i>Escherichia coli</i> strains developed for our tunable consortium. This is crucial to optimizing the metabolic potential of the consortium.</b></br></br></font></div></div><br />
<br />
<div id=note><br />
</br></br></br><p align=center><br />
<font face=arial narrow size=5><b>Why the Dsz operon?</b></font></p><font face=arial narrow><br />
<br />
We choose the Dsz operon for a number of reasons. As stated above, bio-desulfurization has the potential to help relieve serious environmental problem associated with DBT content of fossil fuels. However, the Dsz operon also has a number of advantages directly related to our consortium. First, the pathway has previously been expressed functionally in <i>E. coli</i>, allowing us to obtain a working copy of the pathway expressed in an <i>E. coli</i> monoculture [1]. This will let us directly compare the efficiency of the single cell pathway with our distributed metabolic network. Also, the availability of a working pathway in <i>E. coli</i> indicates that all of the required enzymes will be active when recombinantly expressed in our <i>E. coli</i> consortium strains. Finally, the desulfurization activity of the 4S pathway can be easily monitored and quantitated by HPLC analysis [2].</br></div><br />
<br />
<div id=image><img src="https://static.igem.org/mediawiki/2012/f/ff/Dsz_Consortium.png" width=500></div><br />
<br />
<h2>References</h2><br />
<br />
<b>[1]</b> Reichmuth DM <i>et al</i>. Biodesulfurization of Dibenzothiophene in <i>Escherichia coli</i> Is Enhanced by Expression of a <i>Vibrio harveyi</i> Oxidoreductase Gene<br />
<br />
<b>[2]</b> Bhatia S and Sharma DK;Thermophilic desulfurization of dibenzothiophene<br />
and different petroleum oils by Klebsiellasp. 13T, 2011. Environ Sci Pollut Res 19:3491–3497</div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DesulfurizationTeam:British Columbia/Desulfurization2012-10-04T01:59:45Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
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#break {width:950px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}<br />
#image {width:550px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}<br />
#note {width:380px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}<br />
</style><br />
<br />
<div id=image><p align=center><img src="https://static.igem.org/mediawiki/2012/4/4d/Dsz_operon.png" width=400px></p></div><br />
<br />
<div id=note></br><br />
<p align=center><font face=arial narrow size=5><b>Bio-desulfurization: A Real-world Consortial Case Study</b></font></p><br />
<br />
Once we are able to tune our biological consortium it was time to test it on a system of economic relevance: the desulfurization of dibenzothiophene (DBT). DBT is a polyaromatic sulfur compound present in oil crudes whose combustion by oil-powered machines, such as automobiles, causes the release of sulfur dioxide (SO<sub>2</sub>) into the atmosphere. Sulfur dioxide is a principle source of acid rain and air pollution, and thus the removal of this compound from oil crudes provides benefits to the air quality and general environmental health of developed areas. DBT is highly resistant to hydrodesulfurization, the current process of removing sulphur compounds from crude oil, and thus novel methods of removing this compound are desired. Biodesulfurization of DBT is one means of removing such a compound. Specifically, the 4S pathway from Rhodococcus erythropolis IGTS8, which utilizes the gene products of the Dsz operon, is ideally suited for such a task because it removes DBT from crude oil with minimal impact on the energy density of the crude (i.e. the desirable compounds in the crude that actually serve as fuel, are preserved). As a result of the 4S pathway, DBT is converted to hydroxybiphenyl (HPB, Figure 1).</br></br><br />
</div><div id=break></br><br />
<div align="left"><font face=arial narrow size=4><b>To create a distributed metabolic network, the three catalytic genes of the Dsz operon were separated into three <i>Escherichia coli</i> strains developed for our tunable consortium. This is crucial to optimizing the metabolic potential of the consortium.</b></br></br></font></div></div><br />
<br />
<div id=note><br />
</br></br></br><p align=center><br />
<font face=arial narrow size=5><b>Why the Dsz operon?</b></font></p><font face=arial narrow><br />
<br />
We choose the Dsz operon for a number of reasons. As stated above, bio-desulfurization has the potential to help relieve serious environmental problem associated with DBT content of fossil fuels. However, the Dsz operon also has a number of advantages directly related to our consortium. First, the pathway has previously been expressed functionally in <i>E. coli</i>, allowing us to obtain a working copy of the pathway expressed in an <i>E. coli</i> monoculture (REF). This will let us directly compare the efficiency of the single cell pathway with our distributed metabolic network. Also, the availability of a working pathway in <i>E. coli</i> indicates that all of the required enzymes will be active when recombinantly expressed in our <i>E. coli</i> consortium strains. Finally, the desulfurization activity of the 4S pathway can be easily monitored and quantitated by HPLC analysis [2].</br></div><br />
<br />
<div id=image><img src="https://static.igem.org/mediawiki/2012/f/ff/Dsz_Consortium.png" width=500></div><br />
<br />
<h2>References</h2><br />
<br />
'''[1]'''<br />
<br />
'''[2]''' Bhatia S and Sharma DK;Thermophilic desulfurization of dibenzothiophene<br />
and different petroleum oils by Klebsiellasp. 13T, 2011. Environ Sci Pollut Res 19:3491–3497</div>Rsaerhttp://2012.igem.org/Team:British_Columbia/DesulfurizationTeam:British Columbia/Desulfurization2012-10-04T01:58:47Z<p>Rsaer: </p>
<hr />
<div>{{Template:Team:British_Columbia_Header}}<br />
<html><br />
<style><br />
#break {width:950px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}<br />
#image {width:550px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}<br />
#note {width:380px;float:left; background-color: white; margin-left: 8px; margin-top:10px;}<br />
</style><br />
<br />
<div id=image><p align=center><img src="https://static.igem.org/mediawiki/2012/4/4d/Dsz_operon.png" width=400px></p></div><br />
<br />
<div id=note></br><br />
<p align=center><font face=arial narrow size=5><b>Bio-desulfurization: A Real-world Consortial Case Study</b></font></p><br />
<br />
Once we are able to tune our biological consortium it was time to test it on a system of economic relevance: the desulfurization of dibenzothiophene (DBT). DBT is a polyaromatic sulfur compound present in oil crudes whose combustion by oil-powered machines, such as automobiles, causes the release of sulfur dioxide (SO<sub>2</sub>) into the atmosphere. Sulfur dioxide is a principle source of acid rain and air pollution, and thus the removal of this compound from oil crudes provides benefits to the air quality and general environmental health of developed areas. DBT is highly resistant to hydrodesulfurization, the current process of removing sulphur compounds from crude oil, and thus novel methods of removing this compound are desired. Biodesulfurization of DBT is one means of removing such a compound. Specifically, the 4S pathway from Rhodococcus erythropolis IGTS8, which utilizes the gene products of the Dsz operon, is ideally suited for such a task because it removes DBT from crude oil with minimal impact on the energy density of the crude (i.e. the desirable compounds in the crude that actually serve as fuel, are preserved). As a result of the 4S pathway, DBT is converted to hydroxybiphenyl (HPB, Figure 1).</br></br><br />
</div><div id=break></br><br />
<div align="left"><font face=arial narrow size=4><b>To create a distributed metabolic network, the three catalytic genes of the Dsz operon were separated into three <i>Escherichia coli</i> strains developed for our tunable consortium. This is crucial to optimizing the metabolic potential of the consortium.</b></br></br></font></div></div><br />
<br />
<div id=note><br />
</br></br></br><p align=center><br />
<font face=arial narrow size=5><b>Why the Dsz operon?</b></font></p><font face=arial narrow><br />
<br />
We choose the Dsz operon for a number of reasons. As stated above, bio-desulfurization has the potential to help relieve serious environmental problem associated with DBT content of fossil fuels. However, the Dsz operon also has a number of advantages directly related to our consortium. First, the pathway has previously been expressed functionally in <i>E. coli</i>, allowing us to obtain a working copy of the pathway expressed in an <i>E. coli</i> monoculture (REF). This will let us directly compare the efficiency of the single cell pathway with our distributed metabolic network. Also, the availability of a working pathway in <i>E. coli</i> indicates that all of the required enzymes will be active when recombinantly expressed in our <i>E. coli</i> consortium strains. Finally, the desulfurization activity of the 4S pathway can be easily monitored and quantitated by HPLC analysis [2].</br></div><br />
<br />
<div id=image><img src="https://static.igem.org/mediawiki/2012/f/ff/Dsz_Consortium.png" width=500></div><br />
<br />
==References==<br />
<br />
'''[1]'''<br />
<br />
'''[2]''' Bhatia S and Sharma DK;Thermophilic desulfurization of dibenzothiophene<br />
and different petroleum oils by Klebsiellasp. 13T, 2011. Environ Sci Pollut Res 19:3491–3497</div>Rsaer