http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Achawdhry&year=&month=2012.igem.org - User contributions [en]2024-03-29T01:39:13ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Calgary/Project/OSCAR/DecarboxylationTeam:Calgary/Project/OSCAR/Decarboxylation2012-10-26T01:00:34Z<p>Achawdhry: </p>
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<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Decarboxylation|<br />
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CONTENT=<html><br />
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<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_OSCAR_Decarboxylation_Low-Res.png" style="float: right; padding: 10px; width: 300px;"></img><br />
<h2>Why Decarboxylation?</h2><br />
<br />
<p>Among the toxins found in the tailing ponds, naphthenic acids (NAs) are among the most harmful and the most common. Though there is great diversity within the NAs class of compounds, all share the common chemical feature of a carboxylic acid group. The carboxyl group is the primary cause for their toxicity, allowing these chemicals to traverse cell membranes and react with cellular materials (Frank <i>et al</i>. 2009). NAs are recalcitrant (not easily degraded), potentially harmful to the surrounding ecosystem (Clemente & Fedorak, 2005) and corrosive to extraction and transport equipment of petroleum materials (Slavcheva <i>et al</i>. 1999). Corrosion of pipelines leads to higher maintenance costs as well as the grim possibility of these and other toxins leaking into the environment. <br />
There is a need for methods to degrade NAs that are not prohibitively expensive or that would result in production of other hazardous chemicals.</p> <br />
<br />
<p>The main goal of OSCAR is to turn toxins like these into useable hydrocarbons by removing the carboxylic acid group(s) (Behar & Albrecht, 1984). <br />
<br />
Since NAs from petroleum deposits are a variable mixture, an enzymatic process with broad specificity is necessary. With the removal of the carboxylic acid moiety, we aim to produce alkanes suitable for use as fuel. The goal of this subproject was to find one or more suitable pathways to accomplish the decarboxylation of compounds such as NAs with the broadest specificity.</p><br />
<br />
<br />
<h2>The PetroBrick</h2><br />
<br />
<p>The 2011 Washington iGEM team developed the PetroBrick (<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025.</a>), a BioBrick consisting of two primary genes. These include acyl-ACP reductase (<i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a></i>), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (<i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a></i>), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain (Sukovich, 2010). Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that the fatty acids that the PetroBrick targets, have a very similar structure to NAs.</p><br />
<br />
</html>[[File:UCalgary-Fatty-Acids-vs-NAs.jpg|550px|centre|thumb|Figure 1: A comparison of the structure of fatty acids and naphthenic acids]]<html><br />
<br />
<p>We predicted that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the PetroBrick, we needed to verify that the PetroBrick would work in our hands as it did for the <a href="https://2011.igem.org/Team:Washington">2011 Washington team</a>.<br />
<br />
Figures 2 and 3 demonstrate the function of the PetroBrick.</p><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 2: Gas chromatograph of the differences in peak composition between an <i>E. coli</i> control and the PetroBrick. There was a large increase in a peak with a retention time of 12.25 min, suggesting that the PetroBrick was producing a new hydrocarbon compound.|650px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center|thumb|Figure 3: Mass spectra of the gas chromatograph peak at 12.25 min. This spectra suggests that the PetroBrick is selectively producing a C15 alkane, which resemble the results from the Washington 2011 iGEM team.|750px]]<html><br />
<br />
<p>With the PetroBrick able to successfully produce alkanes, we next wanted to test the PetroBrick on NAs to see if they could be selectively converted into alkanes! This experiment used commercial NAs fractions, which included a large number of different complex NAs compounds. </p><br />
<br />
<a name="Petrobrick"></a><h2>Successful conversion of NAs into hydrocarbons!</h2><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 4: The relative intensity of alkane production from NAs as measured with GC-MS over a retention time in <i>E. coli</i> with and without the PetroBrick . Concentrated NA standard was included for comparison of peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 5: Mass spectrums of alkanes and alkenes produced from NAs with <i>E. coli</i> containing the PetroBrick as in Figure 2. Relative intensity of mass-to -charge ratio (m/z) was compared.]]<html><br />
<br />
<p> Results from Figure 4 and 5 indicate that hydrocarbons were successfully produced from <i>E. coli</i> that contained the PetroBrick construct, as analysed with GC-MS. In Figure 2, <i>E. coli</i> containing the PetroBrick had higher hydrocarbon peaks than the <i>E. coli</i> without the PetroBrick. Not only was the PetroBrick able to degrade NAs into alkanes, the PetroBrick could produce alkenes (Figure 3), indicating that the PetroBrick worked as expected! </p><br />
<br />
<br />
<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)- Can we do better?</h2><br />
<br />
<p>Although we successfully used the PetroBrick to remove carboxyl groups from NAs, we wanted to improve on our results to get a higher yield and/or possibly target other compounds. One of our original concerns in using the PetroBrick to decarboyxlate NAs was that the first enzyme AAR only targeted fatty acids bound to ACP, and non-compatibility with NAs. Therefore, we searched for another enzyme carboxylic acid reductase (CAR) from <i>N. iowensis</i> known to perform a similar task as AAR - converting fatty acids to aldehydes, but with much lower specificity (He <i>et al</i>. 2004). Unlike AAR, CAR does not require covalent attachment to ACP, and likely to have broader substrate specificity. The use of CAR did require a second gene from <i>N. iowensis</i> called <i>Nocardia</i> phosphopantetheinyl transferase (<i>npt</i>) to append a 4’- phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian <i>et al.</i>, 2006).</p><br />
<br />
</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|700px|thumb|Figure 6: Mechanism of CAR catalysis. Taken from He <i>et al.</i>, 2004.]]<html><br />
<br />
<a name="OleT"></a><p>Another enzyme with the potential to remove carboxyl groups from NAs is olefin-forming fatty acid decarboxylase (<i><a href="http://partsregistry.org/Part:BBa_K902048:Experience">OleT</a></i>) from <i>Jeotgalicoccus</i> sp. ATCC 8456. <i>OleT</i> of the cytochrome P450 family acts on fatty acids, but does have low substrate specificity (Rude <i>et al</i>. 2011). Using <i>oleT</i> was beneficial because this single enzyme could do the job of the entire PetroBrick! Given our decarboxylation approach was valid, we started testing and comparing <i>oleT</i> to the PetroBrick.</p><br />
<br />
<h2> Progress so far </h2><br />
<br />
<p>Genes <i>car</i> and <i>npt</i> were cloned from the host organism <i>N. iowensis</i> (NRRL 5646). <i>car</i> was ligated into the pET vector and verified by a restriction digest while <i>npt</i> was cloned into pSB1C3 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902061">BBa_K902061</a>) and similarly verified.</p><br />
<br />
<p><i>car</i> was cloned into pET47b+ plasmid to remove six illegal cut-sites (one XbaI site, two EcoRI sites, and three NotI sites), as it was unsuitable for the BioBrick construction vectors. We first attempted to use a multi-site mutagenesis derived from the QuikChange<sup>®</sup> Multi-Site Directed Mutagenesis Kit, but had little success. Instead, a more time-consuming but effective series of conventional single-site <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis procedure</a> was used with the KAPA Hi-Fi polymerase. The XbaI and EcoRI sites were eliminated first so that <i>car</i> can be moved from the pET Vector and ligated into the PSB1C3 vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902062">BBa_K902062</a>). <br />
<br />
<p>The <i>oleT</i> was successfully amplified from the <i>Jeotgalicoccus</i> sp. ATCC 8456.<br />
<p> </p><br />
<p>Like <i>car</i>, <i>oleT</i> was inserted in a pET47b+ (Novagen) vector before placing it into a BioBrick vector, as two illegal cut sites adjacent to one another needed to be mutagenized. This part is now being ligated into pSB1C3. We are currently in the process of constructing all three parts under control of a <i>tetR</i> promoter and ribosomal binding site (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>), and then constructing these composite parts together as outlined below.</p><br />
<br />
<h2>Final testing constructs</h2><br />
<br />
<p>Final testing constructs are nearly complete. These are illustrated in Figure 7 and will allow us to compare the three different approaches. Unfortunately, Washington only sent us the PetroBrick and not the two individual components, we will have to compare a combination of the PetroBrick and <i>car/npt</i> to the PetroBrick alone and to <i>oleT</i>. </p><br />
<br />
<p></html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|centre|600px]]<html><br />
</html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|centre|280px|thumb|Figure 7: Final constructs required for validating and comparing different decarboxylation approaches]]<html></p><br />
<br />
<a name="TestingOleT"></a><h2> Testing OleT </h2><br />
<br />
<p>One major stumbling block in testing out <i>oleT</i> has been significant difficulty in trying to ligate it into a vector, which has prevented us from submitting it as a BioBrick. As such, we chose to try some assays on the host organism: <i>Jeotgalicoccus</i> sp. ATCC 8456. This way we could at least validate that this gene was functional before we had our BioBricks. We started by trying to verify the results by Rude <i>et al</i>., 2011, namely that OleT could convert fatty acids into alkenes. We grew up cultures based on this <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT_in_Validation_Assay">protocol</a> and used GC-MS to analyze any alkene production (Figure 8 and 9).</p><br />
<br />
<h2> Formation of alkanes by <i>Jeotgalicoccus</i> sp. ATCC 8456</h2><br />
<br />
</html>[[File:UofC_OleT_Assay_1.png|centre|650px|thumb|Figure 8. Gas chromatograph demonstrating the production of olefins (alkenes) from fatty acids as shown from the increase in the peak with a retention time of 14.7 min. The dramatic change in peak intensity at this point suggests that we are producing hydrocarbons.]]<br />
[[File:UofC_OleT_2nd_Assay.png|centre|700px|thumb|Figure 9. Mass spectra of the peak in Figure 8 at retention time 14.7 min. Demonstrating that this peak is an olefin, which is known to be produced in <i>Micrococcus</i>. This verifies our proof-of-concept that the <i>Micrococcus</i> species can degrade fatty acids into olefins. ]]<html><br />
<br />
<p>Based on the additional peak we saw in the gas chromatograph, we could show that our <i>E. coli</i> can produce alkenes with the <i>oleT</i>. This is may be an improvement over the PetroBrick since OleT is only one enzyme instead of two; however, future testing is still needed. Now that we have validated the function of OleT in producing alkenes, the next step is to test it out on complex naphthenic acids in order to compare it to the PetroBrick. This testing is still underway.</p><br />
<br />
</html><br />
<br />
}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Project/OSCAR/DecarboxylationTeam:Calgary/Project/OSCAR/Decarboxylation2012-10-26T00:59:00Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Decarboxylation|<br />
<br />
CONTENT=<html><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_OSCAR_Decarboxylation_Low-Res.png" style="float: right; padding: 10px; width: 300px;"></img><br />
<h2>Why Decarboxylation?</h2><br />
<br />
<p>Among the toxins found in the tailing ponds, naphthenic acids (NAs) are among the most harmful and the most common. Though there is great diversity within the NAs class of compounds, all share the common chemical feature of a carboxylic acid group. The carboxyl group is the primary cause for their toxicity, allowing these chemicals to traverse cell membranes and react with cellular materials (Frank <i>et al</i>. 2009). NAs are recalcitrant (not easily degraded), potentially harmful to the surrounding ecosystem (Clemente & Fedorak, 2005) and corrosive to extraction and transport equipment of petroleum materials (Slavcheva <i>et al</i>. 1999). Corrosion of pipelines leads to higher maintenance costs as well as the grim possibility of these and other toxins leaking into the environment. <br />
There is a need for methods to degrade NAs that are not prohibitively expensive or that would result in production of other hazardous chemicals.</p> <br />
<br />
<p>The main goal of OSCAR is to turn toxins like these into useable hydrocarbons by removing the carboxylic acid group(s) (Behar & Albrecht, 1984). <br />
<br />
Since NAs from petroleum deposits are a variable mixture, an enzymatic process with broad specificity is necessary. With the removal of the carboxylic acid moiety, we aim to produce alkanes suitable for use as fuel. The goal of this subproject was to find one or more suitable pathways to accomplish the decarboxylation of compounds such as NAs with the broadest specificity.</p><br />
<br />
<br />
<h2>The PetroBrick</h2><br />
<br />
<p>The 2011 Washington iGEM team developed the PetroBrick (<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025.</a>), a BioBrick consisting of two primary genes. These include acyl-ACP reductase (<i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a></i>), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (<i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a></i>), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain (Sukovich, 2010). Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that the fatty acids that the PetroBrick targets, have a very similar structure to NAs.</p><br />
<br />
</html>[[File:UCalgary-Fatty-Acids-vs-NAs.jpg|550px|centre|thumb|Figure 1: A comparison of the structure of fatty acids and naphthenic acids]]<html><br />
<br />
<p>We predicted that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the PetroBrick, we needed to verify that the PetroBrick would work in our hands as it did for the <a href="https://2011.igem.org/Team:Washington">2011 Washington team</a>.<br />
<br />
Figures 2 and 3 demonstrate the function of the PetroBrick.</p><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 2: Gas chromatograph of the differences in peak composition between an <i>E. coli</i> control and the PetroBrick. There was a large increase in a peak with a retention time of 12.25 min, suggesting that the PetroBrick was producing a new hydrocarbon compound.|650px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center|thumb|Figure 3: Mass spectra of the gas chromatograph peak at 12.25 min. This spectra suggests that the PetroBrick is selectively producing a C15 alkane, which resemble the results from the Washington 2011 iGEM team.|750px]]<html><br />
<br />
<p>With the PetroBrick able to successfully produce alkanes, we next wanted to test the PetroBrick on NAs to see if they could be selectively converted into alkanes! This experiment used commercial NAs fractions, which included a large number of different complex NAs compounds. </p><br />
<br />
<a name="Petrobrick"></a><h2>Successful conversion of NAs into hydrocarbons!</h2><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 4: The relative intensity of alkane production from NAs as measured with GC-MS over a retention time in <i>E. coli</i> with and without the PetroBrick . Concentrated NA standard was included for comparison of peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 5: Mass spectrums of alkanes and alkenes produced from NAs with <i>E. coli</i> containing the PetroBrick as in Figure 2. Relative intensity of mass-to -charge ratio (m/z) was compared.]]<html><br />
<br />
<p> Results from Figure 4 and 5 indicate that hydrocarbons were successfully produced from <i>E. coli</i> that contained the PetroBrick construct, as analysed with GC-MS. In Figure 2, <i>E. coli</i> containing the PetroBrick had higher hydrocarbon peaks than the <i>E. coli</i> without the PetroBrick. Not only was the PetroBrick able to degrade NAs into alkanes, the PetroBrick could produce alkenes (Figure 3), indicating that the PetroBrick worked as expected! </p><br />
<br />
<br />
<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)- Can we do better?</h2><br />
<br />
<p>Although we successfully used the PetroBrick to remove carboxyl groups from NAs, we wanted to improve on our results to get a higher yield and/or possibly target other compounds. One of our original concerns in using the PetroBrick to decarboyxlate NAs was that the first enzyme AAR only targeted fatty acids bound to ACP, and non-compatibility with NAs. Therefore, we searched for another enzyme carboxylic acid reductase (CAR) from <i>N. iowensis</i> known to perform a similar task as AAR - converting fatty acids to aldehydes, but with much lower specificity (He <i>et al</i>. 2004). Unlike AAR, CAR does not require covalent attachment to ACP, and likely to have broader substrate specificity. The use of CAR did require a second gene from <i>N. iowensis</i> called <i>Nocardia</i> phosphopantetheinyl transferase (<i>npt</i>) to append a 4’- phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian <i>et al.</i>, 2006).</p><br />
<br />
</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|700px|thumb|Figure 6: Mechanism of CAR catalysis. Taken from He <i>et al.</i>, 2004.]]<html><br />
<br />
<a name="OleT"></a><p>Another enzyme with the potential to remove carboxyl groups from NAs is olefin-forming fatty acid decarboxylase (<i><a href="http://partsregistry.org/Part:BBa_K902048:Experience">OleT</a></i>) from <i>Jeotgalicoccus</i> sp. ATCC 8456. OleT of the cytochrome P450 family acts on fatty acids, but does have low substrate specificity (Rude <i>et al</i>. 2011). Using <i>oleT</i> was beneficial because this single enzyme could do the job of the entire PetroBrick! Given our decarboxylation approach was valid, we started testing and comparing <i>oleT</i> to the PetroBrick.</p><br />
<br />
<h2> Progress so far </h2><br />
<br />
<p>Genes <i>car</i> and <i>npt</i> were cloned from the host organism <i>N. iowensis</i> (NRRL 5646). <i>car</i> was ligated into the pET vector and verified by a restriction digest while <i>npt</i> was cloned into pSB1C3 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902061">BBa_K902061</a>) and similarly verified.</p><br />
<br />
<p><i>car</i> was cloned into pET47b+ plasmid to remove six illegal cut-sites (one XbaI site, two EcoRI sites, and three NotI sites), as it was unsuitable for the BioBrick construction vectors. We first attempted to use a multi-site mutagenesis derived from the QuikChange<sup>®</sup> Multi-Site Directed Mutagenesis Kit, but had little success. Instead, a more time-consuming but effective series of conventional single-site <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis procedure</a> was used with the KAPA Hi-Fi polymerase. The XbaI and EcoRI sites were eliminated first so that <i>car</i> can be moved from the pET Vector and ligated into the PSB1C3 vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902062">BBa_K902062</a>). <br />
<br />
<p>The <i>oleT</i> was successfully amplified from the <i>Jeotgalicoccus</i> sp. ATCC 8456.<br />
<p> </p><br />
<p>Like <i>car</i>, <i>oleT</i> was inserted in a pET47b+ (Novagen) vector before placing it into a BioBrick vector, as two illegal cut sites adjacent to one another needed to be mutagenized. This part is now being ligated into pSB1C3. We are currently in the process of constructing all three parts under control of a <i>tetR</i> promoter and ribosomal binding site (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>), and then constructing these composite parts together as outlined below.</p><br />
<br />
<h2>Final testing constructs</h2><br />
<br />
<p>Final testing constructs are nearly complete. These are illustrated in Figure 7 and will allow us to compare the three different approaches. Unfortunately, Washington only sent us the PetroBrick and not the two individual components, we will have to compare a combination of the PetroBrick and <i>car/npt</i> to the PetroBrick alone and to <i>oleT</i>. </p><br />
<br />
<p></html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|centre|600px]]<html><br />
</html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|centre|280px|thumb|Figure 7: Final constructs required for validating and comparing different decarboxylation approaches]]<html></p><br />
<br />
<a name="TestingOleT"></a><h2> Testing OleT </h2><br />
<br />
<p>One major stumbling block in testing out <i>oleT</i> has been significant difficulty in trying to ligate it into a vector, which has prevented us from submitting it as a BioBrick. As such, we chose to try some assays on the host organism: <i>Jeotgalicoccus</i> sp. ATCC 8456. This way we could at least validate that this gene was functional before we had our BioBricks. We started by trying to verify the results by Rude <i>et al</i>., 2011, namely that OleT could convert fatty acids into alkenes. We grew up cultures based on this <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT_in_Validation_Assay">protocol</a> and used GC-MS to analyze any alkene production (Figure 8 and 9).</p><br />
<br />
<h2> Formation of alkanes by <i>Jeotgalicoccus</i> sp. ATCC 8456</h2><br />
<br />
</html>[[File:UofC_OleT_Assay_1.png|centre|650px|thumb|Figure 8. Gas chromatograph demonstrating the production of olefins (alkenes) from fatty acids as shown from the increase in the peak with a retention time of 14.7 min. The dramatic change in peak intensity at this point suggests that we are producing hydrocarbons.]]<br />
[[File:UofC_OleT_2nd_Assay.png|centre|700px|thumb|Figure 9. Mass spectra of the peak in Figure 8 at retention time 14.7 min. Demonstrating that this peak is an olefin, which is known to be produced in <i>Micrococcus</i>. This verifies our proof-of-concept that the <i>Micrococcus</i> species can degrade fatty acids into olefins. ]]<html><br />
<br />
<p>Based on the additional peak we saw in the gas chromatograph, we could show that our <i>E. coli</i> can produce alkenes with the <i>oleT</i>. This is may be an improvement over the PetroBrick since OleT is only one enzyme instead of two; however, future testing is still needed. Now that we have validated the function of OleT in producing alkenes, the next step is to test it out on complex naphthenic acids in order to compare it to the PetroBrick. This testing is still underway.</p><br />
<br />
</html><br />
<br />
}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Project/OSCAR/DecarboxylationTeam:Calgary/Project/OSCAR/Decarboxylation2012-10-26T00:58:23Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Decarboxylation|<br />
<br />
CONTENT=<html><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_OSCAR_Decarboxylation_Low-Res.png" style="float: right; padding: 10px; width: 300px;"></img><br />
<h2>Why Decarboxylation?</h2><br />
<br />
<p>Among the toxins found in the tailing ponds, naphthenic acids (NAs) are among the most harmful and the most common. Though there is great diversity within the NAs class of compounds, all share the common chemical feature of a carboxylic acid group. The carboxyl group is the primary cause for their toxicity, allowing these chemicals to traverse cell membranes and react with cellular materials (Frank <i>et al</i>. 2009). NAs are recalcitrant (not easily degraded), potentially harmful to the surrounding ecosystem (Clemente & Fedorak, 2005) and corrosive to extraction and transport equipment of petroleum materials (Slavcheva <i>et al</i>. 1999). Corrosion of pipelines leads to higher maintenance costs as well as the grim possibility of these and other toxins leaking into the environment. <br />
There is a need for methods to degrade NAs that are not prohibitively expensive or that would result in production of other hazardous chemicals.</p> <br />
<br />
<p>The main goal of OSCAR is to turn toxins like these into useable hydrocarbons by removing the carboxylic acid group(s) (Behar & Albrecht, 1984). <br />
<br />
Since NAs from petroleum deposits are a variable mixture, an enzymatic process with broad specificity is necessary. With the removal of the carboxylic acid moiety, we aim to produce alkanes suitable for use as fuel. The goal of this subproject was to find one or more suitable pathways to accomplish the decarboxylation of compounds such as NAs with the broadest specificity.</p><br />
<br />
<br />
<h2>The PetroBrick</h2><br />
<br />
<p>The 2011 Washington iGEM team developed the PetroBrick (<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025.</a>), a BioBrick consisting of two primary genes. These include acyl-ACP reductase (<i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a></i>), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (<i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a></i>), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain (Sukovich, 2010). Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that the fatty acids that the PetroBrick targets, have a very similar structure to NAs.</p><br />
<br />
</html>[[File:UCalgary-Fatty-Acids-vs-NAs.jpg|550px|centre|thumb|Figure 1: A comparison of the structure of fatty acids and naphthenic acids]]<html><br />
<br />
<p>We predicted that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the PetroBrick, we needed to verify that the PetroBrick would work in our hands as it did for the <a href="https://2011.igem.org/Team:Washington">2011 Washington team</a>.<br />
<br />
Figures 2 and 3 demonstrate the function of the PetroBrick.</p><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 2: Gas chromatograph of the differences in peak composition between an <i>E. coli</i> control and the PetroBrick. There was a large increase in a peak with a retention time of 12.25 min, suggesting that the PetroBrick was producing a new hydrocarbon compound.|650px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center|thumb|Figure 3: Mass spectra of the gas chromatograph peak at 12.25 min. This spectra suggests that the PetroBrick is selectively producing a C15 alkane, which resemble the results from the Washington 2011 iGEM team.|750px]]<html><br />
<br />
<p>With the PetroBrick able to successfully produce alkanes, we next wanted to test the PetroBrick on NAs to see if they could be selectively converted into alkanes! This experiment used commercial NAs fractions, which included a large number of different complex NAs compounds. </p><br />
<br />
<a name="Petrobrick"></a><h2>Successful conversion of NAs into hydrocarbons!</h2><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 4: The relative intensity of alkane production from NAs as measured with GC-MS over a retention time in <i>E. coli</i> with and without the PetroBrick . Concentrated NA standard was included for comparison of peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 5: Mass spectrums of alkanes and alkenes produced from NAs with <i>E. coli</i> containing the PetroBrick as in Figure 2. Relative intensity of mass-to -charge ratio (m/z) was compared.]]<html><br />
<br />
<p> Results from Figure 4 and 5 indicate that hydrocarbons were successfully produced from <i>E. coli</i> that contained the PetroBrick construct, as analysed with GC-MS. In Figure 2, <i>E. coli</i> containing the PetroBrick had higher hydrocarbon peaks than the <i>E. coli</i> without the PetroBrick. Not only was the PetroBrick able to degrade NAs into alkanes, the PetroBrick could produce alkenes (Figure 3), indicating that the PetroBrick worked as expected! </p><br />
<br />
<br />
<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)- Can we do better?</h2><br />
<br />
<p>Although we successfully used the PetroBrick to remove carboxyl groups from NAs, we wanted to improve on our results to get a higher yield and/or possibly target other compounds. One of our original concerns in using the PetroBrick to decarboyxlate NAs was that the first enzyme AAR only targeted fatty acids bound to ACP, and non-compatibility with NAs. Therefore, we searched for another enzyme carboxylic acid reductase (CAR) from <i>N. iowensis</i> known to perform a similar task as AAR - converting fatty acids to aldehydes, but with much lower specificity (He <i>et al</i>. 2004). Unlike AAR, CAR does not require covalent attachment to ACP, and likely to have broader substrate specificity. The use of CAR did require a second gene from <i>N. iowensis</i> called <i>Nocardia</i> phosphopantetheinyl transferase (<i>npt</i>) to append a 4’- phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian <i>et al.</i>, 2006).</p><br />
<br />
</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|700px|thumb|Figure 6: Mechanism of CAR catalysis. Taken from He <i>et al.</i>, 2004.]]<html><br />
<br />
<a name="OleT"></a><p>Another enzyme with the potential to remove carboxyl groups from NAs is olefin-forming fatty acid decarboxylase (<i><a href="http://partsregistry.org/Part:BBa_K902048:Experience">OleT</a></i>) from <i>Jeotgalicoccus</i> sp. ATCC 8456. OleT of the cytochrome P450 family acts on fatty acids, but does have low substrate specificity (Rude <i>et al</i>. 2011). Using <i>oleT<i> was beneficial because this single enzyme could do the job of the entire PetroBrick! Given our decarboxylation approach was valid, we started testing and comparing <i>oleT</i> to the PetroBrick.</p><br />
<br />
<h2> Progress so far </h2><br />
<br />
<p>Genes <i>car</i> and <i>npt</i> were cloned from the host organism <i>N. iowensis</i> (NRRL 5646). <i>car</i> was ligated into the pET vector and verified by a restriction digest while <i>npt</i> was cloned into pSB1C3 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902061">BBa_K902061</a>) and similarly verified.</p><br />
<br />
<p><i>car</i> was cloned into pET47b+ plasmid to remove six illegal cut-sites (one XbaI site, two EcoRI sites, and three NotI sites), as it was unsuitable for the BioBrick construction vectors. We first attempted to use a multi-site mutagenesis derived from the QuikChange<sup>®</sup> Multi-Site Directed Mutagenesis Kit, but had little success. Instead, a more time-consuming but effective series of conventional single-site <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis procedure</a> was used with the KAPA Hi-Fi polymerase. The XbaI and EcoRI sites were eliminated first so that <i>car</i> can be moved from the pET Vector and ligated into the PSB1C3 vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902062">BBa_K902062</a>). <br />
<br />
<p>The <i>oleT</i> was successfully amplified from the <i>Jeotgalicoccus</i> sp. ATCC 8456.<br />
<p> </p><br />
<p>Like <i>car</i>, <i>oleT</i> was inserted in a pET47b+ (Novagen) vector before placing it into a BioBrick vector, as two illegal cut sites adjacent to one another needed to be mutagenized. This part is now being ligated into pSB1C3. We are currently in the process of constructing all three parts under control of a <i>tetR</i> promoter and ribosomal binding site (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>), and then constructing these composite parts together as outlined below.</p><br />
<br />
<h2>Final testing constructs</h2><br />
<br />
<p>Final testing constructs are nearly complete. These are illustrated in Figure 7 and will allow us to compare the three different approaches. Unfortunately, Washington only sent us the PetroBrick and not the two individual components, we will have to compare a combination of the PetroBrick and <i>car/npt</i> to the PetroBrick alone and to <i>oleT</i>. </p><br />
<br />
<p></html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|centre|600px]]<html><br />
</html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|centre|280px|thumb|Figure 7: Final constructs required for validating and comparing different decarboxylation approaches]]<html></p><br />
<br />
<a name="TestingOleT"></a><h2> Testing OleT </h2><br />
<br />
<p>One major stumbling block in testing out <i>oleT</i> has been significant difficulty in trying to ligate it into a vector, which has prevented us from submitting it as a BioBrick. As such, we chose to try some assays on the host organism: <i>Jeotgalicoccus</i> sp. ATCC 8456. This way we could at least validate that this gene was functional before we had our BioBricks. We started by trying to verify the results by Rude <i>et al</i>., 2011, namely that OleT could convert fatty acids into alkenes. We grew up cultures based on this <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT_in_Validation_Assay">protocol</a> and used GC-MS to analyze any alkene production (Figure 8 and 9).</p><br />
<br />
<h2> Formation of alkanes by <i>Jeotgalicoccus</i> sp. ATCC 8456</h2><br />
<br />
</html>[[File:UofC_OleT_Assay_1.png|centre|650px|thumb|Figure 8. Gas chromatograph demonstrating the production of olefins (alkenes) from fatty acids as shown from the increase in the peak with a retention time of 14.7 min. The dramatic change in peak intensity at this point suggests that we are producing hydrocarbons.]]<br />
[[File:UofC_OleT_2nd_Assay.png|centre|700px|thumb|Figure 9. Mass spectra of the peak in Figure 8 at retention time 14.7 min. Demonstrating that this peak is an olefin, which is known to be produced in <i>Micrococcus</i>. This verifies our proof-of-concept that the <i>Micrococcus</i> species can degrade fatty acids into olefins. ]]<html><br />
<br />
<p>Based on the additional peak we saw in the gas chromatograph, we could show that our <i>E. coli</i> can produce alkenes with the <i>oleT</i>. This is may be an improvement over the PetroBrick since OleT is only one enzyme instead of two; however, future testing is still needed. Now that we have validated the function of OleT in producing alkenes, the next step is to test it out on complex naphthenic acids in order to compare it to the PetroBrick. This testing is still underway.</p><br />
<br />
</html><br />
<br />
}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Project/OSCAR/DecarboxylationTeam:Calgary/Project/OSCAR/Decarboxylation2012-10-26T00:56:35Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Decarboxylation|<br />
<br />
CONTENT=<html><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_OSCAR_Decarboxylation_Low-Res.png" style="float: right; padding: 10px; width: 300px;"></img><br />
<h2>Why Decarboxylation?</h2><br />
<br />
<p>Among the toxins found in the tailing ponds, naphthenic acids (NAs) are among the most harmful and the most common. Though there is great diversity within the NAs class of compounds, all share the common chemical feature of a carboxylic acid group. The carboxyl group is the primary cause for their toxicity, allowing these chemicals to traverse cell membranes and react with cellular materials (Frank <i>et al</i>. 2009). NAs are recalcitrant (not easily degraded), potentially harmful to the surrounding ecosystem (Clemente & Fedorak, 2005) and corrosive to extraction and transport equipment of petroleum materials (Slavcheva <i>et al</i>. 1999). Corrosion of pipelines leads to higher maintenance costs as well as the grim possibility of these and other toxins leaking into the environment. <br />
There is a need for methods to degrade NAs that are not prohibitively expensive or that would result in production of other hazardous chemicals.</p> <br />
<br />
<p>The main goal of OSCAR is to turn toxins like these into useable hydrocarbons by removing the carboxylic acid group(s) (Behar & Albrecht, 1984). <br />
<br />
Since NAs from petroleum deposits are a variable mixture, an enzymatic process with broad specificity is necessary. With the removal of the carboxylic acid moiety, we aim to produce alkanes suitable for use as fuel. The goal of this subproject was to find one or more suitable pathways to accomplish the decarboxylation of compounds such as NAs with the broadest specificity.</p><br />
<br />
<br />
<h2>The PetroBrick</h2><br />
<br />
<p>The 2011 Washington iGEM team developed the PetroBrick (<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025.</a>), a BioBrick consisting of two primary genes. These include acyl-ACP reductase (<i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a></i>), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (<i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a></i>), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain (Sukovich, 2010). Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that the fatty acids that the PetroBrick targets, have a very similar structure to NAs.</p><br />
<br />
</html>[[File:UCalgary-Fatty-Acids-vs-NAs.jpg|550px|centre|thumb|Figure 1: A comparison of the structure of fatty acids and naphthenic acids]]<html><br />
<br />
<p>We predicted that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the PetroBrick, we needed to verify that the PetroBrick would work in our hands as it did for the <a href="https://2011.igem.org/Team:Washington">2011 Washington team</a>.<br />
<br />
Figures 2 and 3 demonstrate the function of the PetroBrick.</p><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 2: Gas chromatograph of the differences in peak composition between an <i>E. coli</i> control and the PetroBrick. There was a large increase in a peak with a retention time of 12.25 min, suggesting that the PetroBrick was producing a new hydrocarbon compound.|650px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center|thumb|Figure 3: Mass spectra of the gas chromatograph peak at 12.25 min. This spectra suggests that the PetroBrick is selectively producing a C15 alkane, which resemble the results from the Washington 2011 iGEM team.|750px]]<html><br />
<br />
<p>With the PetroBrick able to successfully produce alkanes, we next wanted to test the PetroBrick on NAs to see if they could be selectively converted into alkanes! This experiment used commercial NAs fractions, which included a large number of different complex NAs compounds. </p><br />
<br />
<a name="Petrobrick"></a><h2>Successful conversion of NAs into hydrocarbons!</h2><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 4: The relative intensity of alkane production from NAs as measured with GC-MS over a retention time in <i>E. coli</i> with and without the PetroBrick . Concentrated NA standard was included for comparison of peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 5: Mass spectrums of alkanes and alkenes produced from NAs with <i>E. coli</i> containing the PetroBrick as in Figure 2. Relative intensity of mass-to -charge ratio (m/z) was compared.]]<html><br />
<br />
<p> Results from Figure 4 and 5 indicate that hydrocarbons were successfully produced from <i>E. coli</i> that contained the PetroBrick construct, as analysed with GC-MS. In Figure 2, <i>E. coli</i> containing the PetroBrick had higher hydrocarbon peaks than the <i>E. coli</i> without the PetroBrick. Not only was the PetroBrick able to degrade NAs into alkanes, the PetroBrick could produce alkenes (Figure 3), indicating that the PetroBrick worked as expected! </p><br />
<br />
<br />
<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)- Can we do better?</h2><br />
<br />
<p>Although we successfully used the PetroBrick to remove carboxyl groups from NAs, we wanted to improve on our results to get a higher yield and/or possibly target other compounds. One of our original concerns in using the PetroBrick to decarboyxlate NAs was that the first enzyme AAR only targeted fatty acids bound to ACP, and non-compatibility with NAs. Therefore, we searched for another enzyme carboxylic acid reductase (CAR) from <i>N. iowensis</i> known to perform a similar task as AAR - converting fatty acids to aldehydes, but with much lower specificity (He <i>et al</i>. 2004). Unlike AAR, CAR does not require covalent attachment to ACP, and likely to have broader substrate specificity. The use of CAR did require a second gene from <i>N. iowensis</i> called <i>Nocardia</i> phosphopantetheinyl transferase (<i>npt</i>) to append a 4’- phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian <i>et al.</i>, 2006).</p><br />
<br />
</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|700px|thumb|Figure 6: Mechanism of CAR catalysis. Taken from He <i>et al.</i>, 2004.]]<html><br />
<br />
<a name="OleT"></a><p>Another enzyme with the potential to remove carboxyl groups from NAs is olefin-forming fatty acid decarboxylase (<i><a href="http://partsregistry.org/Part:BBa_K902048:Experience">OleT</a></i>) from <i>Jeotgalicoccus</i> sp. ATCC 8456. <i>oleT</i> of the cytochrome P450 family acts on fatty acids, but does have low substrate specificity (Rude <i>et al</i>. 2011). Using <i>oleT<i> was beneficial because this single enzyme could do the job of the entire PetroBrick! Given our decarboxylation approach was valid, we started testing and comparing <i>oleT</i> to the PetroBrick.</p><br />
<br />
<h2> Progress so far </h2><br />
<br />
<p>Genes <i>car</i> and <i>npt</i> were cloned from the host organism <i>N. iowensis</i> (NRRL 5646). <i>car</i> was ligated into the pET vector and verified by a restriction digest while <i>npt</i> was cloned into pSB1C3 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902061">BBa_K902061</a>) and similarly verified.</p><br />
<br />
<p><i>car</i> was cloned into pET47b+ plasmid to remove six illegal cut-sites (one XbaI site, two EcoRI sites, and three NotI sites), as it was unsuitable for the BioBrick construction vectors. We first attempted to use a multi-site mutagenesis derived from the QuikChange<sup>®</sup> Multi-Site Directed Mutagenesis Kit, but had little success. Instead, a more time-consuming but effective series of conventional single-site <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis procedure</a> was used with the KAPA Hi-Fi polymerase. The XbaI and EcoRI sites were eliminated first so that <i>car</i> can be moved from the pET Vector and ligated into the PSB1C3 vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902062">BBa_K902062</a>). <br />
<br />
<p>The <i>oleT</i> was successfully amplified from the <i>Jeotgalicoccus</i> sp. ATCC 8456.<br />
<p> </p><br />
<p>Like <i>car</i>, <i>oleT</i> was inserted in a pET47b+ (Novagen) vector before placing it into a BioBrick vector, as two illegal cut sites adjacent to one another needed to be mutagenized. This part is now being ligated into pSB1C3. We are currently in the process of constructing all three parts under control of a <i>tetR</i> promoter and ribosomal binding site (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>), and then constructing these composite parts together as outlined below.</p><br />
<br />
<h2>Final testing constructs</h2><br />
<br />
<p>Final testing constructs are nearly complete. These are illustrated in Figure 7 and will allow us to compare the three different approaches. Unfortunately, Washington only sent us the PetroBrick and not the two individual components, we will have to compare a combination of the PetroBrick and <i>car/npt</i> to the PetroBrick alone and to <i>oleT</i>. </p><br />
<br />
<p></html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|centre|600px]]<html><br />
</html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|centre|280px|thumb|Figure 7: Final constructs required for validating and comparing different decarboxylation approaches]]<html></p><br />
<br />
<a name="TestingOleT"></a><h2> Testing OleT </h2><br />
<br />
<p>One major stumbling block in testing out <i>oleT</i> has been significant difficulty in trying to ligate it into a vector, which has prevented us from submitting it as a BioBrick. As such, we chose to try some assays on the host organism: <i>Jeotgalicoccus</i> sp. ATCC 8456. This way we could at least validate that this gene was functional before we had our BioBricks. We started by trying to verify the results by Rude <i>et al</i>., 2011, namely that OleT could convert fatty acids into alkenes. We grew up cultures based on this <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT_in_Validation_Assay">protocol</a> and used GC-MS to analyze any alkene production (Figure 8 and 9).</p><br />
<br />
<h2> Formation of alkanes by <i>Jeotgalicoccus</i> sp. ATCC 8456</h2><br />
<br />
</html>[[File:UofC_OleT_Assay_1.png|centre|650px|thumb|Figure 8. Gas chromatograph demonstrating the production of olefins (alkenes) from fatty acids as shown from the increase in the peak with a retention time of 14.7 min. The dramatic change in peak intensity at this point suggests that we are producing hydrocarbons.]]<br />
[[File:UofC_OleT_2nd_Assay.png|centre|700px|thumb|Figure 9. Mass spectra of the peak in Figure 8 at retention time 14.7 min. Demonstrating that this peak is an olefin, which is known to be produced in <i>Micrococcus</i>. This verifies our proof-of-concept that the <i>Micrococcus</i> species can degrade fatty acids into olefins. ]]<html><br />
<br />
<p>Based on the additional peak we saw in the gas chromatograph, we could show that our <i>E. coli</i> can produce alkenes with the <i>oleT</i>. This is may be an improvement over the PetroBrick since OleT is only one enzyme instead of two; however, future testing is still needed. Now that we have validated the function of OleT in producing alkenes, the next step is to test it out on complex naphthenic acids in order to compare it to the PetroBrick. This testing is still underway.</p><br />
<br />
</html><br />
<br />
}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Project/OSCAR/DecarboxylationTeam:Calgary/Project/OSCAR/Decarboxylation2012-10-26T00:55:08Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Decarboxylation|<br />
<br />
CONTENT=<html><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_OSCAR_Decarboxylation_Low-Res.png" style="float: right; padding: 10px; width: 300px;"></img><br />
<h2>Why Decarboxylation?</h2><br />
<br />
<p>Among the toxins found in the tailing ponds, naphthenic acids (NAs) are among the most harmful and the most common. Though there is great diversity within the NAs class of compounds, all share the common chemical feature of a carboxylic acid group. The carboxyl group is the primary cause for their toxicity, allowing these chemicals to traverse cell membranes and react with cellular materials (Frank <i>et al</i>. 2009). NAs are recalcitrant (not easily degraded), potentially harmful to the surrounding ecosystem (Clemente & Fedorak, 2005) and corrosive to extraction and transport equipment of petroleum materials (Slavcheva <i>et al</i>. 1999). Corrosion of pipelines leads to higher maintenance costs as well as the grim possibility of these and other toxins leaking into the environment. <br />
There is a need for methods to degrade NAs that are not prohibitively expensive or that would result in production of other hazardous chemicals.</p> <br />
<br />
<p>The main goal of OSCAR is to turn toxins like these into useable hydrocarbons by removing the carboxylic acid group(s) (Behar & Albrecht, 1984). <br />
<br />
Since NAs from petroleum deposits are a variable mixture, an enzymatic process with broad specificity is necessary. With the removal of the carboxylic acid moiety, we aim to produce alkanes suitable for use as fuel. The goal of this subproject was to find one or more suitable pathways to accomplish the decarboxylation of compounds such as NAs with the broadest specificity.</p><br />
<br />
<br />
<h2>The PetroBrick</h2><br />
<br />
<p>The 2011 Washington iGEM team developed the PetroBrick (<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025.</a>), a BioBrick consisting of two primary genes. These include acyl-ACP reductase (<i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a></i>), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (<i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a></i>), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain (Sukovich, 2010). Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that the fatty acids that the PetroBrick targets, have a very similar structure to NAs.</p><br />
<br />
</html>[[File:UCalgary-Fatty-Acids-vs-NAs.jpg|550px|centre|thumb|Figure 1: A comparison of the structure of fatty acids and naphthenic acids]]<html><br />
<br />
<p>We predicted that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the PetroBrick, we needed to verify that the PetroBrick would work in our hands as it did for the <a href="https://2011.igem.org/Team:Washington">2011 Washington team</a>.<br />
<br />
Figures 2 and 3 demonstrate the function of the PetroBrick.</p><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 2: Gas chromatograph of the differences in peak composition between an <i>E. coli</i> control and the PetroBrick. There was a large increase in a peak with a retention time of 12.25 min, suggesting that the PetroBrick was producing a new hydrocarbon compound.|650px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center|thumb|Figure 3: Mass spectra of the gas chromatograph peak at 12.25 min. This spectra suggests that the PetroBrick is selectively producing a C15 alkane, which resemble the results from the Washington 2011 iGEM team.|750px]]<html><br />
<br />
<p>With the PetroBrick able to successfully produce alkanes, we next wanted to test the PetroBrick on NAs to see if they could be selectively converted into alkanes! This experiment used commercial NAs fractions, which included a large number of different complex NAs compounds. </p><br />
<br />
<a name="Petrobrick"></a><h2>Successful conversion of NAs into hydrocarbons!</h2><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 4: The relative intensity of alkane production from NAs as measured with GC-MS over a retention time in <i>E. coli</i> with and without the PetroBrick . Concentrated NA standard was included for comparison of peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 5: Mass spectrums of alkanes and alkenes produced from NAs with <i>E. coli</i> containing the PetroBrick as in Figure 2. Relative intensity of mass-to -charge ratio (m/z) was compared.]]<html><br />
<br />
<p> Results from Figure 4 and 5 indicate that hydrocarbons were successfully produced from <i>E. coli</i> that contained the PetroBrick construct, as analysed with GC-MS. In Figure 2, <i>E. coli</i> containing the PetroBrick had higher hydrocarbon peaks than the <i>E. coli</i> without the PetroBrick. Not only was the PetroBrick able to degrade NAs into alkanes, the PetroBrick could produce alkenes (Figure 3), indicating that the PetroBrick worked as expected! </p><br />
<br />
<br />
<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)- Can we do better?</h2><br />
<br />
<p>Although we successfully used the PetroBrick to remove carboxyl groups from NAs, we wanted to improve on our results to get a higher yield and/or possibly target other compounds. One of our original concerns in using the PetroBrick to decarboyxlate NAs was that the first enzyme AAR only targeted fatty acids bound to ACP, and non-compatibility with NAs. Therefore, we searched for another enzyme carboxylic acid reductase (CAR) from <i>N. iowensis</i> known to perform a similar task as AAR - converting fatty acids to aldehydes, but with much lower specificity (He <i>et al</i>. 2004). Unlike AAR, CAR does not require covalent attachment to ACP, and likely to have broader substrate specificity. The use of CAR did require a second gene from <i>N. iowensis</i> called <i>Nocardia</i> phosphopantetheinyl transferase (<i>npt</i>) to append a 4’- phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian <i>et al.</i>, 2006).</p><br />
<br />
</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|700px|thumb|Figure 6: Mechanism of CAR catalysis. Taken from He <i>et al.</i>, 2004.]]<html><br />
<br />
<a name="OleT"></a><p>Another enzyme with the potential to remove carboxyl groups from NAs is olefin-forming fatty acid decarboxylase (<i><a href="http://partsregistry.org/Part:BBa_K902048:Experience">OleT</a></i>) from <i>Jeotgalicoccus</i> sp. ATCC 8456. <i>OleT<i> of the cytochrome P450 family acts on fatty acids, but does have low substrate specificity (Rude <i>et al</i>. 2011). Using <i>oleT<i> was beneficial because this single enzyme could do the job of the entire PetroBrick! Given our decarboxylation approach was valid, we started testing and comparing <i>oleT</i> to the PetroBrick.</p><br />
<br />
<h2> Progress so far </h2><br />
<br />
<p>Genes <i>car</i> and <i>npt</i> were cloned from the host organism <i>N. iowensis</i> (NRRL 5646). <i>car</i> was ligated into the pET vector and verified by a restriction digest while <i>npt</i> was cloned into pSB1C3 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902061">BBa_K902061</a>) and similarly verified.</p><br />
<br />
<p><i>car</i> was cloned into pET47b+ plasmid to remove six illegal cut-sites (one XbaI site, two EcoRI sites, and three NotI sites), as it was unsuitable for the BioBrick construction vectors. We first attempted to use a multi-site mutagenesis derived from the QuikChange<sup>®</sup> Multi-Site Directed Mutagenesis Kit, but had little success. Instead, a more time-consuming but effective series of conventional single-site <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis procedure</a> was used with the KAPA Hi-Fi polymerase. The XbaI and EcoRI sites were eliminated first so that <i>car</i> can be moved from the pET Vector and ligated into the PSB1C3 vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902062">BBa_K902062</a>). <br />
<br />
<p>The <i>oleT</i> was successfully amplified from the <i>Jeotgalicoccus</i> sp. ATCC 8456.<br />
<p> </p><br />
<p>Like <i>car</i>, <i>oleT</i> was inserted in a pET47b+ (Novagen) vector before placing it into a BioBrick vector, as two illegal cut sites adjacent to one another needed to be mutagenized. This part is now being ligated into pSB1C3. We are currently in the process of constructing all three parts under control of a <i>tetR</i> promoter and ribosomal binding site (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>), and then constructing these composite parts together as outlined below.</p><br />
<br />
<h2>Final testing constructs</h2><br />
<br />
<p>Final testing constructs are nearly complete. These are illustrated in Figure 7 and will allow us to compare the three different approaches. Unfortunately, Washington only sent us the PetroBrick and not the two individual components, we will have to compare a combination of the PetroBrick and <i>car/npt</i> to the PetroBrick alone and to <i>oleT</i>. </p><br />
<br />
<p></html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|centre|600px]]<html><br />
</html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|centre|280px|thumb|Figure 7: Final constructs required for validating and comparing different decarboxylation approaches]]<html></p><br />
<br />
<a name="TestingOleT"></a><h2> Testing OleT </h2><br />
<br />
<p>One major stumbling block in testing out <i>oleT</i> has been significant difficulty in trying to ligate it into a vector, which has prevented us from submitting it as a BioBrick. As such, we chose to try some assays on the host organism: <i>Jeotgalicoccus</i> sp. ATCC 8456. This way we could at least validate that this gene was functional before we had our BioBricks. We started by trying to verify the results by Rude <i>et al</i>., 2011, namely that OleT could convert fatty acids into alkenes. We grew up cultures based on this <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT_in_Validation_Assay">protocol</a> and used GC-MS to analyze any alkene production (Figure 8 and 9).</p><br />
<br />
<h2> Formation of alkanes by <i>Jeotgalicoccus</i> sp. ATCC 8456</h2><br />
<br />
</html>[[File:UofC_OleT_Assay_1.png|centre|650px|thumb|Figure 8. Gas chromatograph demonstrating the production of olefins (alkenes) from fatty acids as shown from the increase in the peak with a retention time of 14.7 min. The dramatic change in peak intensity at this point suggests that we are producing hydrocarbons.]]<br />
[[File:UofC_OleT_2nd_Assay.png|centre|700px|thumb|Figure 9. Mass spectra of the peak in Figure 8 at retention time 14.7 min. Demonstrating that this peak is an olefin, which is known to be produced in <i>Micrococcus</i>. This verifies our proof-of-concept that the <i>Micrococcus</i> species can degrade fatty acids into olefins. ]]<html><br />
<br />
<p>Based on the additional peak we saw in the gas chromatograph, we could show that our <i>E. coli</i> can produce alkenes with the <i>oleT</i>. This is may be an improvement over the PetroBrick since OleT is only one enzyme instead of two; however, future testing is still needed. Now that we have validated the function of OleT in producing alkenes, the next step is to test it out on complex naphthenic acids in order to compare it to the PetroBrick. This testing is still underway.</p><br />
<br />
</html><br />
<br />
}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Project/OSCAR/DecarboxylationTeam:Calgary/Project/OSCAR/Decarboxylation2012-10-26T00:26:24Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Decarboxylation|<br />
<br />
CONTENT=<html><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_OSCAR_Decarboxylation_Low-Res.png" style="float: right; padding: 10px; width: 300px;"></img><br />
<h2>Why Decarboxylation?</h2><br />
<br />
<p>Among the toxins found in the tailing ponds, naphthenic acids (NAs) are among the most harmful and the most common. Though there is great diversity within the NAs class of compounds, all share the common chemical feature of a carboxylic acid group. The carboxyl group is the primary cause for their toxicity, allowing these chemicals to traverse cell membranes and react with cellular materials (Frank <i>et al</i>. 2009). NAs are recalcitrant (not easily degraded), potentially harmful to the surrounding ecosystem (Clemente & Fedorak, 2005) and corrosive to extraction and transport equipment of petroleum materials (Slavcheva <i>et al</i>. 1999). Corrosion of pipelines leads to higher maintenance costs as well as the grim possibility of these and other toxins leaking into the environment. <br />
There is a need for methods to degrade NAs that are not prohibitively expensive or that would result in production of other hazardous chemicals.</p> <br />
<br />
<p>The main goal of OSCAR is to turn toxins like these into useable hydrocarbons by removing the carboxylic acid group(s) (Behar & Albrecht, 1984). <br />
<br />
Since NAs from petroleum deposits are a variable mixture, an enzymatic process with broad specificity is necessary. With the removal of the carboxylic acid moiety, we aim to produce alkanes suitable for use as fuel. The goal of this subproject was to find one or more suitable pathways to accomplish the decarboxylation of compounds such as NAs with the broadest specificity.</p><br />
<br />
<br />
<h2>The PetroBrick</h2><br />
<br />
<p>The 2011 Washington iGEM team developed the PetroBrick (<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025.</a>), a BioBrick consisting of two primary genes. These include acyl-ACP reductase (<i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a></i>), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (<i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a></i>), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain (Sukovich, 2010). Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that the fatty acids that the PetroBrick targets, have a very similar structure to NAs.</p><br />
<br />
</html>[[File:UCalgary-Fatty-Acids-vs-NAs.jpg|550px|centre|thumb|Figure 1: A comparison of the structure of fatty acids and naphthenic acids]]<html><br />
<br />
<p>We predicted that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the PetroBrick, we needed to verify that the PetroBrick would work in our hands as it did for the <a href="https://2011.igem.org/Team:Washington">2011 Washington team</a>.<br />
<br />
Figures 2 and 3 demonstrate the function of the PetroBrick.</p><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 2: Gas chromatograph of the differences in peak composition between an <i>E. coli</i> control and the PetroBrick. There was a large increase in a peak with a retention time of 12.25 min, suggesting that the PetroBrick was producing a new hydrocarbon compound.|650px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center|thumb|Figure 3: Mass spectra of the gas chromatograph peak at 12.25 min. This spectra suggests that the PetroBrick is selectively producing a C15 alkane, which resemble the results from the Washington 2011 iGEM team.|750px]]<html><br />
<br />
<p>With the PetroBrick able to successfully produce alkanes, we next wanted to test the PetroBrick on NAs to see if they could be selectively converted into alkanes! This experiment used commercial NAs fractions, which included a large number of different complex NAs compounds. </p><br />
<br />
<a name="Petrobrick"></a><h2>Successful conversion of NAs into hydrocarbons!</h2><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 4: The relative intensity of alkane production from NAs as measured with GC-MS over a retention time in <i>E. coli</i> with and without the PetroBrick . Concentrated NA standard was included for comparison of peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 5: Mass spectrums of alkanes and alkenes produced from NAs with <i>E. coli</i> containing the PetroBrick as in Figure 2. Relative intensity of mass-to -charge ratio (m/z) was compared.]]<html><br />
<br />
<p> Results from Figure 4 and 5 indicate that hydrocarbons were successfully produced from <i>E. coli</i> that contained the PetroBrick construct, as analysed with GC-MS. In Figure 2, <i>E. coli</i> containing the PetroBrick had higher hydrocarbon peaks than the <i>E. coli</i> without the PetroBrick. Not only was the PetroBrick able to degrade NAs into alkanes, the PetroBrick could produce alkenes (Figure 3), indicating that the PetroBrick worked as expected! </p><br />
<br />
<br />
<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)- Can we do better?</h2><br />
<br />
<p>Although we successfully used the PetroBrick to remove carboxyl groups from NAs, we wanted to improve on our results to get a higher yield and/or possibly target other compounds. One of our original concerns in using the PetroBrick to decarboyxlate NAs was that the first enzyme AAR only targeted fatty acids bound to ACP, and non-compatibility with NAs. Therefore, we searched for another enzyme carboxylic acid reductase (CAR) from <i>N. iowensis</i> known to perform a similar task as AAR - converting fatty acids to aldehydes, but with much lower specificity (He <i>et al</i>. 2004). Unlike AAR, CAR does not require covalent attachment to ACP, and likely to have broader substrate specificity. The use of CAR did require a second gene from <i>N. iowensis</i> called <i>Nocardia</i> phosphopantetheinyl transferase (<i>npt</i>) to append a 4’- phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian <i>et al.</i>, 2006).</p><br />
<br />
</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|700px|thumb|Figure 6: Mechanism of CAR catalysis. Taken from He <i>et al.</i>, 2004.]]<html><br />
<br />
<a name="OleT"></a><p>Another enzyme with the potential to remove carboxyl groups from NAs is olefin-forming fatty acid decarboxylase (OleT) from <i>Jeotgalicoccus</i> sp. ATCC 8456. OleT of the cytochrome P450 family acts on fatty acids, but does have low substrate specificity (Rude <i>et al</i>. 2011). Using OleT was beneficial because this single enzyme could do the job of the entire PetroBrick! Given our decarboxylation approach was valid, we started testing and comparing <i>oleT</i> to the PetroBrick.</p><br />
<br />
<h2> Progress so far </h2><br />
<br />
<p>Genes <i>car</i> and <i>npt</i> were cloned from the host organism <i>N. iowensis</i> (NRRL 5646). <i>car</i> was ligated into the pET vector and verified by a restriction digest while <i>npt</i> was cloned into pSB1C3 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902061">BBa_K902061</a>) and similarly verified.</p><br />
<br />
<p><i>car</i> was cloned into pET47b+ plasmid to remove six illegal cut-sites (one XbaI site, two EcoRI sites, and three NotI sites), as it was unsuitable for the BioBrick construction vectors. We first attempted to use a multi-site mutagenesis derived from the QuikChange<sup>®</sup> Multi-Site Directed Mutagenesis Kit, but had little success. Instead, a more time-consuming but effective series of conventional single-site <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis procedure</a> was used with the KAPA Hi-Fi polymerase. The XbaI and EcoRI sites were eliminated first so that <i>car</i> can be moved from the pET Vector and ligated into the PSB1C3 vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902062">BBa_K902062</a>). <br />
<br />
<p>The <i>oleT</i> was successfully amplified from the <i>Jeotgalicoccus</i> sp. ATCC 8456.<br />
<p> </p><br />
<p>Like <i>car</i>, <i>oleT</i> was inserted in a pET47b+ (Novagen) vector before placing it into a BioBrick vector, as two illegal cut sites adjacent to one another needed to be mutagenized. This part is now being ligated into pSB1C3. We are currently in the process of constructing all three parts under control of a <i>tetR</i> promoter and ribosomal binding site (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>), and then constructing these composite parts together as outlined below.</p><br />
<br />
<h2>Final testing constructs</h2><br />
<br />
<p>Final testing constructs are nearly complete. These are illustrated in Figure 7 and will allow us to compare the three different approaches. Unfortunately, Washington only sent us the PetroBrick and not the two individual components, we will have to compare a combination of the PetroBrick and <i>car/npt</i> to the PetroBrick alone and to <i>oleT</i>. </p><br />
<br />
<p></html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|centre|600px]]<html><br />
</html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|centre|280px|thumb|Figure 7: Final constructs required for validating and comparing different decarboxylation approaches]]<html></p><br />
<br />
<a name="TestingOleT"></a><h2> Testing OleT </h2><br />
<br />
<p>One major stumbling block in testing out <i>oleT</i> has been significant difficulty in trying to ligate it into a vector, which has prevented us from submitting it as a BioBrick. As such, we chose to try some assays on the host organism: <i>Jeotgalicoccus</i> sp. ATCC 8456. This way we could at least validate that this gene was functional before we had our BioBricks. We started by trying to verify the results by Rude <i>et al</i>., 2011, namely that OleT could convert fatty acids into alkenes. We grew up cultures based on this <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT_in_Validation_Assay">protocol</a> and used GC-MS to analyze any alkene production (Figure 8 and 9).</p><br />
<br />
<h2> Formation of alkanes by <i>Jeotgalicoccus</i> sp. ATCC 8456</h2><br />
<br />
</html>[[File:UofC_OleT_Assay_1.png|centre|650px|thumb|Figure 8. Gas chromatograph demonstrating the production of olefins (alkenes) from fatty acids as shown from the increase in the peak with a retention time of 14.7 min. The dramatic change in peak intensity at this point suggests that we are producing hydrocarbons.]]<br />
[[File:UofC_OleT_2nd_Assay.png|centre|700px|thumb|Figure 9. Mass spectra of the peak in Figure 8 at retention time 14.7 min. Demonstrating that this peak is an olefin, which is known to be produced in <i>Micrococcus</i>. This verifies our proof-of-concept that the <i>Micrococcus</i> species can degrade fatty acids into olefins. ]]<html><br />
<br />
<p>Based on the additional peak we saw in the gas chromatograph, we could show that our <i>E. coli</i> can produce alkenes with the <i>oleT</i>. This is may be an improvement over the PetroBrick since OleT is only one enzyme instead of two; however, future testing is still needed. Now that we have validated the function of OleT in producing alkenes, the next step is to test it out on complex naphthenic acids in order to compare it to the PetroBrick. This testing is still underway.</p><br />
<br />
</html><br />
<br />
}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Project/OSCAR/DecarboxylationTeam:Calgary/Project/OSCAR/Decarboxylation2012-10-26T00:25:54Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Decarboxylation|<br />
<br />
CONTENT=<html><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_OSCAR_Decarboxylation_Low-Res.png" style="float: right; padding: 10px; width: 300px;"></img><br />
<h2>Why Decarboxylation?</h2><br />
<br />
<p>Among the toxins found in the tailing ponds, naphthenic acids (NAs) are among the most harmful and the most common. Though there is great diversity within the NAs class of compounds, all share the common chemical feature of a carboxylic acid group. The carboxyl group is the primary cause for their toxicity, allowing these chemicals to traverse cell membranes and react with cellular materials (Frank <i>et al</i>. 2009). NAs are recalcitrant (not easily degraded), potentially harmful to the surrounding ecosystem (Clemente & Fedorak, 2005) and corrosive to extraction and transport equipment of petroleum materials (Slavcheva <i>et al</i>. 1999). Corrosion of pipelines leads to higher maintenance costs as well as the grim possibility of these and other toxins leaking into the environment. <br />
There is a need for methods to degrade NAs that are not prohibitively expensive or that would result in production of other hazardous chemicals.</p> <br />
<br />
<p>The main goal of OSCAR is to turn toxins like these into useable hydrocarbons by removing the carboxylic acid group(s) (Behar & Albrecht, 1984). <br />
<br />
Since NAs from petroleum deposits are a variable mixture, an enzymatic process with broad specificity is necessary. With the removal of the carboxylic acid moiety, we aim to produce alkanes suitable for use as fuel. The goal of this subproject was to find one or more suitable pathways to accomplish the decarboxylation of compounds such as NAs with the broadest specificity.</p><br />
<br />
<br />
<h2>The PetroBrick</h2><br />
<br />
<p>The 2011 Washington iGEM team developed the PetroBrick (<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025.</a>), a BioBrick consisting of two primary genes. These include acyl-ACP reductase (<i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a></i>), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (<i>adc</i>), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain (Sukovich, 2010). Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that the fatty acids that the PetroBrick targets, have a very similar structure to NAs.</p><br />
<br />
</html>[[File:UCalgary-Fatty-Acids-vs-NAs.jpg|550px|centre|thumb|Figure 1: A comparison of the structure of fatty acids and naphthenic acids]]<html><br />
<br />
<p>We predicted that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the PetroBrick, we needed to verify that the PetroBrick would work in our hands as it did for the <a href="https://2011.igem.org/Team:Washington">2011 Washington team</a>.<br />
<br />
Figures 2 and 3 demonstrate the function of the PetroBrick.</p><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 2: Gas chromatograph of the differences in peak composition between an <i>E. coli</i> control and the PetroBrick. There was a large increase in a peak with a retention time of 12.25 min, suggesting that the PetroBrick was producing a new hydrocarbon compound.|650px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center|thumb|Figure 3: Mass spectra of the gas chromatograph peak at 12.25 min. This spectra suggests that the PetroBrick is selectively producing a C15 alkane, which resemble the results from the Washington 2011 iGEM team.|750px]]<html><br />
<br />
<p>With the PetroBrick able to successfully produce alkanes, we next wanted to test the PetroBrick on NAs to see if they could be selectively converted into alkanes! This experiment used commercial NAs fractions, which included a large number of different complex NAs compounds. </p><br />
<br />
<a name="Petrobrick"></a><h2>Successful conversion of NAs into hydrocarbons!</h2><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 4: The relative intensity of alkane production from NAs as measured with GC-MS over a retention time in <i>E. coli</i> with and without the PetroBrick . Concentrated NA standard was included for comparison of peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 5: Mass spectrums of alkanes and alkenes produced from NAs with <i>E. coli</i> containing the PetroBrick as in Figure 2. Relative intensity of mass-to -charge ratio (m/z) was compared.]]<html><br />
<br />
<p> Results from Figure 4 and 5 indicate that hydrocarbons were successfully produced from <i>E. coli</i> that contained the PetroBrick construct, as analysed with GC-MS. In Figure 2, <i>E. coli</i> containing the PetroBrick had higher hydrocarbon peaks than the <i>E. coli</i> without the PetroBrick. Not only was the PetroBrick able to degrade NAs into alkanes, the PetroBrick could produce alkenes (Figure 3), indicating that the PetroBrick worked as expected! </p><br />
<br />
<br />
<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)- Can we do better?</h2><br />
<br />
<p>Although we successfully used the PetroBrick to remove carboxyl groups from NAs, we wanted to improve on our results to get a higher yield and/or possibly target other compounds. One of our original concerns in using the PetroBrick to decarboyxlate NAs was that the first enzyme AAR only targeted fatty acids bound to ACP, and non-compatibility with NAs. Therefore, we searched for another enzyme carboxylic acid reductase (CAR) from <i>N. iowensis</i> known to perform a similar task as AAR - converting fatty acids to aldehydes, but with much lower specificity (He <i>et al</i>. 2004). Unlike AAR, CAR does not require covalent attachment to ACP, and likely to have broader substrate specificity. The use of CAR did require a second gene from <i>N. iowensis</i> called <i>Nocardia</i> phosphopantetheinyl transferase (<i>npt</i>) to append a 4’- phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian <i>et al.</i>, 2006).</p><br />
<br />
</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|700px|thumb|Figure 6: Mechanism of CAR catalysis. Taken from He <i>et al.</i>, 2004.]]<html><br />
<br />
<a name="OleT"></a><p>Another enzyme with the potential to remove carboxyl groups from NAs is olefin-forming fatty acid decarboxylase (OleT) from <i>Jeotgalicoccus</i> sp. ATCC 8456. OleT of the cytochrome P450 family acts on fatty acids, but does have low substrate specificity (Rude <i>et al</i>. 2011). Using OleT was beneficial because this single enzyme could do the job of the entire PetroBrick! Given our decarboxylation approach was valid, we started testing and comparing <i>oleT</i> to the PetroBrick.</p><br />
<br />
<h2> Progress so far </h2><br />
<br />
<p>Genes <i>car</i> and <i>npt</i> were cloned from the host organism <i>N. iowensis</i> (NRRL 5646). <i>car</i> was ligated into the pET vector and verified by a restriction digest while <i>npt</i> was cloned into pSB1C3 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902061">BBa_K902061</a>) and similarly verified.</p><br />
<br />
<p><i>car</i> was cloned into pET47b+ plasmid to remove six illegal cut-sites (one XbaI site, two EcoRI sites, and three NotI sites), as it was unsuitable for the BioBrick construction vectors. We first attempted to use a multi-site mutagenesis derived from the QuikChange<sup>®</sup> Multi-Site Directed Mutagenesis Kit, but had little success. Instead, a more time-consuming but effective series of conventional single-site <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis procedure</a> was used with the KAPA Hi-Fi polymerase. The XbaI and EcoRI sites were eliminated first so that <i>car</i> can be moved from the pET Vector and ligated into the PSB1C3 vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902062">BBa_K902062</a>). <br />
<br />
<p>The <i>oleT</i> was successfully amplified from the <i>Jeotgalicoccus</i> sp. ATCC 8456.<br />
<p> </p><br />
<p>Like <i>car</i>, <i>oleT</i> was inserted in a pET47b+ (Novagen) vector before placing it into a BioBrick vector, as two illegal cut sites adjacent to one another needed to be mutagenized. This part is now being ligated into pSB1C3. We are currently in the process of constructing all three parts under control of a <i>tetR</i> promoter and ribosomal binding site (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>), and then constructing these composite parts together as outlined below.</p><br />
<br />
<h2>Final testing constructs</h2><br />
<br />
<p>Final testing constructs are nearly complete. These are illustrated in Figure 7 and will allow us to compare the three different approaches. Unfortunately, Washington only sent us the PetroBrick and not the two individual components, we will have to compare a combination of the PetroBrick and <i>car/npt</i> to the PetroBrick alone and to <i>oleT</i>. </p><br />
<br />
<p></html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|centre|600px]]<html><br />
</html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|centre|280px|thumb|Figure 7: Final constructs required for validating and comparing different decarboxylation approaches]]<html></p><br />
<br />
<a name="TestingOleT"></a><h2> Testing OleT </h2><br />
<br />
<p>One major stumbling block in testing out <i>oleT</i> has been significant difficulty in trying to ligate it into a vector, which has prevented us from submitting it as a BioBrick. As such, we chose to try some assays on the host organism: <i>Jeotgalicoccus</i> sp. ATCC 8456. This way we could at least validate that this gene was functional before we had our BioBricks. We started by trying to verify the results by Rude <i>et al</i>., 2011, namely that OleT could convert fatty acids into alkenes. We grew up cultures based on this <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT_in_Validation_Assay">protocol</a> and used GC-MS to analyze any alkene production (Figure 8 and 9).</p><br />
<br />
<h2> Formation of alkanes by <i>Jeotgalicoccus</i> sp. ATCC 8456</h2><br />
<br />
</html>[[File:UofC_OleT_Assay_1.png|centre|650px|thumb|Figure 8. Gas chromatograph demonstrating the production of olefins (alkenes) from fatty acids as shown from the increase in the peak with a retention time of 14.7 min. The dramatic change in peak intensity at this point suggests that we are producing hydrocarbons.]]<br />
[[File:UofC_OleT_2nd_Assay.png|centre|700px|thumb|Figure 9. Mass spectra of the peak in Figure 8 at retention time 14.7 min. Demonstrating that this peak is an olefin, which is known to be produced in <i>Micrococcus</i>. This verifies our proof-of-concept that the <i>Micrococcus</i> species can degrade fatty acids into olefins. ]]<html><br />
<br />
<p>Based on the additional peak we saw in the gas chromatograph, we could show that our <i>E. coli</i> can produce alkenes with the <i>oleT</i>. This is may be an improvement over the PetroBrick since OleT is only one enzyme instead of two; however, future testing is still needed. Now that we have validated the function of OleT in producing alkenes, the next step is to test it out on complex naphthenic acids in order to compare it to the PetroBrick. This testing is still underway.</p><br />
<br />
</html><br />
<br />
}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Project/OSCAR/DecarboxylationTeam:Calgary/Project/OSCAR/Decarboxylation2012-10-25T23:57:23Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Decarboxylation|<br />
<br />
CONTENT=<html><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_OSCAR_Decarboxylation_Low-Res.png" style="float: right; padding: 10px; width: 300px;"></img><br />
<h2>Why Decarboxylation?</h2><br />
<br />
<p>Among the toxins found in the tailing ponds, naphthenic acids (NAs) are among the most harmful and the most common. Though there is great diversity within the NAs class of compounds, all share the common chemical feature of a carboxylic acid group. The carboxyl group is the primary cause for their toxicity, allowing these chemicals to traverse cell membranes and react with cellular materials (Frank <i>et al</i>. 2009). NAs are recalcitrant (not easily degraded), potentially harmful to the surrounding ecosystem (Clemente & Fedorak, 2005) and corrosive to extraction and transport equipment of petroleum materials (Slavcheva <i>et al</i>. 1999). Corrosion of pipelines leads to higher maintenance costs as well as the grim possibility of these and other toxins leaking into the environment. <br />
There is a need for methods to degrade NAs that are not prohibitively expensive or that would result in production of other hazardous chemicals.</p> <br />
<br />
<p>The main goal of OSCAR is to turn toxins like these into useable hydrocarbons by removing the carboxylic acid group(s) (Behar & Albrecht, 1984). <br />
<br />
Since NAs from petroleum deposits are a variable mixture, an enzymatic process with broad specificity is necessary. With the removal of the carboxylic acid moiety, we aim to produce alkanes suitable for use as fuel. The goal of this subproject was to find one or more suitable pathways to accomplish the decarboxylation of compounds such as NAs with the broadest specificity.</p><br />
<br />
<br />
<h2>The PetroBrick</h2><br />
<br />
<p>The 2011 Washington iGEM team developed the PetroBrick (<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025.</a>), a BioBrick consisting of two primary genes. These include acyl-ACP reductase (<i>aar</i>), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (<i>adc</i>), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain (Sukovich, 2010). Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that the fatty acids that the PetroBrick targets, have a very similar structure to NAs.</p><br />
<br />
</html>[[File:UCalgary-Fatty-Acids-vs-NAs.jpg|550px|centre|thumb|Figure 1: A comparison of the structure of fatty acids and naphthenic acids]]<html><br />
<br />
<p>We predicted that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the PetroBrick, we needed to verify that the PetroBrick would work in our hands as it did for the <a href="https://2011.igem.org/Team:Washington">2011 Washington team</a>.<br />
<br />
Figures 2 and 3 demonstrate the function of the PetroBrick.</p><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 2: Gas chromatograph of the differences in peak composition between an <i>E. coli</i> control and the PetroBrick. There was a large increase in a peak with a retention time of 12.25 min, suggesting that the PetroBrick was producing a new hydrocarbon compound.|650px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center|thumb|Figure 3: Mass spectra of the gas chromatograph peak at 12.25 min. This spectra suggests that the PetroBrick is selectively producing a C15 alkane, which resemble the results from the Washington 2011 iGEM team.|750px]]<html><br />
<br />
<p>With the PetroBrick able to successfully produce alkanes, we next wanted to test the PetroBrick on NAs to see if they could be selectively converted into alkanes! This experiment used commercial NAs fractions, which included a large number of different complex NAs compounds. </p><br />
<br />
<a name="Petrobrick"></a><h2>Successful conversion of NAs into hydrocarbons!</h2><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 4: The relative intensity of alkane production from NAs as measured with GC-MS over a retention time in <i>E. coli</i> with and without the PetroBrick . Concentrated NA standard was included for comparison of peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 5: Mass spectrums of alkanes and alkenes produced from NAs with <i>E. coli</i> containing the PetroBrick as in Figure 2. Relative intensity of mass-to -charge ratio (m/z) was compared.]]<html><br />
<br />
<p> Results from Figure 4 and 5 indicate that hydrocarbons were successfully produced from <i>E. coli</i> that contained the PetroBrick construct, as analysed with GC-MS. In Figure 2, <i>E. coli</i> containing the PetroBrick had higher hydrocarbon peaks than the <i>E. coli</i> without the PetroBrick. Not only was the PetroBrick able to degrade NAs into alkanes, the PetroBrick could produce alkenes (Figure 3), indicating that the PetroBrick worked as expected! </p><br />
<br />
<br />
<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)- Can we do better?</h2><br />
<br />
<p>Although we successfully used the PetroBrick to remove carboxyl groups from NAs, we wanted to improve on our results to get a higher yield and/or possibly target other compounds. One of our original concerns in using the PetroBrick to decarboyxlate NAs was that the first enzyme AAR only targeted fatty acids bound to ACP, and non-compatibility with NAs. Therefore, we searched for another enzyme carboxylic acid reductase (CAR) from <i>N. iowensis</i> known to perform a similar task as AAR - converting fatty acids to aldehydes, but with much lower specificity (He <i>et al</i>. 2004). Unlike AAR, CAR does not require covalent attachment to ACP, and likely to have broader substrate specificity. The use of CAR did require a second gene from <i>N. iowensis</i> called <i>Nocardia</i> phosphopantetheinyl transferase (<i>npt</i>) to append a 4’- phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian <i>et al.</i>, 2006).</p><br />
<br />
</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|700px|thumb|Figure 6: Mechanism of CAR catalysis. Taken from He <i>et al.</i>, 2004.]]<html><br />
<br />
<a name="OleT"></a><p>Another enzyme with the potential to remove carboxyl groups from NAs is olefin-forming fatty acid decarboxylase (OleT) from <i>Jeotgalicoccus</i> sp. ATCC 8456. OleT of the cytochrome P450 family acts on fatty acids, but does have low substrate specificity (Rude <i>et al</i>. 2011). Using OleT was beneficial because this single enzyme could do the job of the entire PetroBrick! Given our decarboxylation approach was valid, we started testing and comparing <i>oleT</i> to the PetroBrick.</p><br />
<br />
<h2> Progress so far </h2><br />
<br />
<p>Genes <i>car</i> and <i>npt</i> were cloned from the host organism <i>N. iowensis</i> (NRRL 5646). <i>car</i> was ligated into the pET vector and verified by a restriction digest while <i>npt</i> was cloned into pSB1C3 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902061">BBa_K902061</a>) and similarly verified.</p><br />
<br />
<p><i>car</i> was cloned into pET47b+ plasmid to remove six illegal cut-sites (one XbaI site, two EcoRI sites, and three NotI sites), as it was unsuitable for the BioBrick construction vectors. We first attempted to use a multi-site mutagenesis derived from the QuikChange<sup>®</sup> Multi-Site Directed Mutagenesis Kit, but had little success. Instead, a more time-consuming but effective series of conventional single-site <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis procedure</a> was used with the KAPA Hi-Fi polymerase. The XbaI and EcoRI sites were eliminated first so that <i>car</i> can be moved from the pET Vector and ligated into the PSB1C3 vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902062">BBa_K902062</a>). <br />
<br />
<p>The <i>oleT</i> was successfully amplified from the <i>Jeotgalicoccus</i> sp. ATCC 8456.<br />
<p> </p><br />
<p>Like <i>car</i>, <i>oleT</i> was inserted in a pET47b+ (Novagen) vector before placing it into a BioBrick vector, as two illegal cut sites adjacent to one another needed to be mutagenized. This part is now being ligated into pSB1C3. We are currently in the process of constructing all three parts under control of a <i>tetR</i> promoter and ribosomal binding site (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>), and then constructing these composite parts together as outlined below.</p><br />
<br />
<h2>Final testing constructs</h2><br />
<br />
<p>Final testing constructs are nearly complete. These are illustrated in Figure 7 and will allow us to compare the three different approaches. Unfortunately, Washington only sent us the PetroBrick and not the two individual components, we will have to compare a combination of the PetroBrick and <i>car/npt</i> to the PetroBrick alone and to <i>oleT</i>. </p><br />
<br />
<p></html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|centre|600px]]<html><br />
</html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|centre|280px|thumb|Figure 7: Final constructs required for validating and comparing different decarboxylation approaches]]<html></p><br />
<br />
<a name="TestingOleT"></a><h2> Testing OleT </h2><br />
<br />
<p>One major stumbling block in testing out <i>oleT</i> has been significant difficulty in trying to ligate it into a vector, which has prevented us from submitting it as a BioBrick. As such, we chose to try some assays on the host organism: <i>Jeotgalicoccus</i> sp. ATCC 8456. This way we could at least validate that this gene was functional before we had our BioBricks. We started by trying to verify the results by Rude <i>et al</i>., 2011, namely that OleT could convert fatty acids into alkenes. We grew up cultures based on this <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT_in_Validation_Assay">protocol</a> and used GC-MS to analyze any alkene production (Figure 8 and 9).</p><br />
<br />
<h2> Formation of alkanes by <i>Jeotgalicoccus</i> sp. ATCC 8456</h2><br />
<br />
</html>[[File:UofC_OleT_Assay_1.png|centre|650px|thumb|Figure 8. Gas chromatograph demonstrating the production of olefins (alkenes) from fatty acids as shown from the increase in the peak with a retention time of 14.7 min. The dramatic change in peak intensity at this point suggests that we are producing hydrocarbons.]]<br />
[[File:UofC_OleT_2nd_Assay.png|centre|700px|thumb|Figure 9. Mass spectra of the peak in Figure 8 at retention time 14.7 min. Demonstrating that this peak is an olefin, which is known to be produced in <i>Micrococcus</i>. This verifies our proof-of-concept that the <i>Micrococcus</i> species can degrade fatty acids into olefins. ]]<html><br />
<br />
<p>Based on the additional peak we saw in the gas chromatograph, we could show that our <i>E. coli</i> can produce alkenes with the <i>oleT</i>. This is may be an improvement over the PetroBrick since OleT is only one enzyme instead of two; however, future testing is still needed. Now that we have validated the function of OleT in producing alkenes, the next step is to test it out on complex naphthenic acids in order to compare it to the PetroBrick. This testing is still underway.</p><br />
<br />
</html><br />
<br />
}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Project/OSCAR/DecarboxylationTeam:Calgary/Project/OSCAR/Decarboxylation2012-10-25T23:54:56Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Decarboxylation|<br />
<br />
CONTENT=<html><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_OSCAR_Decarboxylation_Low-Res.png" style="float: right; padding: 10px; width: 300px;"></img><br />
<h2>Why Decarboxylation?</h2><br />
<br />
<p>Among the toxins found in the tailing ponds, naphthenic acids (NAs) are among the most harmful and the most common. Though there is great diversity within the NAs class of compounds, all share the common chemical feature of a carboxylic acid group. The carboxyl group is the primary cause for their toxicity, allowing these chemicals to traverse cell membranes and react with cellular materials (Frank <i>et al</i>. 2009). NAs are recalcitrant (not easily degraded), potentially harmful to the surrounding ecosystem (Clemente & Fedorak, 2005) and corrosive to extraction and transport equipment of petroleum materials (Slavcheva <i>et al</i>. 1999). Corrosion of pipelines leads to higher maintenance costs as well as the grim possibility of these and other toxins leaking into the environment. <br />
There is a need for methods to degrade NAs that are not prohibitively expensive or that would result in production of other hazardous chemicals.</p> <br />
<br />
<p>The main goal of OSCAR is to turn toxins like these into useable hydrocarbons by removing the carboxylic acid group(s) (Behar & Albrecht, 1984). <br />
<br />
Since NAs from petroleum deposits are a variable mixture, an enzymatic process with broad specificity is necessary. With the removal of the carboxylic acid moiety, we aim to produce alkanes suitable for use as fuel. The goal of this subproject was to find one or more suitable pathways to accomplish the decarboxylation of compounds such as NAs with the broadest specificity.</p><br />
<br />
<br />
<h2>The PetroBrick</h2><br />
<br />
<p>The 2011 Washington iGEM team developed the PetroBrick (<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025.</a>), a BioBrick consisting of two primary genes. These include acyl-ACP reductase (<i>aar</i>), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (<i>adc</i>), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain (Sukovich, 2010). Essentially this allows for hydrocarbons to be produced from glucose. What we realized though, is that the fatty acids that the PetroBrick targets, have a very similar structure to NAs.</p><br />
<br />
</html>[[File:UCalgary-Fatty-Acids-vs-NAs.jpg|550px|centre|thumb|Figure 1: A comparison of the structure of fatty acids and naphthenic acids]]<html><br />
<br />
<p>We predicted that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the PetroBrick, we needed to verify that the PetroBrick would work in our hands as it did for the <a href="https://2011.igem.org/Team:Washington">2011 Washington team</a>.<br />
<br />
Figures 2 and 3 demonstrate the function of the PetroBrick.</p><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 2: Gas chromatograph of the differences in peak composition between an <i>E. coli</i> control and the PetroBrick. There was a large increase in a peak with a retention time of 12.25 min, suggesting that the PetroBrick was producing a new hydrocarbon compound.|650px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center|thumb|Figure 3: Mass spectra of the gas chromatograph peak at 12.25 min. This spectra suggests that the PetroBrick is selectively producing a C15 alkane, which resemble the results from the Washington 2011 iGEM team.|750px]]<html><br />
<br />
<p>With the PetroBrick able to successfully produce alkanes, we next wanted to test the PetroBrick on NAs to see if they could be selectively converted into alkanes! This experiment used commercial NAs fractions, which included a large number of different complex NAs compounds. </p><br />
<br />
<a name="Petrobrick"></a><h2>Successful conversion of NAs into hydrocarbons!</h2><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 4: The relative intensity of alkane production from NAs as measured with GC-MS over a retention time in <i>E. coli</i> with and without the PetroBrick . Concentrated NA standard was included for comparison of peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 5: Mass spectrums of alkanes and alkenes produced from NAs with <i>E. coli</i> containing the PetroBrick as in Figure 2. Relative intensity of mass-to -charge ratio (m/z) was compared.]]<html><br />
<br />
<p> Results from Figure 4 and 5 indicate that hydrocarbons were successfully produced from <i>E. coli</i> that contained the PetroBrick construct, as analysed with GC-MS. In Figure 2, <i>E. coli</i> containing the PetroBrick had higher hydrocarbon peaks than the <i>E. coli</i> without the PetroBrick. Not only was the PetroBrick able to degrade NAs into alkanes, the PetroBrick could produce alkenes (Figure 3), indicating that the PetroBrick worked as expected! </p><br />
<br />
<br />
<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)- Can we do better?</h2><br />
<br />
<p>Although we successfully used the PetroBrick to remove carboxyl groups from NAs, we wanted to improve on our results to get a higher yield and/or possibly target other compounds. One of our original concerns in using the PetroBrick to decarboyxlate NAs was that the first enzyme AAR only targeted fatty acids bound to ACP, and non-compatibility with NAs. Therefore, we searched for another enzyme carboxylic acid reductase (CAR) from <i>N. iowensis</i> known to perform a similar task as AAR - converting fatty acids to aldehydes, but with much lower specificity (He <i>et al</i>. 2004). Unlike AAR, CAR does not require covalent attachment to ACP, and likely to have broader substrate specificity. The use of CAR did require a second gene from <i>N. iowensis</i> called <i>Nocardia</i> phosphopantetheinyl transferase (<i>npt</i>) to append a 4’- phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian <i>et al.</i>, 2006).</p><br />
<br />
</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|700px|thumb|Figure 6: Mechanism of CAR catalysis. Taken from He <i>et al.</i>, 2004.]]<html><br />
<br />
<a name="OleT"></a><p>Another enzyme with the potential to remove carboxyl groups from NAs is olefin-forming fatty acid decarboxylase (OleT) from <i>Jeotgalicoccus</i> sp. ATCC 8456. OleT of the cytochrome P450 family acts on fatty acids, but does have low substrate specificity (Rude <i>et al</i>. 2011). Using OleT was beneficial because this single enzyme could do the job of the entire PetroBrick! Given our decarboxylation approach was valid, we started testing and comparing <i>oleT</i> to the PetroBrick.</p><br />
<br />
<h2> Progress so far </h2><br />
<br />
<p>Genes <i>car</i> and <i>npt</i> were cloned from the host organism <i>N. iowensis</i> (NRRL 5646). <i>car</i> was ligated into the pET vector and verified by a restriction digest while <i>npt</i> was cloned into pSB1C3 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902061">BBa_K902061</a>) and similarly verified.</p><br />
<br />
<p><i>car</i> was cloned into pET47b+ plasmid to remove six illegal cut-sites (one XbaI site, two EcoRI sites, and three NotI sites), as it was unsuitable for the BioBrick construction vectors. We first attempted to use a multi-site mutagenesis derived from the QuikChange<sup>®</sup> Multi-Site Directed Mutagenesis Kit, but had little success. Instead, a more time-consuming but effective series of conventional single-site <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis procedure</a> was used with the KAPA Hi-Fi polymerase. The XbaI and EcoRI sites were eliminated first so that <i>car</i> can be moved from the pET Vector and ligated into the PSB1C3 vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902062">BBa_K902062</a>). <br />
<br />
<p>The <i>oleT</i> was successfully amplified from the <i>Jeotgalicoccus</i> sp. ATCC 8456.<br />
<p> </p><br />
<p>Like <i>car</i>, <i>oleT</i> was inserted in a pET47b+ (Novagen) vector before placing it into a BioBrick vector, as two illegal cut sites adjacent to one another needed to be mutagenized. This part is now being ligated into pSB1C3. We are currently in the process of constructing all three parts under control of a <i>tetR</i> promoter and ribosomal binding site (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>), and then constructing these composite parts together as outlined below.</p><br />
<br />
<h2>Final testing constructs</h2><br />
<br />
<p>Final testing constructs are nearly complete. These are illustrated in Figure 7 and will allow us to compare the three different approaches. Unfortunately, Washington only sent us the PetroBrick and not the two individual components, we will have to compare a combination of the PetroBrick and <i>car/npt</i> to the PetroBrick alone and to <i>oleT</i>. </p><br />
<br />
<p></html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|centre|600px]]<html><br />
</html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|centre|280px|thumb|Figure 7: Final constructs required for validating and comparing different decarboxylation approaches]]<html></p><br />
<br />
<a name="TestingOleT"></a><h2> Testing OleT </h2><br />
<br />
<p>One major stumbling block in testing out <i>oleT</i> has been significant difficulty in trying to ligate it into a vector, which has prevented us from submitting it as a BioBrick. As such, we chose to try some assays on the host organism: <i>Jeotgalicoccus</i> sp. ATCC 8456. This way we could at least validate that this gene was functional before we had our BioBricks. We started by trying to verify the results by Rude <i>et al</i>., 2011, namely that OleT could convert fatty acids into alkenes. We grew up cultures according to this <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT_in_Validation_Assay">protocol</a> and used GC-MS to analyze any alkene production (Figure 8 and 9).</p><br />
<br />
<h2> Formation of alkanes by <i>Jeotgalicoccus</i> sp. ATCC 8456</h2><br />
<br />
</html>[[File:UofC_OleT_Assay_1.png|centre|650px|thumb|Figure 8. Gas chromatograph demonstrating the production of olefins (alkenes) from fatty acids as shown from the increase in the peak with a retention time of 14.7 min. The dramatic change in peak intensity at this point suggests that we are producing hydrocarbons.]]<br />
[[File:UofC_OleT_2nd_Assay.png|centre|700px|thumb|Figure 9. Mass spectra of the peak in Figure 8 at retention time 14.7 min. Demonstrating that this peak is an olefin, which is known to be produced in <i>Micrococcus</i>. This verifies our proof-of-concept that the <i>Micrococcus</i> species can degrade fatty acids into olefins. ]]<html><br />
<br />
<p>Based on the additional peak we saw in the gas chromatograph, we could show that our <i>E. coli</i> can produce alkenes with the <i>oleT</i>. This is may be an improvement over the PetroBrick since OleT is only one enzyme instead of two; however, future testing is still needed. Now that we have validated the function of OleT in producing alkenes, the next step is to test it out on complex naphthenic acids in order to compare it to the PetroBrick. This testing is still underway.</p><br />
<br />
</html><br />
<br />
}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-03T05:58:57Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
<br />
CONTENT=<html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. Eight potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">"PetroBrick"</a> (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) and aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). We have planned to verify the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> with carboxylic acid reductase (CAR) from <i>Nocardia iowensis</i>, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into <i>E.coli</i>, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in <i>Nocardia iowensis</i>, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called <i>NRRL 5646</i>) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from <a href="http://www.dsmz.de">DSMZ</a>. In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
</html><br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the PetroBrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html><br />
<br />
<br />
<br />
<p> These results suggest that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> <i>E.coli</i> Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with <i>E.coli</i> from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> sequencing results with a complementarity of 87%, as compared to the theoretical <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">General Production Protocol</a> that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed eight overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> has worked on <a href="https://2011.igem.org/Team:Washington/Alkanes/Future/Vector">system optimization</a>, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> that is preventing effective operation. We may need to design inner primers to sequence the entire <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "<a href="https://2011.igem.org/Team:Washington/Alkanes/Future/Vector">system optimization</a>" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) was missing. This explained why our sequencing results for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) gene and no aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). Either the parts registry did not have the proper <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> (containing both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>. It was determined that the latter was true. As such, the transformation for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> (submitted by the University of Washington). This involved transforming the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> into <i>E.coli</i>, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">production media protocol</a> in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">production media protocol</a> to attempt to produce hydrocarbons because we are not certain as to whether the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> sent to us in the 2012 kit plate does not contain a proper functional <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, b) transformation and isolation of the aldehyde decarbonylate (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) and c) culturing the <i>Nocardia strain NRRL 5646</i>. With regards to the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). The <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a> was transformed into the <i>E.coli</i>,and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) being ligated with aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). Upon successfully forming this biobrick (ideally the same as a functional <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> that was being used in 2011. <br />
With regards to culturing <i>Nocardia</i>, a “brain and heart infusion (BHI)” broth was used to inoculate the <i>Nocardia</i> in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the <i>Nocardia</i>, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample <i>Nocardia iowensis</i>growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
</html><br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/<a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a> gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> gene to produce a <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>. The Washington iGEM team agreed to send us a plasmid stock of their <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with twelve colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. Eight overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, and NPT (X/P) with <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
</html>[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with eight different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent <i>E.coli</i> cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent <i>E.coli</i>. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with <i>E.coli</i> on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the<a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>vector. Following standard <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> would be unable to degrade naphthenic acids, we found that bacteria containing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both <i>E.coli</i> that contain the PetroBrick, and in <i>E.coli</i> that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from <i>E.coli</i> containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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</html><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-03T05:57:27Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
<br />
CONTENT=<html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. Eight potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">"PetroBrick"</a> (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) and aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). We have planned to verify the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> with carboxylic acid reductase (CAR) from <i>Nocardia iowensis</i>, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into <i>E.coli</i>, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in <i>Nocardia iowensis</i>, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called <i>NRRL 5646</i>) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from <a href="http://www.dsmz.de">DSMZ</a>. In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
</html><br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the PetroBrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html><br />
<br />
<br />
<br />
<p> These results suggest that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> <i>E.coli</i> Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with <i>E.coli</i> from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> sequencing results with a complementarity of 87%, as compared to the theoretical <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">General Production Protocol</a> that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed eight overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> has worked on <a href="https://2011.igem.org/Team:Washington/Alkanes/Future/Vector">system optimization</a>, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> that is preventing effective operation. We may need to design inner primers to sequence the entire <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "<a href="https://2011.igem.org/Team:Washington/Alkanes/Future/Vector">system optimization</a>" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) was missing. This explained why our sequencing results for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) gene and no aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). Either the parts registry did not have the proper <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> (containing both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>. It was determined that the latter was true. As such, the transformation for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> (submitted by the University of Washington). This involved transforming the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> into <i>E.coli</i>, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">production media protocol</a> in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">production media protocol</a> to attempt to produce hydrocarbons because we are not certain as to whether the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> sent to us in the 2012 kit plate does not contain a proper functional <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, b) transformation and isolation of the aldehyde decarbonylate (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) and c) culturing the <i>Nocardia strain NRRL 5646</i>. With regards to the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). The <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a> was transformed into the <i>E.coli</i>,and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) being ligated with aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). Upon successfully forming this biobrick (ideally the same as a functional <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the <i>Nocardia</i> in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the <i>Nocardia</i>, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample <i>Nocardia iowensis</i>growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
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[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html><br />
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<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/<a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a> gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> gene to produce a <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>. The Washington iGEM team agreed to send us a plasmid stock of their <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>. </p><br />
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<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with twelve colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. Eight overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, and NPT (X/P) with <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
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</html>[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
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<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
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<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with eight different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
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<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent <i>E.coli</i> cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent <i>E.coli</i>. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with <i>E.coli</i> on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
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<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the<a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>vector. Following standard <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> would be unable to degrade naphthenic acids, we found that bacteria containing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
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</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both <i>E.coli</i> that contain the PetroBrick, and in <i>E.coli</i> that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
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</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from <i>E.coli</i> containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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<h2>Why Decarboxylation?</h2><br />
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<p>Though there is great diversity between the many possible compounds termed “naphthenic acids” found in tailings ponds, all share the common chemical feature of a carboxylic acid group. This particular group is known to be the primary cause for the toxicity of these compounds, allowing them to enter through cell membranes and destroy cells (Frank et al, 2009). Naphthenic acids are a very problematic component of the tailings ponds, and are not easily degraded under regular circumstances. They are known to leak into local environments and cause many problems for wildlife (Clemente & Fedorak, 2005), as well as corrosion (Slavcheva et al, 1999).</p> <br />
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<p>Additionally, removing the carboxylic acid group(s) is of great importance in the conversion of these contaminants to biofuel (Behar & Albrecht, 1984). Since naphthenic acids are a widely varying and unspecific mixture of compounds, an enzymatic process with very low substrate specificity is necessary, as particularly specific enzymes would not be able to universally decarboxylate all naphthenic acid substrates found in tailings ponds. Without a carboxylic acid group, and with the removal of sulphur and nitrogen contaminants, an alkane suitable for use as fuel can be obtained. The goal of this subproject was to find a suitable pathway to accomplish the decarboxylation of naphthenic acids with the broadest specificity possible.</p><br />
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<h2>The PetroBrick</h2><br />
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<p>The iGEM Washington team’s PetroBrick is a BioBrick consisting of two primary genes. These include acyl-ACP reductase (AAR), which reduces fatty acids bound to ACP to fatty aldehydes, and a second gene called aldehyde decarbonylase (ADC), which subsequently cleaves the entire aldehyde group and results in a hydrocarbon chain. Essentially, fuel is produced from glucose. The issue that we foresaw in using the PetroBrick to decarboyxlation naphthenic acids is that the first enzyme AAR is thought to be highly specific for fatty acids bound to ACP. Thus, it was not considered to be particularly compatible with naphthenic acids, and so we sought another enzyme called CAR that would perform a similar task with much lower specificity. However, in performing PetroBrick assays, not only did we have successful production of alkanes from glucose, but we were able to optimize the PetroBrick to produce alkane and alkene hydrocarbons from napthenic acids.</p><br />
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<p>Initially the ability for the Petrobrick to produce alkanes from glucose was analyzed in order to verify that the Petrobrick was working as described by the 2011 Washington team. This was demonstrated in Figure 1 and 2. <br />
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<br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 1: Gas Chromatograph demonstrating the differences in peak composition between an <i>E.coli</i> control and the Petrobrick. There was a large increase in a peak with a retention time of 12.25 min. suggesting that the Petrobrick was producing a new compound.|500px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center||thumb|Figure 2: Mass Spectra of the gas chromatograph peak at 12.25 min. The spectra suggests that the Petrobrick is selectively producing a C15 alkane. This is what was expected as determined by the Washington 2011 iGEM team.|500px]]<html><br />
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<p>With the Petrobrick shown to be able to successfully produce alkanes, it was then tested to see if naphthenic acids could be selectively converted into alkanes. This experiment used commercially available naphthenic acid fractions including a large number of different naphthenic acid compounds. <br />
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<h2> Results of the PetroBrick</h2><br />
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<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 3: The relative intensity of alkane production over a retention time in both <i>E.coli</i> that contain the PetroBrick, and in <i>E.coli</i> that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 4: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from <i>E.coli</i> containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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<p> The above graphs indicate that hydrocarbons were successfully produced from <i>E.coli</i> that contained the PetroBrick plasmid, as analysed with GC-MS. In Figure 1, <i>E.coli</i> containing the PetroBrick had significantly higher hydrocarbon peaks than in a control of <i>E.coli</i> that did not contain the PetroBrick plasmid. Not only was the PetroBrick able to degrade naphthenic acids into alkanes, but it was also able to produce alkenes as shown by Figure 2, indicating that the PetroBrick had more potential than previously indicated. </p><br />
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<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)</h2><br />
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<p>In order to model our part after the PetroBrick, an enzyme was needed to replace AAR that would not require covalent attachment to ACP and would have low enough specificity to accommodate diverse naphthenic acids. The enzyme chosen for this purpose was carboxylic acid reductase (CAR), found in Nocardia iowensis. CAR was selected for its remarkably low specificity in converting carboxylic acids to aldehydes (He et al, 2004). It was determined that a second gene from <i>N. iowensis</i>, called Nocardia phosphopantetheinyl transferase (NPT) was also necessary to append a 4’-phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian et al, 2006). </p><br />
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<p><i>N. iowensis</i> (NRRL 5646) was purchased from DSMZ and rehydrated, then grown in both solid and liquid brain heart infusion (BHI) media. CAR and NPT were successfully cloned out and amplified, and CAR was verified after ligation with the PET vector by a restriction digest and subsequent gel. NPT was likewise verified.</p><br />
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<p>CAR was also found to have six cut sites deemed illegal for BioBrick construction: one XpaI site, two EcoRI sites, and three NotI sites. These were first to be addressed by a multi-site mutagenesis derived from the QuikChange® Multi Site Directed Mutagenesis Kit, but this showed little success. Instead, a more time-consuming series of conventional single-site mutagenesis procedures was favoured, using the KAPA amplification system. A PET vector was chosen as an alternate vector to pSB1C3 to ligate with CAR for the duration of mutagenesis, while NPT was still ligated with pSB1C3. The XpaI and EcoRI sites were eliminated first, and following these steps and subsequent verification of their success, the mutated CAR+PET plasmid was successfully amplified with BioBrick primers after multiple attempts in preparation for the final construct. NPT was ligated in a construct following part J13002 from the iGEM parts registry, containing a ribosomal binding site and a TetR promoter.</p><br />
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<h2>How do the PetroBrick and CAR+NPT work together?</h2><br />
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<p>Our team decided to created a BioBrick construction containing three major parts. These included: the PetroBrick (as received from Washington), the CAR gene, and the NPT gene. This construction can be seen below.</p><br />
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</html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|center|500px]]<html><br />
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<p>Both CAR and NPT use the part JI3002, which consists of pTetR (a constitutive promotor), as well as an RBS (ribosomal binding site). This constitutive promoter allows the gene to always be expressed, so that decarboxylation may consistently occur. The construction also includes Washington’s PetroBrick, which contributes the genes ADC and AAR. While AAR is useful to help convert some carboxylic acids and fatty acids to aldehydes, it is not known to be highly specific like the CAR enzyme. We are primarily interested in using the PetroBrick for its ADC gene, which is responsible for producing the only enzyme in our system that is capable of converting aldehydes into hydrocarbons. Meanwhile, both AAR and CAR in our construct will work to convert the initial carboxylic acids into the aldehydes required to produce hydrocarbons.</p><br />
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</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|500px]]<html><br />
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<h2>What about an alternative? (Jeotgalicoccus olefin-forming fatty acid decarboxylase, or OleT)</h2><br />
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<p> In the early stages of work with CAR, an alternate approach to naphthenic acid decarboxylation was proposed that was entirely distinct from the PetroBrick+CAR/NPT system. The idea behind this method was to use olefin-forming fatty acid decarboxylase (OleT) from <i>Jeotgalicoccus sp. ATCC 8456</i>, a decarboxylase of the cytochrome P450 family acting on fatty acids, but with low substrate specificity (Rude et al, 2011). We chose to investigate OleT to determine if it was capable of directly decarboxylating naphthenic acids. </p><br />
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<p> OleT was successfully amplified from the <i>Jeotgalicoccus sp. ATCC 8456</i>. Like CAR, OleT was inserted in a PET vector before placing it into a BioBrick, as two illegal cut sites adjacent to one another needed to be mutagenized. The final construct for OleT is very simple, containing the J13002 promotor/RBS, and the OleT gene. This simple construct should be able to convert carboxylic acids to terminal alkenes on its own. </p><br />
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</html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|center|500px]]<html><br />
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<h2>Week 1 (May 1-4)</h2><br />
<p>&nbsp;&nbsp;This was the first week where we met with other team members and summarized the primary subprojects the team will be tackling this coming summer.</p><br />
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<h2>Week 2 (May 7-11)</h2><br />
<p>&nbsp;&nbsp;During this week literature searches were performed.</p><br />
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<h2>Week 3 (May 14-18)</h2><br />
<p>&nbsp;&nbsp;During this week literature searches were performed.</p><br />
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<h2>Week 4 (May 22-25)</h2><br />
<p>&nbsp;&nbsp;During this week, strains of <i> Pseudomonas fluorescens</i> PF-5 were obtained. Two cultures were started by adding 500 &micro;L stock to 10 mL LB media containing 50 mg/L ACROS Naphthenic Acids. These cultures were grown at 30&deg;C overnight, shaking at 110 rpm.</p><br />
<p>&nbsp;&nbsp;Overnight cultures were then streaked on LB agar the following day with various types and concentrations of antibiotics in order to determine the susceptibility profile of the organism. This was necessary in order to determine what marker could be used on a transposon to allow for selection of organisms with sucessful transposon insertions. These plates were grown overnight to look for death or growth, and the following results were obtained; </p><br />
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<center><table><br />
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<td><b>Gentamycin</b></td><br />
<td><b>Kanamycin</b></td><br />
<td><b>Chloramphenicol</b></td><br />
<td><b>Tetracycline</b></td><br />
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<td>25 &micro;g/ml = no growth</td><br />
<td>5 &micro;g/ml = slight growth</td><br />
<td>5 &micro;g/ml = growth</td><br />
<td>50 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td>50 &micro;g/ml = no growth</td><br />
<td>10 &micro;g/ml = slight growth</td><br />
<td>10 &micro;g/ml = growth</td><br />
<td>100 &micro;g/ml = no growth</td><br />
</tr><br />
<tr><br />
<td>100 &micro;g/ml = no growth</td><br />
<td>25 &micro;g/ml = no growth</td><br />
<td>25 &micro;g/ml = growth</td><br />
<td>200 &micro;g/ml = no growth</td><br />
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<tr><br />
<td> </td><br />
<td>50 &micro;g/ml = no growth</td><br />
<td>50 &micro;g/ml = growth</td><br />
<td> </td><br />
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<br />
<p><br />
<br></br> &nbsp;&nbsp;Based on these results, it was determined that Kanamycin, Gentamycin, and Tetracycline could be used as the selectable marker on the transposon, while Chloramphenicol could not as the strain is naturally resistant. Glycerol stocks of the strains were also made at this time from a fresh overnight culture.</p><br />
<br />
<h2>Week 5 (May 28-June 1)</h2><br />
<p>&nbsp;&nbsp;At the beginning of this week we spoke to Dr. Michael Hynes, who was able to give us <i> E. coli </i> SM10 and SM17-1 cells containing the plasmid pOT182. This plasmid contains an <i> E. coli </i> origin of replication, allowing it to act as a suicide vector when transferred to a different bacterial species. pOT182 contains a Tn5 transposon element containing a promotorless <i>lacZ</i> gene, genes for tetracycline resistance as well as a beta-lactamase, transposase and an <i>E. coli</i> origin of replication. These elements are bordered by insertion element sequences which are recognised by the transposase. When transferred to a different host through conjugation, the plasmid itself can no longer replicate. The transposase however can recognise and transfer the sequence between the insertion elements in a cut-and-paste fashion randomly into the genome. In this fashion, the tetracycline and beta-lactam resistant traits would only persist in cells in which the transposon has jumped into the genome, allowing these antibiotics to select for transposon positive cells. The lacZ protein will only be produced if the transposon jumps in frame downstream of a promotor, and in this case would allow for a lacZ based assay of the promotors response. The <i>E. coli</i> origin of replication present in the transposon allows for the self-cloning of the transposon in a plasmid format after genomic digestion and circularization and transformation into <i>E. coli</i>, allowing for the sequence of bordering gene fragments to be determined easily and therefore mapping the transposon in the genome.</P><br />
<br />
<br />
<br />
<p>&nbsp;&nbsp;Cultures of <i>E. coli</i> SM10 and <i>P. fluorescens</i> PF-5 were grown up overnight in shakers at 37&deg;C and 30&deg;C respectively. SM10 was grown in LB + 10 &micro;g/ml Tet, and PF-5 was grown in LB + 50 mg/L ACROS naphthenic acids. In the morning, the SM10 culture was subcultured (1/4) into LB without antibiotics and allowed to grow for an additional 4h. After this, 3 replicates of mating mixtures were made with 500 &micro;l of each culture were mixed, and the cells were spun down and resuspended in 50 &micro;l of LB. These samples were then plated in separate spots on LB agar. Additional spots were made in the same fashion with just PF-5 culture and just SM10 culture as controls, and the plate was incubated at 37&deg;C overnight. In the morning, each spot was resuspended in 500&micro;l sterile water + 25 &micro;g/ml Tet, and dilutions of 1, 1/10, 1/100, and 1/1000 of each mating mixture was plated on Pseudomonas Isolation Agar (PIA) + 10&micro;g/l Tet + 50 mg/L ACROS naphthenic acids. These cultures were allowed to grow at 30&deg;C over the weekend. </p><br />
<p>&nbsp;&nbsp;The purpose of the PIA is to selectively allow the growth of the PF-5 strain, while killing off the donor SM10 strain. The tetracycline is designed to select for the positive transposon mutants in the PF-5 strain, as the only way that tetracycline resistance would be acquired (barring spontaneous mutation events) would be if the transposable element had jumped into the genome of the cell. We decided to use 10 &micro;g/ml as the concentration of the tetracycline in the plates because we believed that 50 &micro;g/ml would be too high for even strains carrying resistance to survive. Seeing as 10 &micro;g/ml was effective for <i>E. coli</i>, we chose to try this. After streaking the PF-5 culture on the plate to test for its resistance however, it was found that the strain without the transposon was able to grow slightly on the plates, meaning that the concentration of antibiotic is not high enough to properly select for transposon mutants v.s. untransformed cells. In order to try to remedy this, the mating spots were resuspended in a mixture containing a higher dose of antibiotics. The results of this experiment are pending. </p><br />
<br />
<h2>Week 6 (June 4-8)</h2><br />
<br />
<p>&nbsp;&nbsp;When the plates from last week were examined, it was found that though the PIA was sucessful in inhibiting the growth of the <i>E. coli</i> donor strain, the original PF-5 strain was capable of growth on the media. Because of this, the plates were not selective towards cells containing the transposon insertion, and thus lawns of bacteria were seen on each of the plates.</p><br />
<p>&nbsp;&nbsp;Because of this, new selective media plates were prepared. These contained LB agar + 50 mg/L ACROS NA's + 50 &micro;g each chloramphenicol and tetracycline. The chloramphenicol was used in order to kill off the SM10 donor strain, while the tetracycline was used at a concentration previously shown to kill off the PF-5 cells that did not contain a transposon insertion. The conjugation procedure previously described was repeated, with 1 replicate mating spot being plated in 4 different dilutions on the new selective media. These plates were grown overnight at 30&deg;C. It was found that both SM10 and unmodified PF-5 were not capable of growth on the new selective plates, and colonies were found growing on all 4 of the dilutions for the mating spot.<br />
<br />
</html>[[File:Ucalgary2012_TMscreenpractice.png|center|thumb|400px|Figure 1: Transposon selective plates. WT Pf-5 and the donor strain are not capable of growth, however colonies of Pf-5 containing the transposon are capable of growth.]]<html><br />
<br />
<p>&nbsp;&nbsp;Because the host and the untransformed cells were not capable of growth on the selective plates, it is believed that these colonies must represent sucessful transposition events, as this would be the only way that the tetracycline resistance would be transfered to the PF-5 cells (chloramphenicol would have no effect, as PF-5 cells are naturally resistant at this concentration, as previously shown). Because the cell density was too high, the 1 and 1/10 dilution plates were discarded, while the 1/100 and 1/1000 plates were stored at 4&deg;C until the next step of the procedure, in which screening for naphthenic acid response will be performed. </p><br />
<br />
<h2>Week 7 (June 11-15) </h2> <br />
<br />
<p>&nbsp;&nbsp;In order to test for a naphthenic acid response, a lacZ reporter system in the transposon will be utilized. Because the lacZ enzyme is capable of degrading X-gal, a dissacharide sugar containing lactose, into lactose, and an insoluble sugar (5,5'-dibromo-4,4'-dichloro-indigo), which appears blue. Therefore, in response to activation of a native gene promotor, in frame transposon insertions will produce lacZ at levels corresponding to the activation level of the gene, and these colonies will be able to utilize lactose as a carbon source as well as utilize Xgal as a substrate. Cells responding to naphthenic acids will therefore show blue pigmentation and be capable of growth on lactose. Those that do not respond should remain white, and perish when lactose is given as the only sugar source.</p><br />
<p>&nbsp;&nbsp;The concentration of naphthenic acids used in the test plates will be 4x less than the minimal inhibitory concentration (MIC). Therefore, a MIC assay for naphthenic acids with PF-5 cells was carried out overnight at 30&deg;C at 2%, 1%, 0.5%, 0.25%, and 0.125% concentrations in LB media. It was found that PF-5 grew well at all these concentrations; therefore 1% was arbitrarily picked for the test conditions for naphthenic acid response plates.</p><br />
<p><br />
Plates were made as follows, with Xgal spread on top:</p><br />
<br />
<center><table><br />
<table border="1" CELLPADDING=3 CELLSPACING=1 <br />
RULES=NONE FRAME=BOX table width=600px height=150px><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Wild-Type PF-5 Controls</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media Alone, M9 Minimal Media + 0.4% Glucose, M9 Minimal Media + 0.4% Lactose, M9 Minimal Media + 1% NA's</td><br />
</tr><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Transposon Replica Plates</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media + 0.4% Glucose + Chlor, M9 Minimal Media + 0.4% Glucose + Chlor + 1% NA's, M9 Minimal Media + 0.4% Lactose + Tet, M9 Minimal Media + 0.4% Lactose + Tet + 1% NA's </td><br />
</tr><br />
</table></center><br />
<br />
<br />
<p><br />
<br></br><br />
&nbsp;&nbsp;Colonies from the 1/1000 transposon plate were replica stamped using velvet cloth onto the transposon test plates, and WT PF-5 was streaked onto the controls. The plates were allowed to grow over the weekend at 30 &deg;C. </p><br />
<br />
<h2>Week 8 (June 18-22)</h2><br />
<br />
<p>&nbsp;&nbsp;The plates from the previous week were observed on Monday. WT PF-5 grew on M9 + Glucose, but not on any of the other controls, indicating that the wild-type strain could not utilize lactose, indicating that this could possibly be used as a selection measure for responses from the transposon mutants. </p><br />
<p>&nbsp;&nbsp;No growth was observed on any of the transposon test plates. This was believed to be likely from poor transfer during stamping, resulting from a poor contact with the plates in addition to a grease layer formed by the undissolved naphthenic acids. Because of this, a new method of creating plates had to be determined.</P><br />
<p>&nbsp;&nbsp;We tried to dissolve NAs in DMF and spread this mixture on top of agar plates as well as mix it into the liquid agar before pouring plates. Though this helped to solubilize the naphthenic acids, it was not as effective as increasing the pH of the media (pH 8 was found to be effective). We made M9 media at pH 8 before making the same set of test plates as before. It was found that the NAs stayed in solution when mixed, however came out of solution when the plates dried, leading to a grease layer on the plates as previously seen. </P><br />
<p>&nbsp;&nbsp;In another attempt to raise the pH, stock solutions of NAs dissolved in NaOH at pH 12 were made, and these were autoclaved alongside the media. When NAs were added to the NaOH, the solutions became cloudy and uniform, indicating they were in solution. When the NA stocks were added to the media before plate pouring, the NAs remained in solution even after the plates dried, indicating that this method would be sucessful in making test plates. Using this method, the following plates were created:<br />
<br />
<center><table><br />
<table border="1" CELLPADDING=3 CELLSPACING=1 <br />
RULES=NONE FRAME=BOX table width=600px height=150px><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Wild-Type PF-5 Controls</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media Alone, M9 Minimal Media + 0.2% Glucose, M9 Minimal Media + 0.2% Lactose</td><br />
</tr><br />
<tr><br />
<tr title="You are looking at Row 1" bgcolor="66ff66"><br />
<td><b>Transposon Replica Plates</b></td><br />
</tr><br />
<tr><br />
<td>M9 Minimal Media + 0.2% Glucose + Chlor, M9 Minimal Media + 0.2% Glucose + Chlor + 0.05% NA's, M9 Minimal Media + 0.2% Glucose + Chlor + 1% NA's, M9 Minimal Media + 0.2% Lactose + Tet, 0.2% Lactose + Tet + 0.05% NA's, M9 Minimal Media + 0.2% Lactose + Tet + 1% NA's </td><br />
</tr><br />
</table></center><br />
<br />
<p><br />
<br></br><br />
&nbsp;&nbsp;Xgal was spread on all the plates with NA's, however it was left out of the controls by mistake. Stamping was carried out as previously described from both the previously created 1/100 and 1/1000 dilution plates of transposon mutants (A new round of transposon mutagenesis was initiated, but the mating mixture was incubated at 30&deg;C instead of 37&deg;C, and no mutants were obtained on the selective plates). These plates were allowed to grow over the weekend at 30&deg;C.</P><br />
<br />
<h2>Week 9 (June 25-June 29)</h2><br />
<p>Growth was observed from the selection plates incubated over the weekend at 30&deg;C. Screening replica plates are made as before. The 1/1000 diluted transposon plate was used for the replica plating on all the screening conditions. The screening plates are incubated overnight at 30&deg;C. The next day, no growth was observed on the negative control plates (M9 alone, M9 with 0.05% NAs), and no growth was observed on the positive control plates containing glucose. Similarly, no growth was observed on the M9+lactose (+Tet, with/without X-GAL, with/without 0.05% NAs). This suggests that either a large number of matings is needed before a desirable transconjugant is seen, or that the media condition requires modification. The plate surface is not as greasy as before, which suggests that the replica plating transfer process works. However, the pH of the media may affect the growth of transconjugants. Plans are made to modify the pH of the media before replica plating. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>Replica plating screening plates are made so that NA stock solutions (1% at pH 12 diluted to 0.05% final concentration) were added to the M9 agar solution. The pH is adjusted to 7.4 before autoclaving. The pH prior to adjusting is approximately 9.0. This may explain the poor growth observed in the previous trial. A new series of mating were started (2 mating spots), and incubated overnight at 37&deg;C. The mating spots are scraped and serial diluted up to 1/1000, plated on LB+Chlor50+Tet50, and incubated overnight at 30&deg;C. This time, the results are consistent with the previous attempt, where no growth was observed on the negative control plates (M9 alone, M9 with 0.05% NAs), and ample growth was observed on the positive control plates containing glucose. However, still no growth was observed on the M9+lactose (+Tet, with/without X-GAL, with/without 0.05% NAs)plates. This seems to confirm with previous hypothesis that a large number of matings are needed to screen the genome for NA sensitive elements. Alternatively, perhaps the NA concentration used here is too high for proper selection of the transconjugants that have a promoter element upstream of the transposon insertion. If the NA concentration used for the screen is too high, the promoter may be suppressed, and the lacZ is not expressed for cell survival. In fact, the lowest NA concentration used up to this point is 0.05% or 500 mg/L. The culturing conditions required to maintain Pseudomonas Pf-5's NA degrading abilities is LB+50mg/L. Therefore, to allow both cell survival and screen for the most robust and sensitive system, maybe the NA concentration should be lowered.</p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The exact same experiment from the previous week has been repeated with two more mating mixtures (and their dilutions). However, we obtained the same results, with the positive and negative controls yielding predicted growth and no growth, respectively. But no growth was observed on the lactose plates. Furthermore, upon close examination of the selection plates after the conjugating/bipartite mating reaction (LB+Chlor+Tet), two colony morphologies can be seen: drier and larger colonies (resembling Pseudomonas), and smaller moist colonies (resembling E. coli). This suggests perhaps the LB+Chlor+Tet plates may be useful for selecting Pseudomonas transconjugants, but it does not prevent the donor E. coli to be efficiently killed. A final replica plating experiment is designed for next week, and Pseudomonas Isolation agar (PIA, just received)will be used for selection instead. </p> <br />
<br />
<h2>Week 12 (July 16-July 20)</h2><br />
<p>Another mating reaction was set up, and plated on PIA+Tet50 to select for positive transconjugants. The colonies from the resulting mutants show only a single colony morphology consistent with Pseudomonas. From this, we know the selection is appropriate. The 1/1000 dilution of the mating reaction dilution is replica-plated on two sets of screening plates. The first set is prepared the same way as the previous week (NAs added and the media pH adjusted prior to autoclaving); the second set is the same except the NAs are added by spreading 100uL of a 50mg/L sterile stock NA solution in NaOH (pH12) prior to replica plating. The 1/1000 dilution plate of the mating reaction is replica plated on both plates (with the same controls as before). However, the same results are obtained as before, where no growth is observed on the M9+0.2% lactose+X-GAL or the M9+0.2% lactose+X-GAL+50mg/L NAs plates. Lastly, we had an interesting observation where overnight growth of the lactose screening plates showed no growth, if they are left for up to 48hr, some colonies can be observed; however, none of the colonies are blue. The problem may be that the M9 media conditions are not suitable for selecting the Tn mutants, or that the replica plating approach we used cannot effectively transfer all colonies. At this point, since no NA-sensitive strains are isolated, a new approach needs to be taken in order to find a tranposon insertion mutant that has an NA-sensitive promoter upstream. Since all possible approaches have been taken using the replica plating strategy, another mass screening method is needed. </p><br />
<br />
<h2>Week 13 (July 23-July 27)</h2><br />
<p> Even though, the previous screening method is quick and convenient, the waiting period is too long, and no real time data in terms of growth or lactose utilization can be observed/discerned. Therefore, a 96-well mass screening method is devised, where the conjugation mating reaction, and the selection on PIA+Tet50 are the same, but the screening step is different. The screening media is a M9 minimal media with 50 µg/mL tetracycline to maintain the genomic Tn insertions, 0.2% lactose to select for lactose utilization due to Tn insertions, and 50 mg/L NAs to maintain the strain's ability to use/degrade NAs (in short the media is M9+0.2%lactose+50µg/mL Tet+50mg/L NAs). On each 96-well plate, 94 colonies are inoculated and screened; the other two wells are used for negative and positive controls. The negative control well has the same media (M9+0.2%lactose+50µg/mL Tet+50mg/L NAs), with no colonies inoculated. The positive control has M9+0.2%glucose+50µg/mL Tet+50mg/L NAs, and is inoculated with a random colony.<br />
<br />
</p><p><br />
<br />
Using this new method, any inoculated colony that grows in the liquid media can utilize lactose, and this should be because of a transposon insertion downstream of a promoter. To select for a NA sensitive promoter, each colony is inoculated into two plates with the same media components (as described above), except one has NAs, and the other does not. If a promoter is NA-sensitive, then it should not grow if NAs are absent from the media, as the NA mixture would activate the promoter. When NAs are present however, the NA-sensitive promoter would turn on, allowing it to utilize lactose, survive, and grow. <br />
<br />
As a first run of this method, a plate is set up using mutant colonies from a previous mating reaction. This plate contained M9+0.2%lactose+50µg/mL Tet+50mg/L NAs. The appropriate control plate with no NAs was not included as this is only to test feasibility of this approach. The plate is incubated over the weekend for 60hrs in a plate reader at 30&deg;C. </p><br />
<br />
<h2>Week 14 (July 30-August 3)</h2><br />
<p>The data from the plate incubated from last week was collected. First, since the lid of the 96-well plate was kept on the plate to prevent dehydration of the media, there was significant condensation on the middle of the lid (covering about 30 wells), which prevented proper absorbance measurements of those wells (data from these wells were omitted). However, six wells/colonies demonstrated growth from a baseline. The following table is a display of the 96-well plate at the end point, with the colonies/wells that grow were highlighted.</p><br />
<br />
</html>[[File:Ucalgary2012 0801data1.png|center|thumb|400px|Figure 2: A 60hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media]]<html><br />
<br />
<p>The cultures from these six wells are washed with M9+0.2%lactose+50µg/mL Tet (no NAs) three times, and resuspended in 1 mL of M9+0.2%lactose+50µg/mL Tet. 500µl of each colony was inoculated into 5mL cultures with M9+0.2%lactose+50µg/mL Tet or M9+0.2%lactose+50µg/mL Tet+50mg/L NAs. These cultures are grown over 48 hours to observe growth (any strain that grows only when NAs are added would be noted). Also, the colonies are restreaked on PIA+Tet50 plates to ensure that the absorbance increase actually indicated growth (since the increase was so small at around +0.200). The results were not promising, on the restreaked plates, only 2 colonies (from H12 and B1) showed growth, this suggests some of the measurements were not very accurate. Also, in the culturing experiment, only 1 set of cultures (from 1 colony, H12) showed growth, but in both media conditions, which is not desirable.</p><br />
<br />
<p>Even though, no colonies were shown as NA sensitive, this result is still very promising, as this experiment shows that some colonies from the transposon mutagenesis can actually utilize lacZ, which demonstrates that the transposable element approach is an appropriate and feasible approach for screen promoters sensitive to environmental stimuli. (Please note, that mating reactions and mutant selection are conducted regularly every week, usually twice per week, with two mating spots each time, to provide mutants for these screens.)</p><br />
<br />
<h2>Week 15 (August 6-August 10)</h2><br />
<p><br />
The same experiment from the previous week was repeated. However, the plate was not incubated in the plate reader, but incubated in a 30&deg;C shaker, at 140rpm. At the baseline (beginning of incubation), 24hr, and 48hr, timepoint measurements were made. The results are as follows.<br />
</p><p><br />
</html>[[File:Ucalgary2012_080312.png|center|thumb|400px|Figure 3: A 48hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media at time points (baseline, 24hr, and 48hr)]]<html><br />
</p> <br />
<br />
<p><br />
However, these measurements were made with the lid of the plate on. Even though there was no clear trend in terms of the growth in the absorbance data, it is possible to observe some wells become cloudy by eye. For instance, well F9 was cloudy at the end of the 48hr incubation, but the data showed no change in absorbance. The lid should be taken off in the future when making timepoint measurements. Also, the absorbance data can support the visual data.<br />
</p><br />
<br />
<p><br />
Also, this week, two runs of the screening experiment was conducted with two plates run in parallel, one with NAs and the other without (the proper screening setup). However, it was found that the lactose solution (which was filter sterilized) used to make the M9 media for the screen was contaminated with yeast. Therefore, new media solutions and lactose solutions needed to be made. These data are not used because of this.<br />
</p><br />
<br />
<h2>Week 16 (August 13-August 17)</h2><br />
<p><br />
New lactose and glucose stock solutions as well as new M9 screening media were prepared. The experiments from last week were repeated using proper solutions. Also, to prevent fogging of the plate lid and cross-contamination, all media solutions and plates were pre-warmed in a 30&deg;C incubator before dispensing the media into the plates and colony inoculation. The new trial, again, has two plates, with the same wells (e.g. A1) inoculated with the same colony, where one plate has M9 screening media (with Tet and lactose) alone and the other plate containing 50mg/L NAs. Two runs were completed this week. The data from one of those trials are shown below.<br />
</p><p><br />
</html>[[File:Ucalgary2012 081612.png|center|thumb|800px| Figure 4: A 48hr incubation of 94 Pseudomonas fluorescens pf-5 transposon insertion mutants measuring absorbance at 600nm in screening media at time points (baseline, 24hr, and 48hr), comparing the same colonies in M9 screening media with/without 50mg/L NAs]]<html><br />
<br />
<p><br />
In this particular run, only two colonies showed growth (D8 and H11), these were subcultured in 5mL of M9 screening media with NAs. An interesting observation is that there is more growth in the lactose+NAs plate than the lactose alone plate. This suggests that the NAs may play a role in activating the lacZ gene, and improving the survival rates. These results are consistently observed in the other trials conducted. <br />
<br />
<p><br />
At this point, since time is limited, the entire mutant selection process needs to be more efficient. Therefore, the mating spots are diluted, and plated on a more selective media, containing PIA+50ug/mL tetracycline+50mg/L NAs+20 μg/mL X-Gal. This way, the NA-responsive and/or lactose-utilizing Pseudomonas transconjugants can be selected as they would appear blue on the media. This reduces the number of colonies to be inoculated, and improves the efficiency of the whole process. (Note: Wildtype Pseudomonas fluorescens pf-5 are always plated as positive control to observe the natural forward mutation rates, and to ensure that the amount of spontaneous tetracycline resistant Pf-5 mutants are at a reasonable level.<br />
</p><br />
<br />
<p><br />
At this point, a large number of matings should be conducted and mutants selected, in order to provide enough of lactose-utilizing and NA-sensitive Tn-insertion mutants, and to cover as much of the Pseudomonas genome as possible<br />
</p><br />
<br />
<h2>Week 17 (August 20-August 24)</h2><br />
<p>No work on the transposon library was done this week, other than planning for the upcoming screen.</p><br />
<br />
<h2>Week 18 (August 27-August 31)</h2><br />
<p>This week, a large screen was accomplished. First, cultures of <i>P. fluorescens</i> Pf-5 and <i>E. coli</i> SM10 were grown up overnight at 30&deg;C and 37&deg;C respectively to a high optical density. The following morning, the <i>E. coli</i> was subcultured in a 1/4 dilution to dilute out the tetracycline, and grown for approximately 2 more hours. After this, 100 &micro;L of each culture were mixed, spun down, and resuspended in 20 &micro;L of media before being spotted onto LB plates.</p><br />
<p>In total, 500 separate mating spots were plated. These spots were allowed to grow at 37&deg;C overnight. The following day, 2 mating spots were scraped up, combined, and resuspended in 500 &micro;L PBS. 1/400 dilutions of these resuspensions were made, and 100 &micro;L of these was plated onto selective plates consisting of PIA, 100mg/L NAs, and tetracycline. These plates had 40 &micro;L of 20 mg/mL X-gal spread on their surface in order to allow for blue-white screening. 250 plates were made in total.</p><br />
<br />
<p></html>[[File:2012-08-29 21-32-07 236 Calgary.jpg|center|500px|thumb|Figure 5: Plates in incubator]]<html> <p><br />
<br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<br />
<p>Plates were left to grow for 2 days, after which blue colonies (24 in total) were selected and pinned in duplicate into 96-well plates for response testing. Initially, minimal media + lactose with and without NAs was used, however no growth in any of the wells was observed. Because of this, the screening protocol was altered such that LB with or without NAs was used instead of minimal media, and X-gal was used instead of lactose for screening- the idea being that if naphthenic acids were sensed, a blue color change would be observed relative to the negative LB control.</p><br />
<br />
<p></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 6: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16h. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><br />
<br />
<p>When results were observed it was found that 4 colonies showed clear differential regulation in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. Therefore, these colonies will be used in further screening to test the specificity of the response.</p><br />
<br />
<br />
<h2>Week 19 (September 10- September 14)</h2><br />
<p>This week, further screens on the previously identified four hits were performed. These involved the use of different toxins at environmentally relevant concentrations to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used in one of the media samples in order to attempt to rule out a general stress response by the cell.<br />
<br />
<p></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 7: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:170-1data.png|thumb|600px|center|Figure 8: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 9: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 10: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p>Due to these results, further screens on these two colonies will be performed, using lower hydrogen peroxide concentrations to rule out a general stress response, and decanoic acid to rule out a response to fatty acids.</p><br />
<br />
<h2>Week 20 (September 17- September 21)</h2><p><br />
Further screens were conducted, however due to cells drying out in the plate the results were invalidated. Because of this, the screen will be repeated, and explained in further detail when this is done.</p><br />
<br />
<h2>Week 21 (September 24- September 28)</h2><p><br />
Genomes of 66-1 and 170-1 were isolated, digested with BglII and with XbaI, and religated before being transformed into <i>E.coli</i>. Cells were plated onto tetracycline plates to isolate cells containing a ligation product with the transposon present. These colonies have been miniprepped, and are awaiting being sent for sequencing to determine which genes the transposon has been inserted into. </p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/BiosensorTeam:Calgary/Notebook/Biosensor2012-10-03T05:05:35Z<p>Achawdhry: </p>
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<div>{{Team:Calgary/TemplateNotebookGreen|<br />
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CONTENT =<br />
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<!--<br />
NOTE: This is a template for entering things for the time being. All dates should be enclosed in <h2> tags and all paragraphs should be enclosed in <p> tags. For bulleted lists, <ul> tags will create the list and <li> tags will surround each list item. If there are any questions, please let me know.<br />
<br />
Patrick.<br />
--><br />
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<br />
<h2>Week 1 (May 1-4)</h2><br />
<p>This week we went to Biosafety and WHMIS training, where we learned safety procedures and protocols that will be useful when we get to the lab.</p><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>We read some papers about the potentiostat we will be using for the biosensor, and began designing a circuit to take a triangular wave form from 0 to 5 V, amplify it, and offset it to get a waveform from -2 V to 2 V. A large portion of this week was also spent learning how to use MATLAB and LabVIEW software platforms for data acquisition and analysis.</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The circuit design was finalized this week, and we began implementing it on the breadboard. Upon testing the circuit, we found that it was not giving us the predicted results. After a day of troubleshooting, we determined that the Operational Amplifiers were fried and needed to be replaced. Once the circuit was operational, we began using a DAQ to generate an input waveform and measure an output. We wrote a LabVIEW vi for generating a triangular waveform, but had trouble writing software for the measurement. We are currently getting a 'onboard memory overload' error whenever we try to generate output and take back input at the same time.</p><br />
<br />
<h2>Week 4 (May 21-25)</h2><br />
<p>This week we found the bugs in our software that were giving us an error whenever we ran simultaneous signal generation and measurement with the DAQ. By slowing the sampling rate (for both input and output), and giving a longer buffer period for the data to be transferred, we were able to get the vi to work entirely. We tested our circuit and the potentiostat using screen printed carbon electrodes in a solution of PbS. This yielded a noisy graph vaguely resembling a voltammogram. The noise was filtered with a digital filter, and gave us a much more readable voltammogram. Next, we tested the potentiostat after adding Chlorophenol Red (in 200 uL increments) to the PbS solution. Based on the small peaks that emerged, we are able to detect CPR electrochemically with our potentiostat!</p><br />
<br />
<h2>Week 5 (May 28-June 1)</h2><br />
<p> This week we focused on moving the breadboarded circuit to a prototyping board for ease of use and increased portability. Having little soldering experience, the first board was more of a 'test run', and indeed we ran into several problems, including a short circuit that caused one of the 9V batteries to overheat. The majority of the week was spent refining our technique as well as mapping out a new and improved circuit diagram to ensure success on our next attempt. This involved moving the power supplies to a common ground rather then hooking the batteries up in series away from the board. Another improvement was the quality of user-friendliness. An example of this was to use different colored wiring for the power, ground, and internal wiring on the board. This made navigation much simpler. An improvement that we look forward to next week would be the addition of two hardware filters, as well as higher quality op-amps. We would also like to implement all of this into a fully functional prototype available for field testing, complete with switches, pre-cut holes for usb cords, and easier to use battery compartments.</p><br />
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[[File:Earlyprototype.JPG|thumb|200px|right|Figure 1: an image from the Shire...]]<br />
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<h2>Week 6 (June 4-June 8)</h2><br />
<p> This week we focused on the design of three new hardware filters, instead of the original two we thought of before. The reason for the addition of this extra hardware filter would be to further filter out noise before and after the solution, as well as directly after the DAC input into the board. We also spent some more time on the electrochemistry portion, further making sure that our calculations were correct from the original design. Also, we began learning Maya to digitally prototype the final design for our biosensor. </p><br />
<br />
<h2>Week 7 (June 11-June 15)</h2><br />
<p> This week was spent doing many different things. Firstly we chose our values for the capacitors in the hardware filters, as well as the resistor values. Also, we took a business trip to the Glencoe Club, speaking to many different entrepreneurs. Further work was done using Maya, however this was limited as it is a fairly steep learning curve. All that remains to do now is finalize a design on paper for what we think will be the final prototype, design it, and then implement those designs. </p><br />
<br />
<h2>Week 8 (June 18-June 22)</h2><br />
<p> This week we spent a day in Robert's lab looking at some of his electrochemistry. We also spent some time working on software, with one of us working on MATLAB (for data acquisition) and the other working on Maya (for a visual prototype). We made some changes to the potentiostat circuit in the midst of some issues we had with our counter electrode voltages in solutions of different conductivity. The previous version of the potentiostat had 1 kOhm resistors in the first op-amp circuit, but this design relied on the solution have very low resistance. In order to make the counter electrode voltage independent of the solution resistance, we replaced the 1 kOhm resistors with 100 kOhm resistors. This seems to have solved the problem.</p><br />
<br />
<h2>Week 9 (June 25-June 29)</h2><br />
<p>This week was spent troubleshooting our original circuit, as we seemed to have inconsistent results when measured with the oscilloscope. A more complete model in maya was also worked on. Much time however was spent on checking the prototyping board for any errors.</p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>This week the filter was finally put in, after much painstaking troubleshooting and a meeting with our supervisor. It appears to be working correctly, which means we are able to move on to the other filters (if we require them) next week. Also, the maya model was outsourced to another member of the iGEM team, and is looking quite spectacular thus far. More work on that to come. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The conversion from LabView to MATLAB software for data acquisition is in the works. We are hoping to have a full conversion by next week. There is also and unexplained malfunction on the circuit around the fourth op-amp circuit. It is possible that our original soldering quality may have been the cause.</p> <br />
<br />
<h2>Week 12 (July 16-July 20)</h2><br />
<p>The full conversion to MATLAB is complete! Now the doors are open to superior data analysis capabilities and fewer glitches with the DAQ operation. James is working on an algorithm to measure the peaks in our voltammograms and generate a quantitative measurement of the concentration of solute. The circuit is still giving us trouble, however.</p><br />
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[[File:PHOTO.jpg|thumb|250px|right|The circuit as of July 27th|left]]<br />
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<h2>Week 13 (July 23-July 27)</h2><br />
<p>This week we soldered an entire new board, leaving behind the poor job of the last one. The new one is complete with a low pass filter as well as an anti-aliasing filter to produce even better results. Several tests were run with both PBS solution with CPR, as well as with PDP. The software is nearing completion, and the circuit is almost ready to be put onto a printed board.</p><br />
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<h2>Week 14 (July 30-August 3)</h2><br />
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[[File:Circuit_Schematic.PNG|thumb|250px|left|Circuit Schematic]]<br />
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<br />
<p>This week was spent completing a number of different objectives. Firstly, the software was downloaded to design the schematic for the circuit to be translated onto a PCB. Much of the week was spent doing tutorials and learning the user interface to make a nice circuit. Also, the circuit, complete with switches for the batteries as well as a DIP switch to control resistance, was given to the Biomedical Technology Workshop for manufacturing into a case. This will serve as the first prototype (not the PCB), and steps were taken in order to ensure it was pleasing to the eyes. </p><br />
<br />
<br />
<h2>Week 15 (August 6-August 10)</h2><br />
<p>This week the early prototype was completed. The DAQ and circuitry have been enclosed in a black box with a plexiglass lid. It is complete with battery switches, multi-coloured LEDs, and a variable DIP switch. This portable prototype is ready to test in the Birss lab against the commercial potentiostat.</p> <br />
<br />
<h2>Week 16 (August 13-August 17)</h2><br />
<p>This week we tested our prototype in the Birss lab with Robert. Initially, we tried to use his electrode setup (including a platinum electrode and a SHE) but it appears the resistance of the salt bridge is too much for our potentiostat to handle. Robert converted his setup to use our blue strip electrodes so that we can compare results more easily. Before we could get any results, we ran into trouble with the circuit. It appears there is a short around the battery that is affecting the function of the potentiostat. Nick is going to fix this so we can begin testing early next week.</p><br />
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[[File:Socket.JPG|thumb|200px|right|Figure 3: The culprit... a wet electrode socket]]<br />
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<h2>Week 17 (August 20-August 24)</h2><br />
<p>This week the problem with the short circuit around the battery was repaired, and a new battery holder was put in. A new clip to hold the electrodes was also soldered in. A crucial note is that if any liquid at all enters this clip, there is a short circuit that botches trials. This may explain some previous difficulty we have had with the circuit. Once we obtained access to the Birss lab, results finally came our way. We were able to successfully detect PDP, and it was similar to a commercial potentiostat. Other than that, not much more work was done on the circuit this week, as it was difficult to obtain access to the Birss lab after those couple of days. </p><br />
<br />
<h2>Week 18 (August 27-August 31)</h2><br />
<p>This week we did further testing of the prototype in the Birss lab, where our success was hit and miss. We obtained data. </p><br />
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<h2>Week 19 (August September 3-September 7)</h2><br />
<p>Block week courses :(</p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Outreach/TEDxCalgaryTeam:Calgary/Outreach/TEDxCalgary2012-10-03T04:54:59Z<p>Achawdhry: </p>
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<p> This year our team had the opportunity to partner with TedxCalgary in developing a Ted talk which we will be presenting on October 13th in Calgary. We think this will be a great opportunity to present to not only our city, but also a part of a larger, international event (City 2.0). We had a lot of fun thinking about synthetic biology and our project and the best way to share that in a relevant and meaningful way.</p><br />
<br />
<br />
<h2> City 2.0 </h2><br />
<p>Our talk will be part of the City 2.0 event. It is an award winning event that will occur concurrentlly in over 40 cities around the world on October 13th. This event is inspired by many ideas around building better cities, but is trying to move on to the next step: taking action. The themes involve Art, Food, Education, Public Space, Health and more! The ultimate goal is to create the city of the future “inclusive, innovative, healthy, soulful, and thriving!”</p><br />
<br />
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</html>[[File:UofC_City_2.0_Picture.png|centre]]<br />
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<html><br />
<br />
<h2> "Ideas Worth Sharing" </h2><br />
<br />
<p>In accordance with Ted’s mandate, we needed an idea worth spreading. We had a lot of ideas about our project, the iGEM competition and synthetic biology in general. The hard part was coming up with the right theme for our presentation; one that would resonate with the general public both within and outside Alberta. <br />
One of the reasons we chose the project that we did is that it is something important to Alberta but that can also have implications around the globe – environment and energy. There are obvious implications of our project for the oil sands in Alberta, but also to a broader audience in terms of environmental remediation. This was clearly an important theme to us. We felt that the fact that the bacteria we are desigining not only remediate wastes, but can convert them into reusable fuels was particularly important. Every day, the landfills around the world have been growing incessantly. Communicating the potential for applying synthetic biology to clean or recycle the wastes and reclaim these lands would be an interesting focus. What is more exciting is the potential this gives to maintain a self-sustainable environment using this technology. Instead of relying on the city for energy and waste processing, we could use synthetic biology to generate our own energy from waste products.<br></p><br />
<br />
</html>[[File:UofC_CiTYPicture.png|742px|centre]]<html><br />
<br />
<p><br>Outside of the environmental implications of our project, open source was also an interesting theme as it is a major underlying premise of both synthetic biology and iGEM in general. The open access nature coupled with the drive for abstraction in synthetic biology make it something with huge potential for participation, something missing from other scientific fields. The amount of knowledge required to master biology is immense, but building systems with standard parts is not so hard. Just take a look at the things coming out of iGEM each year and you’ll understand. Undergraduates on small budgets can accomplish quite a lot in a summer! This idea of democratizing science was thus another angle worth exploring.</p><br />
<br />
<p>The theme we chose to go with was merging these two. We wanted to emphasize the potential synthetic biology could have in remediation, while showing that this technology can be used by many, allowing people to actively participate in science and synthetic biology, and in turn help improve the environment.<br />
Through our TED talk, we want to emphasize the democratization of science and generate new discussions and ideas for improving our health and our cities’ health through better environment, raising more questions on how to build a self-sustainable city!<br />
</p><br />
<br />
<h2> Check it out! </h2><br />
<p><a href="http://tedxcalgary.ca/events/city-2-0" target="_blank">Find our talk after Jamboree at TEDxCalgary website.</a></p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Outreach/TEDxCalgaryTeam:Calgary/Outreach/TEDxCalgary2012-10-03T04:53:08Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateOutreach|<br />
TITLE=TEDxCalgary|<br />
CONTENT=<br />
<html><br />
<br />
<p> This year our team had the opportunity to partner with TedxCalgary in developing a Ted talk which we will be presenting on October 13th in Calgary. We think this will be a great opportunity to present to not only our city, but also a part of a larger, international event (City 2.0). We had a lot of fun thinking about synthetic biology and our project and the best way to share that in a relevant and meaningful way.</p><br />
<br />
<br />
<h2> City 2.0 </h2><br />
<p>Our talk will be part of the City 2.0 event. It is an award winning event that will occur concurrentlly in over 40 cities around the world on October 13th. This event is inspired by many ideas around building better cities, but is trying to move on to the next step: taking action. The themes involve Art, Food, Education, Public Space, Health and more! The ultimate goal is to create the city of the future “inclusive, innovative, healthy, soulful, and thriving!”</p><br />
<br />
<br />
</html>[[File:UofC_City_2.0_Picture.png|centre]]<br />
<br />
<html><br />
<br />
<h2> "Ideas Worth Sharing" </h2><br />
<br />
<p>In accordance with Ted’s mandate, we needed an idea worth spreading. We had a lot of ideas about our project, the iGEM competition and synthetic biology in general. The hard part was coming up with the right theme for our presentation; one that would resonate with the general public both within and outside Alberta. <br />
One of the reasons we chose the project that we did is that it is something important to Alberta but that can also have implications around the globe – environment and energy. There are obvious implications of our project for the oil sands in Alberta, but also to a broader audience in terms of environmental remediation. This was clearly an important theme to us. We felt that the fact that the bacteria we are desigining not only remediate wastes, but can convert them into reusable fuels was particularly important. Every day, the landfills around the world have been growing incessantly. Communicating the potential for applying synthetic biology to clean or recycle the wastes and reclaim these lands would be an interesting focus. What is more exciting is the potential this gives to maintain a self-sustainable environment using this technology. Instead of relying on the city for energy and waste processing, we could use synthetic biology to generate our own energy from waste products.<br></p><br />
<br />
</html>[[File:UofC_CiTYPicture.png|742px|centre]]<html><br />
<br />
<p><br>Outside of the environmental implications of our project, open source was also an interesting theme as it is a major underlying premise of both synthetic biology and iGEM in general. The open access nature coupled with the drive for abstraction in synthetic biology make it something with huge potential for participation, something missing from other scientific fields. The amount of knowledge required to master biology is immense, but building systems with standard parts is not so hard. Just take a look at the things coming out of iGEM each year and you’ll understand. Undergraduates on small budgets can accomplish quite a lot in a summer! This idea of democratizing science was thus another angle worth exploring.</p><br />
<br />
<p>The theme we chose to go with was merging these two. We wanted to emphasize the potential synthetic biology could have in remediation, while showing that this technology can be used by many, allowing people to actively participate in science and synthetic biology, and in turn help improve the environment.<br />
Through our TED talk, we want to emphasize the democratization science and generate new discussions and ideas for improving our health and our cities’ health through better environment, raising more questions on how to build a self-sustainable city!<br />
</p><br />
<br />
<h2> Check it out! </h2><br />
<p><a href="http://tedxcalgary.ca/events/city-2-0" target="_blank">Find our talk after Jamboree at TEDxCalgary website.</a></p><br />
<br />
<br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Outreach/BlogTeam:Calgary/Outreach/Blog2012-10-03T04:52:04Z<p>Achawdhry: </p>
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<p>Genome Alberta sponsored us this year to share with their website's audience some of the interesting things we were doing with genetics and synthetic biology this year. We have authored a number of the blog posts and been featured in one of their writer's blog posts. This was a great way to interface with industry and show a more 'humanistic' side of iGEM. It also provided a great opportunity to connect with Alberta and Canada's scientific community and industries.<br />
<br />
<h2>The Blog Posts</h2><br />
<br />
<h2></html>[http://genomealberta.ca/blogs/tackling-tailings-ponds-the-igem-way.aspx Tackling Tailings Ponds - the iGEM Way]<html></h2><br />
<br />
<p><br />
<br />
<h2></html>[http://genomealberta.ca/blogs/jumpin-at-the-agem-jamboree.aspx Jumpin' at the aGEM Jamboree]<html></h2><br />
<br />
<p><br />
<br />
<h2></html>[http://genomealberta.ca/blogs/sleepless-nights-in-the-lab.aspx Sleepless Nights in the Lab]<html></h2><br />
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<p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Outreach/MindsInMotionTeam:Calgary/Outreach/MindsInMotion2012-10-03T04:49:37Z<p>Achawdhry: </p>
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<br />
<h2>Reaching Out To Our Neighbours</h2><br />
<br />
<p>It may be difficult to admit but the average person often has little to no knowledge about synthetic biology. If people have never heard of this field it is hard to expect them to accept this concept as a viable alternative to products used in society. This is why it is a major objective of our project to be involved in the community. One way we wanted to approach this situation was to target a young audience, especially those enthusiastic about science. The atmosphere when working with kids is unlike that of adults, as kids often have a different opinion and mindset. Therefore, when given the opportunity to work with Minds in Motion we knew that this was a chance to work with an audience unusual for a university setting.</p><br />
<br />
<h2>Exploring Synthetic Biology with Minds in Motion</h2><br />
<br />
<p>Minds in Motion is a student led organization that focuses on providing summer camps to kids of all ages at the University of Calgary. Each year the camp aims to expose youth to the exploration of science, engineering and technology through innovating and hands-on projects. This year our University of Calgary iGEM was very fortunate to be invited several times as a special guest working with kids age 10-12. During this time our goal was to provide our audience with a general understanding of synthetic biology through hands-on activities that showcases proper wet lab techniques. </p><br />
<br />
<h2>The Activities</h2><br />
<br />
<p>Using simple common household items (balloons and yarn), the Minds in Motion campers would follow along as we explain the idea of inserting a gene into a bacteria. This was done through analogies such as blowing up the balloon to represent transformation or popping the balloon to simulate DNA isolation (mini prep). The next activity included using pipettes and simple LB plates to explain the process of preparing bacteria for plating. This activity was meant to explain proper use of equipment, techniques to perform wet lab work and display bacteria with different colour (GFP). For the last hands-on activity, we attempted to inform the campers the basics of acidity and basicity by testing common liquids and determining their pH. </p><br />
<br />
<h2>As Kids See Synthetic Biology</h2><br />
<br />
<p>Working with the participants at Minds in Motion it was evident that the kids were intrigued with the idea of synthetic biology. Throughout the presentation, there was constant active participation from the audience and many engaging question which further the discussion. Lastly at the end of the presentation we asked the kids what is something new that they learned from our visit. Surprisingly, everyone had something to talk about. Some mentioned the neat things that synthetic biologists have done like making rats glow, others talked about proper techniques and use of equipment. Another time, we received a thank you card from the kids at the Minds in Motion as a token of appreciation for being a special guest. </p> <br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Outreach/SparkTeam:Calgary/Outreach/Spark2012-10-03T04:45:28Z<p>Achawdhry: </p>
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<h3> </h3><br />
<h2>Sharing Synthetic Biology</h2><br />
<br />
<p>Telus Spark has given us the amazing opportunity to share and engage with the general public to demonstrate through hands on activities what biology is and how we can use synthetic biology to solve problems. The Science Centre brings together a huge range of people, from young children and their parents, to adults of all ages at their monthly Adults Only Nights. We have shown off our work and an exhibit meant to demonstrate the cool factor of synthetic biology. The first event we ran was Bacterial Art, and the second was an public beta test of our video game.<br />
<br />
<h2>Bacterial Art</h2><br />
<br />
<p>We have been able to let hundreds of people try their hand at making magnificent art by drawing bacteria containing different fluorescent biobricks onto agar plates. Stocks of bacteria with various colors of fluorescent protein plasmids were prepared. Visitors were got to use their imaginations and 'draw' the bacteria onto agar plates to make pieces of art. These agar plates contained a antibiotic to ensure the plasmid containing the fluorescent marker was not shed by the bacteria. These plates were then incubated overnight, before being photographed under a UV light and being posted to Telus Spark's <a href=https://www.facebook.com/media/set/?set=a.10152086146370538.896423.24253660537>facebook page</a> for the public to tag as theirs.<br />
<br />
<p>This event let us go hands on with the public with bacteria that we use everyday in our lab. We got to explain what the bacteria were, how we changed them, and a bit of the biology on how biobricks coding for fluorescent proteins work. Not only that we got to challenge some of the negative press that bacteria have by showing people that there are bacteria out their that do not cause disease and that can do amazing things.<br />
<br />
<p><br />
<br />
<h2>LAB ESCAPE Video Game Premiere</h2><br />
<br />
<p>Our second major event was the first public beta test of the <a href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game</a> we have been developing throughout the summer. We spent two days on a weekend (September 29-30, 2012) letting the general public try out our video game and learn a thing or two about synthetic biology. Players of the game gave us valuable feedback on places where we could improve the game, make it more clear, and most importantly how to make it more fun!<br />
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<h2>Creating LAB ESCAPE: A Synthetic Biology Video Game</h2><br />
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<p>The iGEM 2012 Calgary team ventured into new territory by developing a video game centred on synthetic biology called <b>LAB ESCAPE</b>. As scientists, we are often asked, “What exactly do you do all day?” and “What do you mean you can see DNA?” Our video game provides an opportunity to educate and entertain the player by guiding them through a routine experiment used by the iGEM 2012 Calgary team. LAB ESCAPE focuses on gel electrophoresis – a common molecular biology technique to separate and visualize DNA from PCR and restriction digests. We also incorporated basic laboratory safety procedures to further immerse the player in the research experience. </p><br />
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<p><b><FONT COLOR="FF7A00">LAB ESCAPE is part science, part fun, and all iGEM.</FONT></b></p><br />
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<h2>From Storyboard to Application</h2><br />
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<p>The initial stage in the development of LAB ESCAPE focused on creating an engaging environment resembling a typical synthetic biology research lab. We then created a script outlining the player's actions as they find themselves locked in the lab, progress through the tasks to find the secret code, and (hopefully) escape. We originally wanted to incorporate numerous experimental techniques, but we decided to focus on one common technique - gel electrophoresis. This avoided over-whelming the player with multiple experiments and also kept the time required to an optimal level. Once the scene was set we jumped into the world of video game creation.</p><br />
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<p>The iGEM 2012 Calgary team created all the graphical images for LAB ESCAPE using Adobe Illustrator. Adobe Flash was used as the platform for the programming code creating LAB ESCAPE, with some programming code being modified from open-source repositories. With these tools, we designed a ‘point-and-click’ adventure video game, where the player collects items hidden throughout the virtual environment, then complete a task to obtain the code required to open the locked lab door. We even composed our own musical soundtrack!!</p><br />
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<p>After completing the first build of LAB ESCAPE, we challenged members of the iGEM 2012 Calgary team to <i>escape from the lab</i>. After incorporating the comments from our teammates, we then released the game to our friends, family and other willing volunteers to gather further feedback from people with a wide variety of backgrounds and experiences.</p><br />
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<p>By September 2012, we had a completely functional video game to share with the world. We premiered LAB ESCAPE at the <a href="http://www.sparkscience.ca">TELUS SPARK </a> science centre in Calgary on September 29 and 30, 2012. During the premier event, people of all ages enjoyed LAB ESCAPE and learned about synthetic biology. With our iGEM wiki going live in early October even more people have enjoyed LAB ESCAPE.</p><br />
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<h2>The Challenge: Can you escape LAB ESCAPE?</h2><br />
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<p>Now that you have all the background information on the creation of LAB ESCAPE, it's time to see if you can escape! All you have to do is collect all the misplaced items needed to run your gel electrophoresis, find the code and escape the lab. It may sound easy, but science is often easy - in theory. One hint for those of you that made it this far: remember that many chemicals in research labs can be quite dangerous. Appropriate safety equipment is always required before working on any experiments.</p><br />
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<p>Finally, while good science should never be rushed, competition often leads to greatness. Keep an eye on the timer and see how quickly you can escape LAB ESCAPE. </p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/SafetyTeam:Calgary/Safety2012-10-03T04:34:56Z<p>Achawdhry: </p>
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<h2>The Risks of FRED and OSCAR</h2><br />
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<p>Our project utilizes two types of engineered bacteria to detoxify tailings water. To quantify the amount of the toxic compounds present in the tailings water we are relying on <a href="http:/2012.igem.org/Team:Calgary/Project/FRED/">FRED</a>, a biosensor bacterium, that will work inside a closed environment to detect the toxins. Within our bioreactor system, we intend to introduce <a href="http:/2012.igem.org/Team:Calgary/Project/OSCAR/">OSCAR</a>, a bacterium capable of detoxifying toxic compounds, to large volumes of tailings water. Working with engineered bacteria to detoxify tailings water can pose a certain amount of risk to the native environment.</p><br />
<p>Whenever synthetic bacteria are used, there is an inherent risk that the synthetic bacteria might escape from their containment vessels into the surrounding environment. There is no direct evidence to suggest the genetic systems we have implemented would have a negative impact to the environment. However, the implications of horizontal gene transfer to native microorganisms in tailings ponds and the surrounding environment must be addressed. This issue has been voiced by numerous leaders in the oil industry as well as individuals living near tailing ponds. Our approach to biosafety and addressing the concerns of interested parties was inspired by a comment published in Nature (Dana et al., 2012) which suggested multiple ways to prevent “Synthetic Biology Disaster”. We strongly believe that we must tackle four major safety concerns with our project. First, the synthetically engineered bacteria may be harmful physiologically to natural flora in the environment. Second, the bacteria may not only survive in the tailing pond environment but thrive in it allowing it to outcompete naturally occurring bacteria. Third, genes may be transferred from our synthetic bacteria to native organisms. Fourth, if any of our genetically modified bacteria were to be able to grow in the tailings pond, evolution may allow for mutations which prevent our safety measures from working.</p><br />
<p> To address these four major safety concerns, we have engineered mechanical and biological safety measures that function to contain genetic elements of our synthetic bacteria. The first two concerns have been addressed through mechanical engineered controls by physically separating our organisms from the native environment. The third concern has been addressed through the development of a novel <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">kill switch system</a> to prevent our engineered organisms DNA from spreading to other organisms. The fourth concern will be addressed by producing redundancy in our kill switch system which can be applied in the scale-up process of our project. By integrating these controls, we have taken a proactive approach to the biosafety of FRED and OSCAR.</p><br />
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<h2>Mechanical Engineered Controls</h2><br />
<h3>FRED</h3><br />
<p>Our team has established a series of controls which we hope to implement into our biosensor during field testing and optimization of the final product. The final product will contain FRED within one-time use closed contains, with one-way valves. The operator will insert water samples through the one way valve, isolating FRED from the operator and the environment. Once testing is completed, the operator is instructed to add a pre-installed allotment of bleach by simply twisting the cap of the tube to destroy FRED prior to proper disposal of the container. </p><br />
<h3>OSCAR</h3><br />
<p>The goal of OSCAR is to be able to detoxify tailings ponds waste through the removal of nitrogen, sulfur, and carboxylic acid functional groups. OSCAR has been designed to function in a closed system to which tailings pond water is added, as opposed to adding OSCAR directly into the environment. Hydrocarbon products generated in the bioreactor after microbial remediation, are collected from the culture with a continuous belt skimming device. There is potential for OSCAR in the bioreactor vessel to escape by adhering to the belt. To counter this risk, the belt is treated with UV radiation as it exits the bioreactor solution. This process will destroy OSCAR on the belt while simultaneously maintaining integrity of the generated hydrocarbons. The extracted solution is then sent through fractional distillation, a process which heats the hydrocarbon solution to over 400&deg;C, killing any OSCAR that may have survived UV exposure.</p><br />
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<h2>Biological Engineered Controls</h2><br />
<p>Kill switches that have previously been entered into the registry often rely on methods that cause the induction of cell lysis. In these systems, genetic material is left intact, allowing for the remaining DNA to be taken up by bacteria and introducing the possibility that synthetic genes escape into the environment. We feel that lysis based kill switches are insufficient for use in FRED and OSCAR, necessitating design of novel kill switches. </p><br />
<p>To ensure that synthetic genetic elements cannot escape the closed systems in which FRED and OSCAR will be used, we engineered novel biological kill switches which we named "Ribo-Kill-Switches." These Ribo-Kill-Switches initiate cell death through degradation of genomic and plasmid DNA. Through a unique cell culture condition within the closed bioreactor and biosensor systems the kill switch genes can be suppressed. Should bacteria escape, the lack of the unique suppression conditions enables the kill switch system to become active. </p><br />
<p>Activation of the kill switch system causes the engineered cell to produce micrococcal nuclease and CviAII restriction enzyme. Our kill switch mechanisms are superior to previous nuclease-based kill switches bas we have improved the completeness of DNA degradation. CviAII and micrococcal nuclease work in tandem: the endonuclease CviAII creates DNA double strand breaks at multiple sites while the micrococcal exonuclease activity degrades remaining strands into single nucleotides. The degradative enzymes chosen for our system were specifically selected for their ability to function at low temperatures, in variable pH conditions, and to work quickly to degrade as much of the genetic material as possible. These engineered biological controls ensure that synthetic genetic elements are completely destroyed in the event that FRED or OSCAR escape from the closed bioreactor or biosensor systems.</p><br />
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<h2>Laboratory Personal Safety</h2><br />
<p>All of the students working with the iGEM 2012 Calgary Team received appropriate safety training as described by the University of Calgary’s safety policies. This included a Biosafety course which introduced the students to proper handling of biological materials. In addition, all iGem students were required to attend proper Workplace Hazardous Materials Information System (WHMIS) training sessions. All safety procedures and guidelines of “Level 2 Biohazard Labs” were followed. Students were also supervised at all times by at least one authorized senior member, lab coordinator, teaching assistant, or professor. </p><br />
<p>The bacterial strains (<i>Nocardia</i>, <i>Rhodoccocus</i>, <i>Pseudomonas</i>, and <i>Escherichia</i>) used in the research are lab strains rated as Biosafety Level 1 and do not pose a health risk to laboratory workers, the general public, or the environment. The team also practiced appropriate procedures for working with and the disposal of tailings water samples. Appropriate handling measures were also applied for genetically modified bacteria and materials contaminated with bacteria. All measures outlined in the Material Safety Data Sheets (MSDS) and the biosafety regulations present at the University of Calgary were followed. Through these procedures, none of the genetically modified bacteria could have a chance of being introduced into the environment. The constructs that we have built to test our systems in the laboratory all used a safe, non-pathogenic bacterial strain of <i>E. coli</i> commonly used in labs worldwide. Other bacteria which were used for characterization of their genes, as listed above, are also non-pathogenic.</p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/DesignTeam:Calgary/Project/HumanPractices/Design2012-10-03T04:26:10Z<p>Achawdhry: </p>
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<h2>Bioreactor Design Considerations</h2><br />
<p>Over the summer, much thought was put into the design of our bioreactor in order to optimize functionality, expense, and safety. Although many of the details of our design cannot be worked out due to the time constraint of a four-month period, there are still lots of theoretical aspects that we were able to cover.<br />
</p><br />
<p>The first aspect of our design was choosing what type of bioreactor system to use. For lab scale experiments and design, we chose to use a system that is closest to that of a batch system. This system requires all reactants to be added at time zero, with everything being removed at once when the remediation has come to completion. However, our design uses a belt skimmer to continually remove any products (hydrocarbons) formed either emulsified or found on the top layer. This way, we are able to reuse our culture and remove product until all the naphthenic acids are converted. We then remove everything in the tank and begin the process again. Our skimmed product goes through UV radiation in order to kill any bacteria that happen to be left in the product. <br />
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In addition, our bioreactor bacteria will contain kill genes. When our bacteria are in a glucose-free environment (a.k.a. outside the tank) the bacteria are programmed to self-destruct. Since we have three different intermediate steps for remediation (desulphurization, denitrification, decarboxylation) we will need three tanks with the product from the previous tank acting as the reactant for the next tank in line. The product of the last tank will go through distillation to purify our desired alkane. Distillation will also assist in classifying the different hydrocarbons we formed and ensure bacteria do not escape into the environment. The produced hydrocarbons may have different carbon and hydrogen bonds, thus its boiling and condensation temperature will vary. If the bacteria removed by the belt skimmer were to somehow survive UV radiation and thrive in a glucose-free environment, it would be distilled along with the rest of the skimmed material. In distillation the bacteria would be heated to an extremely high temperature and would consequently die as a result.<br />
</p><br />
<p>To improve the growth and environment of our bacteria, we will keep our bioreactor at ideal growth temperature (if E coli, 37 degreees; if pseudomonas 25 degrees). In addition, we will have an agitator (turbine) and an air sparger to help mix and oxygenate our solution. In order to help maintain these ideal conditions, our bioreactor will be a closed system with our belt skimmer contained inside the tank.</p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/DesignTeam:Calgary/Project/HumanPractices/Design2012-10-03T04:24:07Z<p>Achawdhry: </p>
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<h2>Bioreactor Design Considerations</h2><br />
<p>Over the summer, much thought was put into the design of our bioreactor in order to optimize functionality, expense, and safety. Although many of the details of our design cannot be worked out due to the time constraint of a four-month period, there are still lots of theoretical aspects that we were able to cover.<br />
</p><br />
<p>The first aspect of our design was choosing what type of bioreactor system to use. For lab scale experiments and design, we chose to use a system that is closest to that of a batch system. This system requires all reactants to be added at time zero, with everything being removed at once when the remediation has come to completion. However, our design uses a belt skimmer to continually remove any products (hydrocarbons) formed either emulsified or found on the top layer. This way, we are able to reuse our culture and remove product until all the naphthenic acids are converted. We then remove everything in the tank and begin the process again. Our skimmed product goes through UV radiation in order to kill any bacteria that happen to be left in the product. <br />
<br />
In addition, our bioreactor bacteria will contain kill genes. When our bacteria are in a glucose-free environment (a.k.a. outside the tank) the bacteria are programmed to self-destruct. Since we have three different intermediate steps for remediation (desulphurization, denitrification, decarboxylation) we will need three tanks with the product from the previous tank acting as the reactant for the next tank in line. The product of the last tank will go through distillation to purify our desired alkane. Distillation will also assist in classifying the different hydrocarbons we formed and ensure bacteria do not escape into the environment. The produced hydrocarbons may have different carbon and hydrogen bonds, thus its boiling and condensation temperature will vary. If the bacteria removed by the belt skimmer was some how to survive UV radiation and thrive in a glucose-free environment, it would be distilled along with the rest of the skimmed material. In distillation the bacteria would be heated to an extremely high temperature and would consequently die as a result.<br />
</p><br />
<p>To improve the growth and environment of our bacteria, we will keep our bioreactor at ideal growth temperature (if E coli, 37 degreees; if pseudomonas 25 degrees). In addition, we will have an agitator (turbine) and air sparger to help mix and oxygenate our solution. In order to help maintain these ideal conditions, our bioreactor will be a closed system with our belt skimmer contained inside the tank.</p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/DesignTeam:Calgary/Project/HumanPractices/Design2012-10-03T04:23:10Z<p>Achawdhry: </p>
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<h2>Bioreactor Design Considerations</h2><br />
<p>Over the summer, much thought was put into the design of our bioreactor in order to optimize functionality, expense, and safety. Although many of the details of our design cannot be worked out due to the time constraint of a four-month period, there are still lots of theoretical aspects that we were able to cover.<br />
</p><br />
<p>The first aspect of our design was choosing what type of bioreactor system to use. For lab scale experiments and design, we chose to use a system that is closest to that of a batch system. This system requires all reactants to be added at time zero, with everything being removed at once when the remediation has come to completion. However, our design uses a belt skimmer to continually remove any products (hydrocarbons) formed either emulsified or found on the top layer. This way, we are able to reuse our culture and remove product until all the naphthenic acids are converted. We then remove everything in the tank and begin the process again. Our skimmed product goes through UV radiation in order to kill any bacteria that happen to be left in the product. <br />
<br />
In addition, our bioreactor bacteria will contain kill genes. When our bacteria are in a glucose-free environment (a.k.a. outside the tank) the bacteria are programmed to self-destruct. Since we have three different intermediate steps for remediation (desulphurization, denitrification, decarboxylation) we will need three tanks with the product from the previous tank acting as the reactant for the next tank in line. The product of the last tank will go through distillation to purify our desired alkane. Distillation will also assist in classifying the different hydrocarbons we formed and ensure bacterial do not escape into the environment. The produced hydrocarbons may have different carbon and hydrogen bonds, thus its boiling and condensation temperature will vary. If the bacteria removed by the belt skimmer was some how to survive UV radiation and thrive in a glucose-free environment, it would be distilled along with the rest of the skimmed material. In distillation the bacteria would be heated to an extremely high temperature and would consequently die as a result.<br />
</p><br />
<p>To improve the growth and environment of our bacteria, we will keep our bioreactor at ideal growth temperature (if E coli, 37 degreees; if pseudomonas 25 degrees). In addition, we will have an agitator (turbine) and air sparger to help mix and oxygenate our solution. In order to help maintain these ideal conditions, our bioreactor will be a closed system with our belt skimmer contained inside the tank.</p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-03T04:21:15Z<p>Achawdhry: </p>
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<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team began our project with human practices in mind. While we had established a research objective to produce a biosensor and bioreactor system, we wanted to ensure that our system was relevant to the inudstry it would be employed in. As well, we wanted to ensure that academic, government, and industry professionals' concerns were taken into consideration during the design process of our system. In order to best accomplish this, we conducted interviews with two leaders in oilsands reclamation. We approached a major oilsands company, Suncor, and talked to Christine Daly, an Ecologist who works in Environmental Cleanup. We then approached Ryan Radke, the president of BioAlberta. BioAlberta focuses on bringing biotechnology to our province and develop these in an industrial setting. His experience allowed us to better predict if our project would have any concerns amongst legislators and industrial leaders. <br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Outreach/SparkTeam:Calgary/Outreach/Spark2012-10-03T04:14:13Z<p>Achawdhry: </p>
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<h2>Sharing Synthetic Biology</h2><br />
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<p>Telus Spark has given us the amazing opportunity to share and engage with the general public to demonstrate through hands on activities what biology is and how we can use synthetic biology to solve problems. The Science Centre brings together a huge range people, from young children and their parents, to adults of all ages at their monthly Adults Only Nights. We have shown off our work and an exhibit meant to demonstrate the cool factor of synthetic biology. The first event we ran was Bacterial Art, and the second was an public beta test of our video game.<br />
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<h2>Bacterial Art</h2><br />
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<p>We have been able to let hundreds of people try their hand at making magnificent art by drawing bacteria containing different fluorescent biobricks onto agar plates. Stocks of bacteria with various colors of fluorescent protein plasmids were prepared. Visitors were got to use their imaginations and 'draw' the bacteria onto agar plates to make pieces of art. These agar plates contained a antibiotic to ensure the plasmid containing the fluorescent marker was not shed by the bacteria. These plates were then incubated overnight, before being photographed under a UV light and being posted to Telus Spark's <a href=https://www.facebook.com/media/set/?set=a.10152086146370538.896423.24253660537>facebook page</a> for the public to tag as theirs.<br />
<br />
<p>This event let us go hands on with the public with bacteria that we use everyday in our lab. We got to explain what the bacteria were, how we changed them, and a bit of the biology on how biobricks coding for fluorescent proteins work. Not only that we got to challenge some of the negative press that bacteria have by showing people that their are bacteria out their that do not cause disease and that can do amazing things.<br />
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<h2>LAB ESCAPE Video Game Premiere</h2><br />
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<p>Our second major event was the first public beta test of the <a href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game</a> we have been developing throughout the summer. We spent two days on a weekend (September 29-30, 2012) letting the general public try out our video game and learn a thing or two about synthetic biology. Players of the game gave us valuable feedback on places where we could improve the game, make it more clear, and most importantly how to make it more fun!<br />
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<h3> </h3><br />
<h2>Sharing Synthetic Biology</h2><br />
<br />
<p>Telus Spark has given us the amazing opportunity to share and engage with the general public to demonstrate through hands on activities what biology is and how we can use synthetic biology to solve problems. The Science Center brings together a huge range people, from young children and their parents, to adults of all ages at their monthly Adults Only Nights. We have shown off our work and an exhibit meant to demonstrate the cool factor of synthetic biology. The first event we ran was Bacterial Art, and the second was an public beta test of our video game.<br />
<br />
<h2>Bacterial Art</h2><br />
<br />
<p>We have been able to let hundreds of people try their hand at making magnificent art by drawing bacteria containing different fluorescent biobricks onto agar plates. Stocks of bacteria with various colors of fluorescent protein plasmids were prepared. Visitors were got to use their imaginations and 'draw' the bacteria onto agar plates to make pieces of art. These agar plates contained a antibiotic to ensure the plasmid containing the fluorescent marker was not shed by the bacteria. These plates were then incubated overnight, before being photographed under a UV light and being posted to Telus Spark's <a href=https://www.facebook.com/media/set/?set=a.10152086146370538.896423.24253660537>facebook page</a> for the public to tag as theirs.<br />
<br />
<p>This event let us go hands on with the public with bacteria that we use everyday in our lab. We got to explain what the bacteria were, how we changed them, and a bit of the biology on how biobricks coding for fluorescent proteins work. Not only that we got to challenge some of the negative press that bacteria have by showing people that their are bacteria out their that do not cause disease and that can do amazing things.<br />
<br />
<p><br />
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<h2>LAB ESCAPE Video Game Premiere</h2><br />
<br />
<p>Our second major event was the first public beta test of the <a href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game</a> we have been developing throughout the summer. We spent two days on a weekend (September 29-30, 2012) letting the general public try out our video game and learn a thing or two about synthetic biology. Players of the game gave us valuable feedback on places where we could improve the game, make it more clear, and most importantly how to make it more fun!<br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Outreach/VideoGameTeam:Calgary/Outreach/VideoGame2012-10-03T04:11:42Z<p>Achawdhry: </p>
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<p>The iGEM 2012 Calgary team ventured into new territory by developing a video game centred on synthetic biology called <b>LAB ESCAPE</b>. As scientists, we are often asked, “What exactly do you do all day?” and “What do you mean you can see DNA?” Our video game provides an opportunity to educate and entertain the player by guiding them through a routine experiment used by the iGEM 2012 Calgary team. LAB ESCAPE focuses on gel electrophoresis – a common molecular biology technique to separate and visualize DNA from PCR and restriction digests. We also incorporated basic laboratory safety procedures to further immerse the player in the research experience. </p><br />
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<p><b><FONT COLOR="FF7A00">LAB ESCAPE is part science, part fun, and all iGEM.</FONT></b></p><br />
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<h2>From Storyboard to Application</h2><br />
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<p>The initial stage in the development of LAB ESCAPE focused on creating an engaging environment resembling a typical synthetic biology research lab. We then created a script outlining the players actions as they find themselves locked in the lab, progress through the tasks to find the secret code, and (hopefully) escape. We originally wanted to incorporate numerous experimental techniques, but we decided to focus on one common technique - gel electrophoresis. This avoided over-whelming the player with multiple experiments and also kept the time required to an optimal level. Once the scene was set we jumped into the world of video game creation.</p><br />
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<p>The iGEM 2012 Calgary team created all the graphical images for LAB ESCAPE using Adobe Illustrator. Adobe Flash was used as the platform for the programming code creating LAB ESCAPE, with some programming code being modified from open-source repositories. With these tools, we designed a ‘point-and-click’ adventure video game, where the player collects items hidden throughout the virtual environment, then complete a task to obtain the code required to open the locked lab door. We even composed our own musical soundtrack!!</p><br />
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<p>After completing the first build of LAB ESCAPE, we challenged members of the iGEM 2012 Calgary team to <i>escape from the lab</i>. After incorporating the comments from our teammates, we then released the game to our friends, family and other willing volunteers to gather further feedback from people with a wide variety of backgrounds and experiences.</p><br />
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<p>By September 2012, we had a completely functional video game to share with the world. We premiered LAB ESCAPE at the <a href="http://www.sparkscience.ca">TELUS SPARK </a> science centre in Calgary on September 29 and 30, 2012. During the premier event, people of all ages enjoyed LAB ESCAPE and learned about synthetic biology. With our iGEM wiki going live in early October even more people have enjoyed LAB ESCAPE.</p><br />
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<h2>The Challenge: Can you escape LAB ESCAPE?</h2><br />
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<p>Now that you have all the background information on the creation of LAB ESCAPE, it's time to see if you can escape! All you have to do is collect all the misplaced items needed to run your gel electrophoresis, find the code and escape the lab. It may sound easy, but science is often easy - in theory. One hint for those of you that made it this far: remember that many chemicals in research labs can be quite dangerous. Appropriate safety equipment is always required before working on any experiments.</p><br />
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<p>Finally, while good science should never be rushed, competition often leads to greatness. Keep an eye on the timer and see how quickly you can escape LAB ESCAPE. </p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/OutreachTeam:Calgary/Outreach2012-10-03T04:10:44Z<p>Achawdhry: </p>
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<p> Through our interview process, we realized that many of the diverse experts that we interviewed didn't know too much about synthetic biology. The goal of the outreach part of our project was thus to interface with a variety of different audiences both within our city and outside it. We also wanted to do this in a more interesting and engaging way then your typical outreach initiative. We had five main activities this summer. We targeted youths in the city, through our video game and the minds in motion science camp. We targeted an older audience through our spark science centre evenings. We interfaced with industry and our sponsors through our Genome Alberta blog series. Finally, we looked to a broader audience through the development of our Ted talk for the upcoming City 2.0 event.</p><br />
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<h2> What did our Outreach activities entail? </h2><br />
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<a class="hublink" href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame"><br />
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<h2>LAB ESCAPE Videogame</h2> <br />
<p>We ventured into new territory this year by developing a video game centered on synthetic biology called </html>'''LAB ESCAPE'''<html>. As scientists, we are often asked, “What exactly do you do all day?” Our video game provides an opportunity to educate and entertain the player by guiding them through a routine experiment used by the iGEM 2012 Calgary team.</p><br />
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<a class="hublink" href="https://2012.igem.org/Team:Calgary/Outreach/Spark"> <br />
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<h2>Telus Spark Science</h2><br />
<p>Calgary's local </html> '''science centre'''<html>, Telus Spark, let us share with their visitors hands on activities with synthetic biology during their monthly Adults Only Night. We had two showcases over the summer. The first, showed off the ability of bacteria to be engineered with genes for fluorescent proteins by getting people to draw art on agar plates. The second, was an public beta test of our synthetic biology video game.</p><br />
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<a class="hublink" href="https://2012.igem.org/Team:Calgary/Outreach/MindsInMotion"> <br />
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<h2>Minds In Motion</h2><br />
<p>We partnered with Minds in Motion, a </html>'''science and engineering camp'''<html> for elementary aged students run through the University of Calgary. We hosted 'synthetic biology' afternoons throughout the summer where we kids got to do crafts and run 'experiments' pertaining to synthetic biology. They even got to wear cool 'sciencey' gloves! </p><br />
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<a class="hublink" href="https://2012.igem.org/Team:Calgary/Outreach/Blog"> <br />
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<h2>Genome Alberta Blog Posts</h2><br />
<p>One of our generous sponsors, Genome Alberta, invited us to post </html>'''blog entries'''<html> on their webpage about the life of an iGEM student. We had a great time writing about the humerous and sometimes stressful life of a student on an iGEM team. This was a great way to interface with industry and convey a more humanistic view of synthetic biology. After all, we're all kind of normal...maybe...</p><br />
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<a class="hublink" href="https://2012.igem.org/Team:Calgary/Outreach/TEDxCalgary"> <br />
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<h2>TED City 2.0</h2><br />
<p>Our team was chosen by TedxCalgary to work with them on developing a </html>'''ted talk'''<html> for the upcoming City 2.0 international event in Calgary. This was an awesome challenge for us to present what we're doing to a general audience in a relevant and meaningful way. </p><br />
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<a class="hublink" href="http://www.twitter.com/iGEMCalgary/" target="_blank"> <br />
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<h2>Follow Us</h2><br />
<p>What iGEM team would be complete without </html> '''social media'''<html>, where we can waste hours laughing at iGEM memes (#useful?) and even tweet our city's mayor about our project! Check us out on Twitter!</p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Project/HumanPracticesTeam:Calgary/Project/HumanPractices2012-10-03T04:04:47Z<p>Achawdhry: </p>
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<h2>Overview:</h2><br />
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<p>Have you heard about the environmental debate surrounding oilsands? Well, majority of the toxic byproducts are contained in what are known as tailings ponds in the environment. One of the major toxins in the tailings ponds is naphthenic acids, which is essentially a mixture of many carboxylic acids. Naphthenic acid poses a huge risk factor to the environment, and current methods for its detection are expensive, time-consuming, and labor-intensive. Thus, We have constructed an efficient solution using genetically modified bacteria to better detect and detoxify naphthenic acid in tailings ponds.</p><br />
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<p>The Calgary 2012 iGEM team saw safety, ethics, and human practices as one of the most important elements in clearly developing our project. When it comes to fighting current environmental issues with genetically engineered bacteria, safety is our top priority. We wanted to fix the problem that toxic oil sands byproducts imposes on our environment without escalating current issues with our bacteria. To this end, we have carefully designed a two-step security mechanism strictly prohibiting the genetically engineered bacteria from leaking into the environment. FRED concentrated on effectively detecting these acids while the project OSCAR focused on removing the toxic components of these toxic acids and converting them into usable fuels. </p><br />
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<a class="hublink" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaboration"><br />
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<h2>Collaboration</h2> <br />
<p>Before developing the synthetic biology systems, we aimed to ensure that each component is to be developed with the concerns, priorities, and opinions of oil sands professionals and political leaders in mind. Through various interviews with specialists, outreach activities, the development of physical and genetic safety elements, and the production of a synthetic biology video game, our group complimented the wetlab work with relevance to our community and end users. To read more about this, click here!</p><br />
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<a class="hublink" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"> <br />
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<h2>Interviews With Experts</h2><br />
<p>In order to determine the relevance of producing a device capable of detecting and breaking down naphthenic acids (NAs) in the tailings ponds, our group conducted a series of interviews. Individuals within Alberta's oil and gas sector with various different backgrounds shared their knowledge and experience with us. The purpose of these interviews was to obtain a general impression of what kind of safety or ethical concerns these individuals had. This work allowed us to better define our project for its intended use. Through the interviews conducted, our group concluded that in order for the remediation system to serve in the oil and gas sector, safety and controls have to be incorporated into every part of our design. To read more about this, click here!</p><br />
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<a class="hublink" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design"> <br />
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<h2>Physical/Engineered Design Considerations</h2><br />
<p>Based on findings from our conversations with experts, we wanted to design a contained and safe biosensor and bioreactor. We took care in designing enclosed systems with structural safety mechanisms in place.</p><br />
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<a class="hublink" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch"> <br />
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<h2>Genetic Killswitch</h2><br />
<p>While the physical design considerations served as indispensable first steps to ensuring the containment of our systems, we also felt it necessary to implement genetic safety mechanisms. This took the form of a killswitch, which selectively destroys the genetically engineered bacteria in the rare case of their escape from our biosensor or bioreactor. Keeping both our environment and industry concerns in hand, we developed a novel ribo-killswitch system for containing our organism using exo/endonucleases. </p><br />
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<a class="hublink" href="https://2012.igem.org/Team:Calgary/Safety"> <br />
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<h2>Safety</h2><br />
<p>We take great care in making sure our project is safe for not only the environment, but also to all of the team members working on FRED and OSCAR. There are a number of considerations that we have taken from the sections above in order to ensure that the construction of FRED and OSCAR was done in the safest way possible. </p><br />
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<a class="hublink" href="https://2012.igem.org/Team:Calgary/Outreach"><br />
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<h2>Outreach</h2> <br />
<p>Speaking to many of our industry experts, we learned that many of them knew very little about synthetic biology. We designed a diverse outreach program to interface with our community. We targeted a variety of audiences in fun and creative ways. </p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DenitrogenationTeam:Calgary/Notebook/Denitrogenation2012-10-03T03:59:13Z<p>Achawdhry: </p>
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<h2>Week 1 (May 1-4)</h2><br />
<p>The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrogenation, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a bioreactor capable of not only detecting but also converting naphthenic acids and other toxins to useful or at least innocuous compounds. </p><br />
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<h2>Week 2-3 (May 7-18)</h2><br />
<p>In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the <i>car</i> operon, <i>CarA</i> (<i>CarAaAcAd</i>), <i>CarB</i> (<i>CarBaBb</i>), and <i>CarC</i>. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the <i>ant</i> operon, <i>antA, B, and C</i>, yielding cathecol while releasing CO2 and NH3. The <i>car</i> and <i>ant</i> operons are both regulated by the P<sup>ant</sup> promoter which is induced by the protein, antR. <i>CarAa</i> also has its own promoter which is not induced by <i>antR</i> (Diaz & Garcia, 2010). We have also investigated an alternative pathway using <i>CarA</i> combined with an amidase (<i>amdA</i>) that selectively cleaves NH2 from an intermediate of the car pathway (Xu et al, 2006). This could bypass much of the car/ant pathway and is possibly more efficient.</p><br />
<p>We have decided to use <i>Pseudomonas resinovorans</i> and <i>Rhodococcus erythropolis</i> to derive these genes from. <i>CarABC</i> and <i>AntABC</i> from<i> P. resinovorans</i> has been shown to have a wide range of nitrogen containing substrate specificity. <i>R. erythropolis</i> contains the <i>amdA</i> gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its <i>CarABC</i> pathway.</p><br />
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<h2>Week 4 (May 22-25)</h2><br />
<p>This week we reviewed the primers listed in the database and also designed some new ones. Primers for <i>CarAa</i>,<i> CarAc</i>, and <i>CarAd</i> were designed individually, while primers for <i>CarBaBbC</i> and <i>AntABC</i> were designed to encompass multiple genes in a sequence. These primers were designed to be used on <i>Pseudomonas putida</i> which we decided to use as our gene source since it was available to us as opposed to ordering <i>Pseudomonas resinovorans</i>. We also designed a primer for the<i> AmdA</i> gene from <i>Rhodococcus erthyroplois</i>. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative <i>AmdA</i> pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.</p><br />
<p>We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock <i>Psuedomonas putida</i> on Thursday. We also resuspended the primers for <i>CarAa</i>, <i>CarAc</i>, <i>CarAd</i>,<i> AntA</i>, <i>AntB</i>, and <i>Ant C</i> that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.</p><br />
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<h2>Week 5 (May 28 - June 1)</h2><br />
<p>On Monday and Tuesday we used the database primers to attempt to isolate <i>CarAa</i>, <i>CarAc</i>, <i>CarAd</i>, <i>AntA</i>, <i>AntB</i>, and <i>AntC</i> from the <i>Psuedomonas putida</i> we plated last week. <i>CarAd</i>, <i>AntB</i>, and <i>AntC</i> were all put in the gradient PCR machine to account for the wide range of their primers’ melting points, while the others were done via regular PCR. Unfortunately, no positive results were obtained from these reactions. The PCR on <i>CarAc</i> and <i>AntA</i> was repeated on Wednesday, still not giving positive results. Finally, we attempted to use a salt concentration gradient on the PCR reaction for <i>CarAc</i> and <i>AntA</i>, using concentrations that ranged from 1.0 microlitres/tube to 2.0 microlitres/tube in increments of 0.2 microlitres/tube. This helped as <i>CarAc</i> showed bands in samples that had concentrations of 1.2 and 1.4 microlitres/tube. <i> AntA</i> also showed bands, however they were not the correct size, indicating contamination and/or non-specific annealing of the primer. The positive control also showed bands of the correct size. Earlier in the week we also made overnight cultures of 3 environmental strains (28, 29, 30) of <i>Psuedomonas putida</i> from -80 glycerol stock. On Friday we performed a genomic prep on these cultures, and plan on attempting to amplify genes from the isolated DNA next week. </p><br />
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<h2>Week 6 (June 4 - June 8)</h2><br />
<p> We started off this week by determining the DNA concentration of our genomic prep samples from last week using the nanodrop. DNA concentration for all three<i> P. putida</i> strains was at least 1000 ng/microlitre, well above what was needed for PCR. 1/2 and 1/3 dilutions were prepared for all three strains so as not to have an excess of template DNA in PCR reactions. PCR was performed on all 3 strains using primers for <i>CarAc</i>, <i>CarAd</i>, <i>AntA</i>, <i> AntB</i>, and <i>AntC</i> using 6 replicates per gene. The only successful amplification appeared to be <i>AntB</i> and <i>CarAc</i>, both from strain 28 (with weaker bands in strain 29). We then performed another PCR, just on those two genes with an increased amount of Taq polymerase to hopefully get enough amplified DNA to move forward with. This resulted in strong bands for both at the expected size. We then performed PCR purification using the Qiaquick kit and obtained samples containing 33.5 ng/microlitre of <i>AntB</i> DNA and 129 ng/microlitre of <i>CarAc </i>DNA. These concentrations were both sufficient to begin a restriction digest and ligation of these parts into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Next week we hope to verify the results of the restriction digest, continue to amplify <i>CarAc</i> and <i>AntB</i> from strain 28, and hopefully submit a biobrick for sequencing.</p><br />
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<h2>Week 7 (June 11 - June 15)</h2><br />
<p>The purified PCR products for <i>CarAc</i> and <i>AntB</i> were digested with restriction enzymes EcoRI+SpeI, EcoRI+PstI, and XbaI+PstI. However, only <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmids that had been digested with EcoRI+SpeI and EcoRI+PstI were available to attempt ligation. Gel results were inconclusive on the restriction digest product as the plasmid size appeared much larger than expected. However, transformation was still attempted on the ligation products for both genes (both using EcoRI+SpeI ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid). The transformation products were plated onto chloroamphenicol (chlor) plates to select for colonies that had the chloro resistance gene on the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid. Also this week another round of PCR was performed for the <i> Car</i> and <i> Ant</i> genes, however all, but <i>CarAc</i> showed bands in the negative control lane, possibly indicating contamination or the formation of primer dimers. The <i>CarAc</i> bands were fairly weak and a PCR purification resulted in very low DNA concentration, insufficient to move onto restriction digest. Next week's plan hinges heavily on the result of the transformation from this Friday. </p><br />
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<h2>Week 8 (June 18 - June 22)</h2><br />
<p>The results of our transformation on <i>CarAc</i> and <i>AntB</i> from last week showed mostly red colonies indicating that the ligation was unsuccessful and that the PSB1C3 plasmid simply closed on itself with no insert. However, there were three colonies for <i>CarAc</i> that were white (cut with both X+P and E+P). Colony PCR was performed on these three colonies using biobrick primer sets to verify that the part had been inserted in these colonies, but unfortunately all PCR results were negative indicating an unsuccessful ligation.</p><br />
<p>We also performed a miniprep on <i>Pseudomonas putida</i> strain #28 this week using a home-made protocol rather than the Qiagen kit. Miniprep A had a DNA concentration of 1539.8 ng/μL and Miniprep B had DNA concentration of 1001.2 ng/μL possibly indicating that there may have been a lot of genomic DNA contamination. However miniprep A product was still used as a PCR template for a reaction attempting to isolate <i>CarAc</i>, <i>CarAd</i>, <i>AntA</i>, <i>AntB</i>, and <i>AntC</i>. Special conditions for this PCR included replacing 60 μL of water with 60 μL of betaine heated to 37 degrees Celsius and running a Mg gradient ranging from 0.5 μL - 2.5 μL per tube for each gene. Of these only <i>AntB</i> had bands of the right size in 3 of the lanes (Mg concentrations of 1.5, 2, and 2.5 μL). However, all of the <i>AntB</i> bands (including the negative control) contained a contamination band at around 100 bases, probably due to the formation of primer dimers. This forced us to do a gel extraction rather than a PCR purification. This gel extraction did not work as the nanodrop results indicated high amounts of agarose contamination. Another PCR was performed using the same conditions, but using miniprep B instead as there was no more miniprep A product remaining. Unfortunately there were no bands for <i>AntB</i>, only the 100 base pair contamination bands. </p><br />
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<h2>Week 9 (June 25 - June 29)</h2><br />
<p>This week we modified our PCR protocol to adjust for the homemade Taq polymerase we were using. Template DNA was boiled for 10 minutes on its own to replace the initialization step. This is because this polymerase could not tolerate the 95 degree heat during the regular initialization step. Another modification we made was to boil our primers for 5 minutes prior to adding them to the master mix. This was done to discourage the formation of primer-dimers. Finally we also attempted a touch-down PCR which started at an annealing temperature greater than the Tm of our primers and gradually worked its way down to below the Tm of the primers. This was done to increase the specificity of our reaction and also eliminate the formation of primer-dimers. Unfortunately these techniques still saw the formation of dimers, preventing us from performing a PCR purification. Again, the gel purification procedure did not lead to successful purification of our target gene. Due to the problems we have had so far our goal for next week is to perform an assay experiment to try and determine which of the <i>Pseudomonas putida</i> strains we have access to can actually degrade carbazole and therefore contain the pCAR1 plasmid. </p><br />
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<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The goal for this week was to grow different strains of<i> Pseudomonas</i> in media containing B-N media + glucose with only carbazole as a source of nitrogen. In theory only those strains which contained the pCAR1 plasmid would be able to use carbazole as a nitrogen source and thrive in this media. The strains we tested from glycerol stock included <i>P. putida</i> LD1, <i>P.putida</i> environmental strains 28-30, and <i>P. fluorescens</i> PF5. Each of these strains had two cultures grown; one with carbazole and one without it. <i>E. coli</i> grown with carbazole was used as a negative control as it does not contain the pCAR plasmid. At timepoints of 22, 46, and 117 hours of growth samples were taken to do an OD600 reading to measure bacterial growth and had an ammonia assay performed on it. The detection of ammonia acted as a proxy for detecting carbazole degradation as it is an end-product of the pathway. With this in mind, an additional control strain of LD1 was grown in media containing glucose and ammonia. This is to detect the possibility that <i>Pseudomonas</i> can use ammonia as a source of nitrogen after it has degraded carbazole which would be problematic as far as using ammonia levels to determine carbazole degradation. So far we have been having difficulties reading the results of the ammonia assay with a plate reader and may resort to testing our samples using a different assay protocol next week. The OD600 readings were initially very high at 22 hours and then dropped at 46 hours. </p><br />
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<h2>Week 11 (July 9-July 13)</h2><br />
<p>This week we took our last readings from the experiment that we started last week. The OD600 readings taken at 117 hours showed minimal growth over the weekend for all strains except for environmental strain #29 (with carbazole) which increased from 0.086 to 0.313. No other strain showed an increase of 0.100 or greater. This shows that only strain #29 was able to thrive in the carbazole media, possibly indicating that it contains the pCAR plasmid enabling it to use carbazole as a nitrogen source. To verify this we used a new ammonia assay protocol to measure the production of ammonia in the different cultures and theoretically how much carbazole was degraded. Unlike the previous assay we were able to produce a standard curve with a consistent slope based on samples with known amounts of ammonia. However, the only samples that showed any elevation of ammonia levels above the blank were the LD1 samples that were cultured with ammonia chloride to control for the possibility of pseudomonas using ammonia as a nitrogen source. The ammonia levels in this sample were relatively constant which would indicate that the LD1 was not using ammonia as a nitrogen source. Unfortunately the fact that none of the test samples had elevated ammonia levels did not give us any evidence that these strains contain the pCAR plasmid. </p><br />
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</html>[[File: Standard_curve_for_ammonia_assay_UCalgary2012_denitrification_group.png|500px|thumb|Standard curve for ammonia assay using OD635 absorbance.|center|]]<html><br />
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<h2>Week 12 (July 16 -July 20)</h2><br />
<p>We received a new strain of Pseudomonas, LD2, this week that had previously been used to study the pCAR1 plasmid. It was grown up in PCA media and had its colonies used for colony PCR. We also received our new primers this week, further increasing the chances of successful PCR. Using the new strain and the new primers <i>CarAa</i>, <i>CarAc</i>, and <i>CarAd</i> all showed at least some bands of close to the correct size. After PCR purification we obtained DNA concentrations of 21.3 ng/μL for <i>CarAa</i>, 41.9 ng/ μL for <i>CarAc</i>, and 32.8 ng/μL for<i> CarAd</i>. These products were then restriction digested using EcoRI + PstI sites. </p><br />
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</html>[[File:07.20.2012 CarAa and CarAc.jpg|500px|thumb|Gel of colony PCR from Pseudomonas LD2 for CarAa and CarAc genes. CarAa expected size is 1155 bp and CarAc is 323 bp.|center]]<br />
[[File:07.20.2012 CarAd colony PCR from LD2.jpg|500px|thumb|Gel of colony PCR from Pseudomonas LD2 for CarAd gene. CarAd expected size is 989 bp.|center]] <html><br />
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<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The restriction digested <i>Car</i> genes were ligated into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> cells. White colonies grew for<i> CarAa</i> and <i>CarAc</i> suggesting that ligation and transformation were successful, however no colonies grew for <i>CarAd</i> suggesting that the transformation did not work. Colony PCR was performed to confirm the presence of these genes within the vectors, however only <i>CarAc</i> was positive. The colonies containing the <i>CarAc</i> plasmid were then mini prepped and restriction digested to isolate the plasmid and send for sequencing. Further attempts at transforming<i> CarAa</i> and <i>CarAd</i> were not successful. Another goal was to perform colony PCR on the <i>P. putida</i> to isolate the<i> AntABC</i> genes using both the primers for the entire operon and those for the individual genes. No attempt showed any bands, although salt and temperature gradients were used as well as different types of polymerase. LD2 cultures were grown up to be plasmid prepped and genomic prepped in order to have a higher concentration of template DNA in the PCR master mix and more PCR will be attempted on these genes next week. Mutagenesis primers were also designed this week to remove the illegal restriction cut sites in the<i> CarAd</i> and <i>AntB</i> genes (NotI and 2xEcoRI respectively). Only 1 mutagenesis primer was designed for <i>AntB</i> as one of its EcoRI sites was very close to the beginning of the gene and could be removed using a new primer. During this process it was also discovered that the original Ant primers were designed for the wrong genes and therefore do not match the intended sequence. Although the new <i>AntABC</i> primers were designed using the correct gene the PCR may not be working due to the length of the operon so the design of new individual primers may be necessary. Finally, we also aimed to grow up the freeze dried culture of <i>Rhodococcus erythropolis</i> (source of the<i> AmdA</i> gene) that we received from DSMZ. The reactivation media was produced and autoclaved to begin the process next week.</p><br />
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</html>[[File:07.23.2012.Restriction Digest of Car genes.jpg|500px|thumb|Gel of restriction digest of Car genes. A refers to CarAa, C to CarAc, and D to CarAd. Ladder shows that after purification and digestion DNA is still the correct size.|center]]<br />
[[File:CarAc biobrick confirmation PCR.jpg|500px|thumb|Gel of confirmation PCR done on competent Top 10 E. coli cells in which a PSB1C3 vector containing the CarAc gene had been inserted. Red X marks the 500 bp mark on the weak ladder showing the product to be the expected size (323 bp gene + 2*100 bp biobrick sites on each end).|center]] <html><br />
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<h2>Week 14 (July 30 - August 3)</h2><br />
<p>We received the sequencing results this week for <i>CarAc</i> which came back as a 100% match to the expected nucleotide sequence. We also successfully ligated both<i> CarAa</i> and<i> CarAd</i> into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into top 10 <i>E. coli</i> cells. Colony PCR verified the presence of the biobrick plasmid in these colonies. These were both miniprepped, digested and sent for sequencing by the end of the week. Using a salt gradient, addition of DMSO, and the KAPA buffer specifically designed for GC-rich amplicons we were able to amplify the Ant operon from a genomic prep of LD2. However, all attempts at ligating and transforming this sequence were met with the unfortunate presence of red colonies (indicating failed insertion into the plasmid). Next week we will recut a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector with Xba and Pst to attempt to mitigate this problem. We also reactivated the <i>R. erythropolis</i> using DSMZ media #1 and grew it on plates made from DSMZ media #220 at 28 degrees. Colonies grew very quickly and were used for colony PCR and to make overnight cultures for downstream applications. The colony PCR using the amidase primers was unsuccessful, so a genomic prep was performed on the overnight culture. Unfortunately we lacked some of the required equipment and the genomic prep did not work, but with a few procedural modifications and the right equipment we are confident it will work next week. A glycerol stock of the<i> R. erythropoplis</i> was also made from another overnight culture. We also started construction on the <i>CarAc </i>part this week by inserting a ribosome binding site (B0034) in front of the gene. Finally, since the<i> Ant</i> operon was successfully amplified by PCR we designed mutagenesis primers for the 2nd EcoRI site in the operon rather than making new individual primers. We also designed a mutagenesis primer to remove the SpeI site in <i>AmdA</i>. Now that we have conclusively proven that the LD2 strain contains the pCAR1 plasmid we aim to demonstrate its ability to degrade carbazole and other nitrogen containing heterocycles using an assay experiment similar to the one performed in Week 10.</p><br />
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</html>[[File:07.30.2012. CarAa, CarAd.jpg|500px|thumb|CarAa and CarAd amplified from genomic prep of Pseudomonas LD2. Amplicons are larger than expected, potentially the gel ran diagonally. The 6th lane for each gene was a negative control.|center]]<br />
[[File:07.31.2012.Ant operon.jpg|500px|thumb|Ant operon amplified from genomic prep of Pseudomonas LD2 at the expected size of 3000 bp.|center]]<br />
[[File:08.01.2012 CarAdBB.jpg|500px|thumb|Lanes 2,4,5, and 11 contain CarAd genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent E.Coli.|center]]<br />
[[File:08.01.2012 CarAaBB.jpg|500px|thumb|Lane 9 contains CarAa genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent E.Coli. Although larger than expected, the positive control RFP generator in lane 10 is also larger than its expected size (~1200 bp including biobrick bookends) indicating that the gel may have a slant in it.|center]]<html><br />
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<h2>Week 15 (August 7 - August 10)</h2><br />
<p>This week we received the sequencing results for <i>CarAa</i> and <i>CarAd</i>. <i>CarAd</i> was a match, however <i>CarAa</i> did not match at all and had misplaced biobrick restriction enzyme sites. Due to this we decided to restart the biobricking process for <i>CarAa</i> all the way back to PCR from the plasmid. By the end of the week we had progressed to the point of transforming it into competent <i>E. coli</i> cells, however a thick lawn of bacteria had grown overnight making it impossible to pick colonies. A streak plate was made from this plate to attempt to get less dense growth. Since the sequencing came back positive for <i>CarAd</i> the next step was to perform site directed mutagenesis on its NotI restriction enzyme site using the primers that were designed previously (for site-directed mutagenesis optimization, please refer to Desulfurization Journal Week 14). The PCR was successful, showing very bright bands at 3000 bp, and the PCR product was digested with DpnI to remove parental DNA. Transformation of this product will indicate whether the mutagenesis was successful. We were also able to successfully perform a genomic prep of <i>Rhodococcus erythropolis</i> and amplify the <i>AmdA</i> gene this week. It was ligated into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> by Friday, and a confirmation colony PCR was run on Friday evening to verify that the gene was inserted. These results will be available by Monday. After many attempts the <i>Ant</i> operon was finally ligated and transformed as well this week. Confirmation PCR showed bands of about 3000 bp which is consistent with the size of the operon. By Friday the successful colonies had been grown up and miniprepped, ready to send for sequencing early next week. The <i>CarAc</i>/B0034 construct was also successfully transformed this week and verified by running a PCR using the B0034 forward primer. It was also miniprepped and is ready to send for sequencing next week. </p><br />
<p>In addition to this work constructing biobricks we also started an experiment to demonstrate the function of the pCAR plasmid in LD2. Strains of LD2 were grown up in LB and then washed and placed in B-N media + glucose and various experimental additions. These additions included carbazole, 4-PBAH (a nitrogen containing naphthenic acid), ammonia chloride, and a negative control with no nitrogen source. Strains of <i>E. coli</i> were also grown under the same conditions to compare growth and ammonia production of LD2 to a strain that does not contain the pCAR plasmid. These results should act as a preview of what we expect our final biobrick containing <i>E. col</i>i species to produce as well as allowing us to establish LD2 as a positive control for this experiment. </p><br />
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<br />
</html>[[File:08.07.2012 amdA pcr from genomic prep.jpg|500px|thumb|AmdA amplified from the genomic prep of R. Erythropolis shows bands just greater than 1500 base pairs in lanes 2,4-6,9, and 12-13. Lane 14 was a negative control. The expected size of AmdA is 1566 base pairs.|center]]<br />
[[File:08.09.2012 confirmation pcr of ant operon.jpg|500px|thumb|The two labelled lanes show amplification at 3000 base pairs from a confirmation colony PCR using the biobrick primers. The expected size of the Ant operon with the biobrick bookends is about 3100 base pairs.|center]]<br />
[[File:08.10.2012 carad notI mutagenesis.jpg|500px|thumb|Strong amplification at just above 3000 base pairs from the CarAd PCR using mutagenic primers. Since PSB1C3 is 2070 base pairs and CarAd is 989 base pairs the amplification should run at about 3059 base pairs. 3 different amounts of template DNA were tested in lanes 2-7 as indicated and a positive control showed weaker amplification in lanes 8-9 at about 4000 base pairs.|center]]<br />
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<h2>Week 16 (August 13 - August 17)</h2><br />
<p>The sequencing for the <i>CarAc</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> construct came back with only <i>CarAc</i> and no evidence of a <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> site preceding it. The confirmation PCR that was run last week using the <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> forward primer seemed to indicate that it was inserted, however since <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> is only 12 base pairs the primer may not have been specific enough. The construction on this part was restarted this week. <i>CarAa</i> colony PCR on last week's transformation showed two positive colonies, however upon culturing, miniprepping, and digesting these colonies the plasmid appeared to be too large (3000 base pairs as opposed to the expected 2000). This may be due to a problem of a mislabeled plasmid tube that has been experienced by other team members, causing the gene to be inserted into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a> rather than <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. To test this, a streak plate was made from these colonies and grown on an ampicillin resistant plate. Mutagenized <i>CarAd</i> was successfully transformed into Top 10 <i>E. coli</i> cells, cultured, and miniprepped. The miniprep product was digested with NotI to test whether the NotI site was successfully removed. These results were good, as the plasmid ran at 2000 base pairs and the insert at just under 1000 base pairs in the mutagenized <i>CarAd</i> whereas the non-mutagenized <i>CarAd</i> had its insert cut into 2 bands. This will be sent for sequencing next week to confirm the mutation. Unfortunately the sequencing results came back negative for the <i>Ant</i> operon meaning we will need to restart genomic PCR on it next week. Multiple transformations and confirmation PCRs were run on <i>AmdA</i> this week, however none displayed a band size of 1700-1800 base pairs as expected. We have continued to try different digested plasmids as other team members also had difficulties this week with ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, possibly indicating a problem with our Antarctic Phosphatase stock.</p><br />
<p>The other news from this week was the final results from our carbazole degradation experiment attempting to demonstrate LD2's ability to degrade carbazole and hopefully other nitrogen containing compounds. Unfortunately, these results were very difficult to make sense of as the LD2 that were cultured with carbazole grew, but did not show elevated ammonia levels which would be indicative of carbazole degradation. One of our controls was LD2 cultured with ammonium chloride to test whether LD2 was capable of using ammonia as a nitrogen source. This would pose a problem for us as we were planning to use an increase in ammonia levels as a proxy for carbazole degradation rather than measuring carbazole degradation directly using GC-MS. However, the LD2 showed significantly less ammonia after 44 hours when compared to 26 hours indicating that it may be degraded, either by the LD2 or by an unaccounted for abiotic process. This has lead us to decide to use GC-MS and directly measure carbazole levels in our sample the next time we do this experiment.</p><br />
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<h2>Week 17 (August 20 - August 24)</h2><br />
<p>The <i>CarAa</i> was re-streaked onto ampicillin plates and grew, probably showing that it was ligated into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a>, not <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, which explained the irregularity of our confirmation digest last week. It was sent for sequencing, but in case it was incorrect we also started a new <i>CarAa</i> pipeline by PCRing it from the <i>Pseudomonas</i> genome again and redigesting and ligating. It was transformed into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> over the weekend. <i>CarAc</i> did not see a lot of progress this week as we spent much of the time attempting to construct a RBS (B0034) in front of it, but were unsuccessful. <i>CarAd</i> was also subjected to this construction with B0034 and we were able to send it for sequencing by the end of the week. Several new constructs were also started for <i>CarAd</i> in case the sequencing is negative. The <i>Ant</i> operon was successfully transformed and confirmed by colony PCR this week, after which it was grown up and sent for sequencing. We also began to attempt mutagenesis on one of the EcoRI sites in the operon, but had no amplification of the plasmid. We also re-digested some PCR purified product to start another ligation in case sequencing failed. After many attempts we were also able to finally successfully transform an <i>AmdA</i>/PSB1C3 plasmid into Top 10 cells this week. After a miniprep they showed bands of about 1800 base pairs on the confirmation digest gel, so they were also sent for sequencing this week and had mutagenesis started on the illegal SpeI site. Unfortunately, the sequencing sent early in the week came back with ambiguous results. The forward reaction for<i> AntABC</i> was a match, but its reverse reaction matched to <i>CarAa</i>. Both <i>CarAa</i> reactions matched nothing. Since these two genes are over 20 000 base pairs apart on the template plasmid it is virtually impossible that they were actually combined into one plasmid (PCR extension time would not have been long enough and even if it was the product would have run much above the 3000 base pair mark). This is possibly a mistake on Eurofins end, so we are not 100% sure if we have 1 good gene, both of them, or neither. Also the sequence for mutagenized <i>CarAd</i> showed only about the first 100 base pairs, which were correct however not long enough meaning it was a poor reaction and inconclusive. Finally, we also re-started our carbazole degradation assay using the GC-MS at biological sciences. This process will take 2 weeks to demonstrate LD2's ability to degrade carbazole. We are forcing it to use carbazole as a carbon and a nitrogen source in one sample and as only a nitrogen source in another sample (this sample has had glucose added to it).</p><br />
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<h2>Week 18 (August 27 - August 31)</h2><br />
<p>The new stock of <i>CarAa</i> that we had PCRed from LD2 was sent for sequencing this week and came back as a match, so we have begun constructing a RBS site and a TetR promoter (<a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>) in front of it. At this point all the <i>Car</i> genes have been successfully amplified, inserted into a biobrick plasmid, and sequence verified. We also continued to attempt to construct <i>CarAc</i> with an RBS site (<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>) in front of it, but had no success. On a better note, we confirmed this week that<i> CarAd</i> was mutated properly and we were also able to insert an RBS site in front of it. The next step for this gene will be to attempt a plasmid switch as it is currently in <a href="http://partsregistry.org/Part:BBa_K590025">pSB1A3</a> and needs to be in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> for submission to the parts registry. After sending a new batch of the Ant operon for sequencing we got the same confusing results as we did last week. We therefore hypothesized that our primers were annealing to a homologous region that is similar to the<i> Ant</i> genes and very close to <i>CarAa</i>. This means that we need to restart PCR on this gene as all of our current product is incorrect. The sequencing results for <i>AmdA</i> that came back this week were also somewhat confusing as they did not quite match the sequence we expected. However, they were a 100% match for the <i>AmdA</i> gene in a different strain of <i>Rhodococcus erythropolis</i>. This means that DSMZ probably sent us the wrong strain of bacteria and that we were very lucky that the primers we designed based off of the expected template actually worked to amplify a slightly different gene in another strain. The good news is that this new sequence contains no illegal cut sites, which explains why mutagenesis was not working (there was nothing to mutate) and allows us to immediately begin construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> in front of <i>AmdA</i> this week. </p><br />
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<h2>Week 19 (September 3- September 7)</h2><br />
<p><i>CarAd</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> was successfully plasmid switched into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, making it ready for submission. Construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> and <i>CarAa</i> may have been successful as the digest of the miniprep looked promising. It will be sent for sequencing next week. Unfortunately there was no progress made on the constructions of <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> or <i>CarAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. These will be redigested and religated for next week. We could not amplify the <i>Ant</i> operon despite trying many variations of PCR using high fidelity KAPA polymerase. This is probably due to the multiple, large regions of homology on the operon that the reverse primer binds to making amplification difficult. Because the function of this gene can theoretically be replaced by <i>AmdA</i>, we may scale back efforts dedicated to it, as the chances of successful amplification appear slim. </p><br />
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<h2>Week 20 (September 10 - September 14)</h2><br />
<p>Constructs that continued this week included <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, <i>CarAa</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, and <i>CarAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. Some sequencing was sent, but no positive results were obtained. A new approach to biobrick the <i>Ant</i> operon was implemented this week. A new forward primer was designed that would anneal about 1500 base pairs upstream of the <i>Ant</i> operon, avoiding the region of homology that is repeated at various points on the plasmid. Once this region is successfully amplified we could use this amplified DNA as a template for another PCR reaction which would use the normal <i>Ant</i> primers. Since the template would no longer include regions of homology these primers should be free to work properly and amplify the <i>Ant</i> operon. The PCR using the new primers was started this week, and although amplification at 4500 base pairs was observed as expected, there was also a lot of non-specific amplification. This means that to purify the amplified DNA that we want and avoid the other products we will have to perform a dreaded gel extraction next week. </p><br />
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<h2>Week 21 (September 17-21)</h2><br />
<p>Nothing worked this week, please see above </p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DenitrogenationTeam:Calgary/Notebook/Denitrogenation2012-10-03T03:54:44Z<p>Achawdhry: </p>
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<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Denitrogenation Journal|<br />
CONTENT=<br />
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<h2>Week 1 (May 1-4)</h2><br />
<p>The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrogenation, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a bioreactor capable of not only detecting but also converting naphthenic acids and other toxins to useful or at least innocuous compounds. </p><br />
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<h2>Week 2-3 (May 7-18)</h2><br />
<p>In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the <i>car</i> operon, <i>CarA</i> (<i>CarAaAcAd</i>), <i>CarB</i> (<i>CarBaBb</i>), and <i>CarC</i>. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the <i>ant</i> operon, <i>antA, B, and C</i>, yielding cathecol while releasing CO2 and NH3. The <i>car</i> and <i>ant</i> operons are both regulated by the P<sup>ant</sup> promoter which is induced by the protein, antR. <i>CarAa</i> also has its own promoter which is not induced by <i>antR</i> (Diaz & Garcia, 2010). We have also investigated an alternative pathway using <i>CarA</i> combined with an amidase (<i>amdA</i>) that selectively cleaves NH2 from an intermediate of the car pathway (Xu et al, 2006). This could bypass much of the car/ant pathway and is possibly more efficient.</p><br />
<p>We have decided to use <i>Pseudomonas resinovorans</i> and <i>Rhodococcus erythropolis</i> to derive these genes from. <i>CarABC</i> and <i>AntABC</i> from<i> P. resinovorans</i> has been shown to have a wide range of nitrogen containing substrate specificity. <i>R. erythropolis</i> contains the <i>amdA</i> gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its <i>CarABC</i> pathway.</p><br />
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<h2>Week 4 (May 22-25)</h2><br />
<p>This week we reviewed the primers listed in the database and also designed some new ones. Primers for <i>CarAa</i>,<i> CarAc</i>, and <i>CarAd</i> were designed individually, while primers for <i>CarBaBbC</i> and <i>AntABC</i> were designed to encompass multiple genes in a sequence. These primers were designed to be used on <i>Pseudomonas putida</i> which we decided to use as our gene source since it was available to us as opposed to ordering <i>Pseudomonas resinovorans</i>. We also designed a primer for the<i> AmdA</i> gene from <i>Rhodococcus erthyroplois</i>. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative <i>AmdA</i> pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.</p><br />
<p>We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock <i>Psuedomonas putida</i> on Thursday. We also resuspended the primers for <i>CarAa</i>, <i>CarAc</i>, <i>CarAd</i>,<i> AntA</i>, <i>AntB</i>, and <i>Ant C</i> that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.</p><br />
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<h2>Week 5 (May 28 - June 1)</h2><br />
<p>On Monday and Tuesday we used the database primers to attempt to isolate <i>CarAa</i>, <i>CarAc</i>, <i>CarAd</i>, <i>AntA</i>, <i>AntB</i>, and <i>AntC</i> from the <i>Psuedomonas putida</i> we plated last week. <i>CarAd</i>, <i>AntB</i>, and <i>AntC</i> were all put in the gradient PCR machine to account for the wide range of their primers’ melting points, while the others were done via regular PCR. Unfortunately, no positive results were obtained from these reactions. The PCR on <i>CarAc</i> and <i>AntA</i> was repeated on Wednesday, still not giving positive results. Finally, we attempted to use a salt concentration gradient on the PCR reaction for <i>CarAc</i> and <i>AntA</i>, using concentrations that ranged from 1.0 microlitres/tube to 2.0 microlitres/tube in increments of 0.2 microlitres/tube. This helped as <i>CarAc</i> showed bands in samples that had concentrations of 1.2 and 1.4 microlitres/tube. <i> AntA</i> also showed bands, however they were not the correct size, indicating contamination and/or non-specific annealing of the primer. The positive control also showed bands of the correct size. Earlier in the week we also made overnight cultures of 3 environmental strains (28, 29, 30) of <i>Psuedomonas putida</i> from -80 glycerol stock. On Friday we performed a genomic prep on these cultures, and plan on attempting to amplify genes from the isolated DNA next week. </p><br />
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<h2>Week 6 (June 4 - June 8)</h2><br />
<p> We started off this week by determining the DNA concentration of our genomic prep samples from last week using the nanodrop. DNA concentration for all three<i> P. putida</i> strains was at least 1000 ng/microlitre, well above what was needed for PCR. 1/2 and 1/3 dilutions were prepared for all three strains so as not to have an excess of template DNA in PCR reactions. PCR was performed on all 3 strains using primers for <i>CarAc</i>, <i>CarAd</i>, <i>AntA</i>, <i> AntB</i>, and <i>AntC</i> using 6 replicates per gene. The only successful amplification appeared to be <i>AntB</i> and <i>CarAc</i>, both from strain 28 (with weaker bands in strain 29). We then performed another PCR, just on those two genes with an increased amount of Taq polymerase to hopefully get enough amplified DNA to move forward with. This resulted in strong bands for both at the expected size. We then performed PCR purification using the Qiaquick kit and obtained samples containing 33.5 ng/microlitre of <i>AntB</i> DNA and 129 ng/microlitre of <i>CarAc </i>DNA. These concentrations were both sufficient to begin a restriction digest and ligation of these parts into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Next week we hope to verify the results of the restriction digest, continue to amplify <i>CarAc</i> and <i>AntB</i> from strain 28, and hopefully submit a biobrick for sequencing.</p><br />
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<h2>Week 7 (June 11 - June 15)</h2><br />
<p>The purified PCR products for <i>CarAc</i> and <i>AntB</i> were digested with restriction enzymes EcoRI+SpeI, EcoRI+PstI, and XbaI+PstI. However, only <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmids that had been digested with EcoRI+SpeI and EcoRI+PstI were available to attempt ligation. Gel results were inconclusive on the restriction digest product as the plasmid size appeared much larger than expected. However, transformation was still attempted on the ligation products for both genes (both using EcoRI+SpeI ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid). The transformation products were plated onto chloroamphenicol (chlor) plates to select for colonies that had the chloro resistance gene on the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid. Also this week another round of PCR was performed for the <i> Car</i> and <i> Ant</i> genes, however all, but <i>CarAc</i> showed bands in the negative control lane, possibly indicating contamination or the formation of primer dimers. The <i>CarAc</i> bands were fairly weak and a PCR purification resulted in very low DNA concentration, insufficient to move onto restriction digest. Next week's plan hinges heavily on the result of the transformation from this Friday. </p><br />
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<h2>Week 8 (June 18 - June 22)</h2><br />
<p>The results of our transformation on <i>CarAc</i> and <i>AntB</i> from last week showed mostly red colonies indicating that the ligation was unsuccessful and that the PSB1C3 plasmid simply closed on itself with no insert. However, there were three colonies for <i>CarAc</i> that were white (cut with both X+P and E+P). Colony PCR was performed on these three colonies using biobrick primer sets to verify that the part had been inserted in these colonies, but unfortunately all PCR results were negative indicating an unsuccessful ligation.</p><br />
<p>We also performed a miniprep on <i>Pseudomonas putida</i> strain #28 this week using a home-made protocol rather than the Qiagen kit. Miniprep A had a DNA concentration of 1539.8 ng/μL and Miniprep B had DNA concentration of 1001.2 ng/μL possibly indicating that there may have been a lot of genomic DNA contamination. However miniprep A product was still used as a PCR template for a reaction attempting to isolate <i>CarAc</i>, <i>CarAd</i>, <i>AntA</i>, <i>AntB</i>, and <i>AntC</i>. Special conditions for this PCR included replacing 60 μL of water with 60 μL of betaine heated to 37 degrees Celsius and running a Mg gradient ranging from 0.5 μL - 2.5 μL per tube for each gene. Of these only <i>AntB</i> had bands of the right size in 3 of the lanes (Mg concentrations of 1.5, 2, and 2.5 μL). However, all of the <i>AntB</i> bands (including the negative control) contained a contamination band at around 100 bases, probably due to the formation of primer dimers. This forced us to do a gel extraction rather than a PCR purification. This gel extraction did not work as the nanodrop results indicated high amounts of agarose contamination. Another PCR was performed using the same conditions, but using miniprep B instead as there was no more miniprep A product remaining. Unfortunately there were no bands for <i>AntB</i>, only the 100 base pair contamination bands. </p><br />
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<h2>Week 9 (June 25 - June 29)</h2><br />
<p>This week we modified our PCR protocol to adjust for the homemade Taq polymerase we were using. Template DNA was boiled for 10 minutes on its own to replace the initialization step. This is because this polymerase could not tolerate the 95 degree heat during the regular initialization step. Another modification we made was to boil our primers for 5 minutes prior to adding them to the master mix. This was done to discourage the formation of primer-dimers. Finally we also attempted a touch-down PCR which started at an annealing temperature greater than the Tm of our primers and gradually worked its way down to below the Tm of the primers. This was done to increase the specificity of our reaction and also eliminate the formation of primer-dimers. Unfortunately these techniques still saw the formation of dimers, preventing us from performing a PCR purification. Again, the gel purification procedure did not lead to successful purification of our target gene. Due to the problems we have had so far our goal for next week is to perform an assay experiment to try and determine which of the <i>Pseudomonas putida</i> strains we have access to can actually degrade carbazole and therefore contain the pCAR1 plasmid. </p><br />
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<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The goal for this week was to grow different strains of<i> Pseudomonas</i> in media containing B-N media + glucose with only carbazole as a source of nitrogen. In theory only those strains which contained the pCAR1 plasmid would be able to use carbazole as a nitrogen source and thrive in this media. The strains we tested from glycerol stock included <i>P. putida</i> LD1, <i>P.putida</i> environmental strains 28-30, and <i>P. fluorescens</i> PF5. Each of these strains had two cultures grown; one with carbazole and one without it. <i>E. coli</i> grown with carbazole was used as a negative control as it does not contain the pCAR plasmid. At timepoints of 22, 46, and 117 hours of growth samples were taken to do an OD600 reading to measure bacterial growth and had an ammonia assay performed on it. The detection of ammonia acted as a proxy for detecting carbazole degradation as it is an end-product of the pathway. With this in mind, an additional control strain of LD1 was grown in media containing glucose and ammonia. This is to detect the possibility that <i>Pseudomonas</i> can use ammonia as a source of nitrogen after it has degraded carbazole which would be problematic as far as using ammonia levels to determine carbazole degradation. So far we have been having difficulties reading the results of the ammonia assay with a plate reader and may resort to testing our samples using a different assay protocol next week. The OD600 readings were initially very high at 22 hours and then dropped at 46 hours. </p><br />
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<h2>Week 11 (July 9-July 13)</h2><br />
<p>This week we took our last readings from the experiment that we started last week. The OD600 readings taken at 117 hours showed minimal growth over the weekend for all strains except for environmental strain #29 (with carbazole) which increased from 0.086 to 0.313. No other strain showed an increase of 0.100 or greater. This shows that only strain #29 was able to thrive in the carbazole media, possibly indicating that it contains the pCAR plasmid enabling it to use carbazole as a nitrogen source. To verify this we used a new ammonia assay protocol to measure the production of ammonia in the different cultures and theoretically how much carbazole was degraded. Unlike the previous assay we were able to produce a standard curve with a consistent slope based on samples with known amounts of ammonia. However, the only samples that showed any elevation of ammonia levels above the blank were the LD1 samples that were cultured with ammonia chloride to control for the possibility of pseudomonas using ammonia as a nitrogen source. The ammonia levels in this sample were relatively constant which would indicate that the LD1 was not using ammonia as a nitrogen source. Unfortunately the fact that none of the test samples had elevated ammonia levels did not give us any evidence that these strains contain the pCAR plasmid. </p><br />
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</html>[[File: Standard_curve_for_ammonia_assay_UCalgary2012_denitrification_group.png|500px|thumb|Standard curve for ammonia assay using OD635 absorbance.|center|]]<html><br />
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<h2>Week 12 (July 16 -July 20)</h2><br />
<p>We received a new strain of Pseudomonas, LD2, this week that had previously been used to study the pCAR1 plasmid. It was grown up in PCA media and had its colonies used for colony PCR. We also received our new primers this week, further increasing the chances of successful PCR. Using the new strain and the new primers <i>CarAa</i>, <i>CarAc</i>, and <i>CarAd</i> all showed at least some bands of close to the correct size. After PCR purification we obtained DNA concentrations of 21.3 ng/μL for <i>CarAa</i>, 41.9 ng/ μL for <i>CarAc</i>, and 32.8 ng/μL for<i> CarAd</i>. These products were then restriction digested using EcoRI + PstI sites. </p><br />
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</html>[[File:07.20.2012 CarAa and CarAc.jpg|500px|thumb|Gel of colony PCR from Pseudomonas LD2 for CarAa and CarAc genes. CarAa expected size is 1155 bp and CarAc is 323 bp.|center]]<br />
[[File:07.20.2012 CarAd colony PCR from LD2.jpg|500px|thumb|Gel of colony PCR from Pseudomonas LD2 for CarAd gene. CarAd expected size is 989 bp.|center]] <html><br />
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<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The restriction digested <i>Car</i> genes were ligated into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> cells. White colonies grew for<i> CarAa</i> and <i>CarAc</i> suggesting that ligation and transformation were successful, however no colonies grew for <i>CarAd</i> suggesting that the transformation did not work. Colony PCR was performed to confirm the presence of these genes within the vectors, however only <i>CarAc</i> was positive. The colonies containing the <i>CarAc</i> plasmid were then mini prepped and restriction digested to isolate the plasmid and send for sequencing. Further attempts at transforming<i> CarAa</i> and <i>CarAd</i> were not successful. Another goal was to perform colony PCR on the <i>P. putida</i> to isolate the<i> AntABC</i> genes using both the primers for the entire operon and those for the individual genes. No attempt showed any bands, although salt and temperature gradients were used as well as different types of polymerase. LD2 cultures were grown up to be plasmid prepped and genomic prepped in order to have a higher concentration of template DNA in the PCR master mix and more PCR will be attempted on these genes next week. Mutagenesis primers were also designed this week to remove the illegal restriction cut sites in the<i> CarAd</i> and <i>AntB</i> genes (NotI and 2xEcoRI respectively). Only 1 mutagenesis primer was designed for <i>AntB</i> as one of its EcoRI sites was very close to the beginning of the gene and could be removed using a new primer. During this process it was also discovered that the original Ant primers were designed for the wrong genes and therefore do not match the intended sequence. Although the new <i>AntABC</i> primers were designed using the correct gene the PCR may not be working due to the length of the operon so the design of new individual primers may be necessary. Finally, we also aimed to grow up the freeze dried culture of <i>Rhodococcus erythropolis</i> (source of the<i> AmdA</i> gene) that we received from DSMZ. The reactivation media was produced and autoclaved to begin the process next week.</p><br />
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</html>[[File:07.23.2012.Restriction Digest of Car genes.jpg|500px|thumb|Gel of restriction digest of Car genes. A refers to CarAa, C to CarAc, and D to CarAd. Ladder shows that after purification and digestion DNA is still the correct size.|center]]<br />
[[File:CarAc biobrick confirmation PCR.jpg|500px|thumb|Gel of confirmation PCR done on competent Top 10 E. coli cells in which a PSB1C3 vector containing the CarAc gene had been inserted. Red X marks the 500 bp mark on the weak ladder showing the product to be the expected size (323 bp gene + 2*100 bp biobrick sites on each end).|center]] <html><br />
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<h2>Week 14 (July 30 - August 3)</h2><br />
<p>We received the sequencing results this week for <i>CarAc</i> which came back as a 100% match to the expected nucleotide sequence. We also successfully ligated both<i> CarAa</i> and<i> CarAd</i> into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into top 10 <i>E. coli</i> cells. Colony PCR verified the presence of the biobrick plasmid in these colonies. These were both miniprepped, digested and sent for sequencing by the end of the week. Using a salt gradient, addition of DMSO, and the KAPA buffer specifically designed for GC-rich amplicons we were able to amplify the Ant operon from a genomic prep of LD2. However, all attempts at ligating and transforming this sequence were met with the unfortunate presence of red colonies (indicating failed insertion into the plasmid). Next week we will recut a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector with Xba and Pst to attempt to mitigate this problem. We also reactivated the <i>R. erythropolis</i> using DSMZ media #1 and grew it on plates made from DSMZ media #220 at 28 degrees. Colonies grew very quickly and were used for colony PCR and to make overnight cultures for downstream applications. The colony PCR using the amidase primers was unsuccessful, so a genomic prep was performed on the overnight culture. Unfortunately we lacked some of the required equipment and the genomic prep did not work, but with a few procedural modifications and the right equipment we are confident it will work next week. A glycerol stock of the<i> R. erythropoplis</i> was also made from another overnight culture. We also started construction on the <i>CarAc </i>part this week by inserting a ribosome binding site (B0034) in front of the gene. Finally, since the<i> Ant</i> operon was successfully amplified by PCR we designed mutagenesis primers for the 2nd EcoRI site in the operon rather than making new individual primers. We also designed a mutagenesis primer to remove the SpeI site in <i>AmdA</i>. Now that we have conclusively proven that the LD2 strain contains the pCAR1 plasmid we aim to demonstrate its ability to degrade carbazole and other nitrogen containing heterocycles using an assay experiment similar to the one performed in Week 10.</p><br />
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</html>[[File:07.30.2012. CarAa, CarAd.jpg|500px|thumb|CarAa and CarAd amplified from genomic prep of Pseudomonas LD2. Amplicons are larger than expected, potentially the gel ran diagonally. The 6th lane for each gene was a negative control.|center]]<br />
[[File:07.31.2012.Ant operon.jpg|500px|thumb|Ant operon amplified from genomic prep of Pseudomonas LD2 at the expected size of 3000 bp.|center]]<br />
[[File:08.01.2012 CarAdBB.jpg|500px|thumb|Lanes 2,4,5, and 11 contain CarAd genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent E.Coli.|center]]<br />
[[File:08.01.2012 CarAaBB.jpg|500px|thumb|Lane 9 contains CarAa genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent E.Coli. Although larger than expected, the positive control RFP generator in lane 10 is also larger than its expected size (~1200 bp including biobrick bookends) indicating that the gel may have a slant in it.|center]]<html><br />
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<h2>Week 15 (August 7 - August 10)</h2><br />
<p>This week we received the sequencing results for <i>CarAa</i> and <i>CarAd</i>. <i>CarAd</i> was a match, however <i>CarAa</i> did not match at all and had misplaced biobrick restriction enzyme sites. Due to this we decided to restart the biobricking process for <i>CarAa</i> all the way back to PCR from the plasmid. By the end of the week we had progressed to the point of transforming it into competent <i>E. coli</i> cells, however a thick lawn of bacteria had grown overnight making it impossible to pick colonies. A streak plate was made from this plate to attempt to get less dense growth. Since the sequencing came back positive for <i>CarAd</i> the next step was to perform site directed mutagenesis on its NotI restriction enzyme site using the primers that were designed previously (for site-directed mutagenesis optimization, please refer to Desulfurization Journal Week 14). The PCR was successful, showing very bright bands at 3000 bp, and the PCR product was digested with DpnI to remove parental DNA. Transformation of this product will indicate whether the mutagenesis was successful. We were also able to successfully perform a genomic prep of <i>Rhodococcus erythropolis</i> and amplify the <i>AmdA</i> gene this week. It was ligated into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> by Friday, and a confirmation colony PCR was run on Friday evening to verify that the gene was inserted. These results will be available by Monday. After many attempts the <i>Ant</i> operon was finally ligated and transformed as well this week. Confirmation PCR showed bands of about 3000 bp which is consistent with the size of the operon. By Friday the successful colonies had been grown up and miniprepped, ready to send for sequencing early next week. The <i>CarAc</i>/B0034 construct was also successfully transformed this week and verified by running a PCR using the B0034 forward primer. It was also miniprepped and is ready to send for sequencing next week. </p><br />
<p>In addition to this work constructing biobricks we also started an experiment to demonstrate the function of the pCAR plasmid in LD2. Strains of LD2 were grown up in LB and then washed and placed in B-N media + glucose and various experimental additions. These additions included carbazole, 4-PBAH (a nitrogen containing naphthenic acid), ammonia chloride, and a negative control with no nitrogen source. Strains of <i>E. coli</i> were also grown under the same conditions to compare growth and ammonia production of LD2 to a strain that does not contain the pCAR plasmid. These results should act as a preview of what we expect our final biobrick containing <i>E. col</i>i species to produce as well as allowing us to establish LD2 as a positive control for this experiment. </p><br />
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<br />
</html>[[File:08.07.2012 amdA pcr from genomic prep.jpg|500px|thumb|AmdA amplified from the genomic prep of R. Erythropolis shows bands just greater than 1500 base pairs in lanes 2,4-6,9, and 12-13. Lane 14 was a negative control. The expected size of AmdA is 1566 base pairs.|center]]<br />
[[File:08.09.2012 confirmation pcr of ant operon.jpg|500px|thumb|The two labelled lanes show amplification at 3000 base pairs from a confirmation colony PCR using the biobrick primers. The expected size of the Ant operon with the biobrick bookends is about 3100 base pairs.|center]]<br />
[[File:08.10.2012 carad notI mutagenesis.jpg|500px|thumb|Strong amplification at just above 3000 base pairs from the CarAd PCR using mutagenic primers. Since PSB1C3 is 2070 base pairs and CarAd is 989 base pairs the amplification should run at about 3059 base pairs. 3 different amounts of template DNA were tested in lanes 2-7 as indicated and a positive control showed weaker amplification in lanes 8-9 at about 4000 base pairs.|center]]<br />
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<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>The sequencing for the <i>CarAc</i>/<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> construct came back with only <i>CarAc</i> and no evidence of a <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> site preceding it. The confirmation PCR that was run last week using the <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> forward primer seemed to indicate that it was inserted, however since <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> is only 12 base pairs the primer may not have been specific enough. The construction on this part was restarted this week. <i>CarAa</i> colony PCR on last week's transformation showed two positive colonies, however upon culturing, miniprepping, and digesting these colonies the plasmid appeared to be too large (3000 base pairs as opposed to the expected 2000). This may be due to a problem of a mislabeled plasmid tube that has been experienced by other team members, causing the gene to be inserted into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a> rather than <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. To test this, a streak plate was made from these colonies and grown on an ampicillin resistant plate. Mutagenized <i>CarAd</i> was successfully transformed into Top 10 <i>E. coli</i> cells, cultured, and miniprepped. The miniprep product was digested with NotI to test whether the NotI site was successfully removed. These results were good, as the plasmid ran at 2000 base pairs and the insert at just under 1000 base pairs in the mutagenized <i>CarAd</i> whereas the non-mutagenized <i>CarAd</i> had its insert cut into 2 bands. This will be sent for sequencing next week to confirm the mutation. Unfortunately the sequencing results came back negative for the <i>Ant</i> operon meaning we will need to restart genomic PCR on it next week. Multiple transformations and confirmation PCRs were run on <i>AmdA</i> this week, however none displayed a band size of 1700-1800 base pairs as expected. We have continued to try different digested plasmids as other team members also had difficulties this week with ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, possibly indicating a problem with our Antarctic Phosphatase stock.</p><br />
<p>The other news from this week was the final results from our carbazole degradation experiment attempting to demonstrate LD2's ability to degrade carbazole and hopefully other nitrogen containing compounds. Unfortunately, these results were very difficult to make sense of as the LD2 that were cultured with carbazole grew, but did not show elevated ammonia levels which would be indicative of carbazole degradation. One of our controls was LD2 cultured with ammonium chloride to test whether LD2 was capable of using ammonia as a nitrogen source. This would pose a problem for us as we were planning to use an increase in ammonia levels as a proxy for carbazole degradation rather than measuring carbazole degradation directly using GC-MS. However, the LD2 showed significantly less ammonia after 44 hours when compared to 26 hours indicating that it may be degraded, either by the LD2 or by an unaccounted for abiotic process. This has lead us to decide to use GC-MS and directly measure carbazole levels in our sample the next time we do this experiment.</p><br />
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<h2>Week 17 (August 20 - August 24)</h2><br />
<p>The <i>CarAa</i> was re-streaked onto ampicillin plates and grew, probably showing that it was ligated into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a>, not <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, which explained the irregularity of our confirmation digest last week. It was sent for sequencing, but in case it was incorrect we also started a new <i>CarAa</i> pipeline by PCRing it from the <i>Pseudomonas</i> genome again and redigesting and ligating. It was transformed into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> over the weekend. <i>CarAc</i> did not see a lot of progress this week as we spent much of the time attempting to construct a RBS (B0034) in front of it, but were unsuccessful. <i>CarAd</i> was also subjected to this construction with B0034 and we were able to send it for sequencing by the end of the week. Several new constructs were also started for <i>CarAd</i> in case the sequencing is negative. The <i>Ant</i> operon was successfully transformed and confirmed by colony PCR this week, after which it was grown up and sent for sequencing. We also began to attempt mutagenesis on one of the EcoRI sites in the operon, but had no amplification of the plasmid. We also re-digested some PCR purified product to start another ligation in case sequencing failed. After many attempts we were also able to finally successfully transform an <i>AmdA</i>/PSB1C3 plasmid into Top 10 cells this week. After a miniprep they showed bands of about 1800 base pairs on the confirmation digest gel, so they were also sent for sequencing this week and had mutagenesis started on the illegal SpeI site. Unfortunately, the sequencing sent early in the week came back with ambiguous results. The forward reaction for<i> AntABC</i> was a match, but its reverse reaction matched to <i>CarAa</i>. Both <i>CarAa</i> reactions matched nothing. Since these two genes are over 20 000 base pairs apart on the template plasmid it is virtually impossible that they were actually combined into one plasmid (PCR extension time would not have been long enough and even if it was the product would have run much above the 3000 base pair mark). This is possibly a mistake on Eurofins end, so we are not 100% sure if we have 1 good gene, both of them, or neither. Also the sequence for mutagenized <i>CarAd</i> showed only about the first 100 base pairs, which were correct however not long enough meaning it was a poor reaction and inconclusive. Finally, we also re-started our carbazole degradation assay using the GC-MS at biological sciences. This process will take 2 weeks to demonstrate LD2's ability to degrade carbazole. We are forcing it to use carbazole as a carbon and a nitrogen source in one sample and as only a nitrogen source in another sample (this sample has had glucose added to it).</p><br />
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<h2>Week 18 (August 27 - August 31)</h2><br />
<p>The new stock of <i>CarAa</i> that we had PCRed from LD2 was sent for sequencing this week and came back as a match, so we have begun constructing a RBS site and a TetR promoter (<a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>) in front of it. At this point all the <i>Car</i> genes have been successfully amplified, inserted into a biobrick plasmid, and sequence verified. We also continued to attempt to construct <i>CarAc</i> with an RBS site (<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>) in front of it, but had no success. On a better note, we confirmed this week that<i> CarAd</i> was mutated properly and we were also able to insert an RBS site in front of it. The next step for this gene will be to attempt a plasmid switch as it is currently in <a href="http://partsregistry.org/Part:BBa_K590025">pSB1A3</a> and needs to be in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> for submission to the parts registry. After sending a new batch of the Ant operon for sequencing we got the same confusing results as we did last week. We therefore hypothesized that our primers were annealing to a homologous region that is similar to the<i> Ant</i> genes and very close to <i>CarAa</i>. This means that we need to restart PCR on this gene as all of our current product is incorrect. The sequencing results for <i>AmdA</i> that came back this week were also somewhat confusing as they did not quite match the sequence we expected. However, they were a 100% match for the <i>AmdA</i> gene in a different strain of <i>Rhodococcus erythropolis</i>. This means that DSMZ probably sent us the wrong strain of bacteria and that we were very lucky that the primers we designed based off of the expected template actually worked to amplify a slightly different gene in another strain. The good news is that this new sequence contains no illegal cut sites, which explains why mutagenesis was not working (there was nothing to mutate) and allows us to immediately begin construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> in front of <i>AmdA</i> this week. </p><br />
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<h2>Week 19 (September 3- September 7)</h2><br />
<p><i>CarAd</i>/B0034 was successfully plasmid switched into PSB1C3, making it ready for submission. Construction of J13002 and <i>CarAa</i> may have been successful as the digest of the miniprep looked promising. It will be sent for sequencing next week. Unfortunately there was no progress made on the constructions of <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> or <i>CarAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. These will be redigested and religated for next week. We could not amplify the <i>Ant</i> operon despite trying many variations of PCR using high fidelity KAPA polymerase. This is probably due to the multiple, large regions of homology on the operon that the reverse primer binds to making amplification difficult. Because the function of this gene can theoretically be replaced by <i>AmdA</i>, we may scale back efforts dedicated to it, as the chances of successful amplification appear slim. </p><br />
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<h2>Week 20 (September 10 - September 14)</h2><br />
<p>Constructs that continued this week included <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, <i>CarAa</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, and <i>CarAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. Some sequencing was sent, but no positive results were obtained. A new approach to biobrick the <i>Ant</i> operon was implemented this week. A new forward primer was designed that would anneal about 1500 base pairs upstream of the <i>Ant</i> operon, avoiding the region of homology that is repeated at various points on the plasmid. Once this region is successfully amplified we could use this amplified DNA as a template for another PCR reaction which would use the normal <i>Ant</i> primers. Since the template would no longer include regions of homology these primers should be free to work properly and amplify the <i>Ant</i> operon. The PCR using the new primers was started this week, and although amplification at 4500 base pairs was observed as expected, there was also a lot of non-specific amplification. This means that to purify the amplified DNA that we want and avoid the other products we will have to perform a dreaded gel extraction next week. </p><br />
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<h2>Week 21 (September 17-21)</h2><br />
<p>Nothing worked this week, please see above </p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DenitrogenationTeam:Calgary/Notebook/Denitrogenation2012-10-03T03:50:37Z<p>Achawdhry: </p>
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<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Denitrogenation Journal|<br />
CONTENT=<br />
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<h2>Week 1 (May 1-4)</h2><br />
<p>The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrogenation, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a bioreactor capable of not only detecting but also converting naphthenic acids and other toxins to useful or at least innocuous compounds. </p><br />
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<h2>Week 2-3 (May 7-18)</h2><br />
<p>In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the <i>car</i> operon, <i>CarA</i> (<i>CarAaAcAd</i>), <i>CarB</i> (<i>CarBaBb</i>), and <i>CarC</i>. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the <i>ant</i> operon, <i>antA, B, and C</i>, yielding cathecol while releasing CO2 and NH3. The <i>car</i> and <i>ant</i> operons are both regulated by the P<sup>ant</sup> promoter which is induced by the protein, antR. <i>CarAa</i> also has its own promoter which is not induced by <i>antR</i> (Diaz & Garcia, 2010). We have also investigated an alternative pathway using <i>CarA</i> combined with an amidase (<i>amdA</i>) that selectively cleaves NH2 from an intermediate of the car pathway (Xu et al, 2006). This could bypass much of the car/ant pathway and is possibly more efficient.</p><br />
<p>We have decided to use <i>Pseudomonas resinovorans</i> and <i>Rhodococcus erythropolis</i> to derive these genes from. <i>CarABC</i> and <i>AntABC</i> from<i> P. resinovorans</i> has been shown to have a wide range of nitrogen containing substrate specificity. <i>R. erythropolis</i> contains the <i>amdA</i> gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its <i>CarABC</i> pathway.</p><br />
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<h2>Week 4 (May 22-25)</h2><br />
<p>This week we reviewed the primers listed in the database and also designed some new ones. Primers for <i>CarAa</i>,<i> CarAc</i>, and <i>CarAd</i> were designed individually, while primers for <i>CarBaBbC</i> and <i>AntABC</i> were designed to encompass multiple genes in a sequence. These primers were designed to be used on <i>Pseudomonas putida</i> which we decided to use as our gene source since it was available to us as opposed to ordering <i>Pseudomonas resinovorans</i>. We also designed a primer for the<i> AmdA</i> gene from <i>Rhodococcus erthyroplois</i>. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative <i>AmdA</i> pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.</p><br />
<p>We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock <i>Psuedomonas putida</i> on Thursday. We also resuspended the primers for <i>CarAa</i>, <i>CarAc</i>, <i>CarAd</i>,<i> AntA</i>, <i>AntB</i>, and <i>Ant C</i> that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.</p><br />
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<h2>Week 5 (May 28 - June 1)</h2><br />
<p>On Monday and Tuesday we used the database primers to attempt to isolate <i>CarAa</i>, <i>CarAc</i>, <i>CarAd</i>, <i>AntA</i>, <i>AntB</i>, and <i>AntC</i> from the <i>Psuedomonas putida</i> we plated last week. <i>CarAd</i>, <i>AntB</i>, and <i>AntC</i> were all put in the gradient PCR machine to account for the wide range of their primers’ melting points, while the others were done via regular PCR. Unfortunately, no positive results were obtained from these reactions. The PCR on <i>CarAc</i> and <i>AntA</i> was repeated on Wednesday, still not giving positive results. Finally, we attempted to use a salt concentration gradient on the PCR reaction for <i>CarAc</i> and <i>AntA</i>, using concentrations that ranged from 1.0 microlitres/tube to 2.0 microlitres/tube in increments of 0.2 microlitres/tube. This helped as <i>CarAc</i> showed bands in samples that had concentrations of 1.2 and 1.4 microlitres/tube. <i> AntA</i> also showed bands, however they were not the correct size, indicating contamination and/or non-specific annealing of the primer. The positive control also showed bands of the correct size. Earlier in the week we also made overnight cultures of 3 environmental strains (28, 29, 30) of <i>Psuedomonas putida</i> from -80 glycerol stock. On Friday we performed a genomic prep on these cultures, and plan on attempting to amplify genes from the isolated DNA next week. </p><br />
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<h2>Week 6 (June 4 - June 8)</h2><br />
<p> We started off this week by determining the DNA concentration of our genomic prep samples from last week using the nanodrop. DNA concentration for all three<i> P. putida</i> strains was at least 1000 ng/microlitre, well above what was needed for PCR. 1/2 and 1/3 dilutions were prepared for all three strains so as not to have an excess of template DNA in PCR reactions. PCR was performed on all 3 strains using primers for <i>CarAc</i>, <i>CarAd</i>, <i>AntA</i>, <i> AntB</i>, and <i>AntC</i> using 6 replicates per gene. The only successful amplification appeared to be <i>AntB</i> and <i>CarAc</i>, both from strain 28 (with weaker bands in strain 29). We then performed another PCR, just on those two genes with an increased amount of Taq polymerase to hopefully get enough amplified DNA to move forward with. This resulted in strong bands for both at the expected size. We then performed PCR purification using the Qiaquick kit and obtained samples containing 33.5 ng/microlitre of <i>AntB</i> DNA and 129 ng/microlitre of <i>CarAc </i>DNA. These concentrations were both sufficient to begin a restriction digest and ligation of these parts into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Next week we hope to verify the results of the restriction digest, continue to amplify <i>CarAc</i> and <i>AntB</i> from strain 28, and hopefully submit a biobrick for sequencing.</p><br />
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<h2>Week 7 (June 11 - June 15)</h2><br />
<p>The purified PCR products for <i>CarAc</i> and <i>AntB</i> were digested with restriction enzymes EcoRI+SpeI, EcoRI+PstI, and XbaI+PstI. However, only <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmids that had been digested with EcoRI+SpeI and EcoRI+PstI were available to attempt ligation. Gel results were inconclusive on the restriction digest product as the plasmid size appeared much larger than expected. However, transformation was still attempted on the ligation products for both genes (both using EcoRI+SpeI ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid). The transformation products were plated onto chloroamphenicol (chlor) plates to select for colonies that had the chloro resistance gene on the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid. Also this week another round of PCR was performed for the <i> Car</i> and <i> Ant</i> genes, however all, but <i>CarAc</i> showed bands in the negative control lane, possibly indicating contamination or the formation of primer dimers. The <i>CarAc</i> bands were fairly weak and a PCR purification resulted in very low DNA concentration, insufficient to move onto restriction digest. Next week's plan hinges heavily on the result of the transformation from this Friday. </p><br />
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<h2>Week 8 (June 18 - June 22)</h2><br />
<p>The results of our transformation on <i>CarAc</i> and <i>AntB</i> from last week showed mostly red colonies indicating that the ligation was unsuccessful and that the PSB1C3 plasmid simply closed on itself with no insert. However, there were three colonies for <i>CarAc</i> that were white (cut with both X+P and E+P). Colony PCR was performed on these three colonies using biobrick primer sets to verify that the part had been inserted in these colonies, but unfortunately all PCR results were negative indicating an unsuccessful ligation.</p><br />
<p>We also performed a miniprep on <i>Pseudomonas putida</i> strain #28 this week using a home-made protocol rather than the Qiagen kit. Miniprep A had a DNA concentration of 1539.8 ng/μL and Miniprep B had DNA concentration of 1001.2 ng/μL possibly indicating that there may have been a lot of genomic DNA contamination. However miniprep A product was still used as a PCR template for a reaction attempting to isolate <i>CarAc</i>, <i>CarAd</i>, <i>AntA</i>, <i>AntB</i>, and <i>AntC</i>. Special conditions for this PCR included replacing 60 μL of water with 60 μL of betaine heated to 37 degrees Celsius and running a Mg gradient ranging from 0.5 μL - 2.5 μL per tube for each gene. Of these only <i>AntB</i> had bands of the right size in 3 of the lanes (Mg concentrations of 1.5, 2, and 2.5 μL). However, all of the <i>AntB</i> bands (including the negative control) contained a contamination band at around 100 bases, probably due to the formation of primer dimers. This forced us to do a gel extraction rather than a PCR purification. This gel extraction did not work as the nanodrop results indicated high amounts of agarose contamination. Another PCR was performed using the same conditions, but using miniprep B instead as there was no more miniprep A product remaining. Unfortunately there were no bands for <i>AntB</i>, only the 100 base pair contamination bands. </p><br />
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<h2>Week 9 (June 25 - June 29)</h2><br />
<p>This week we modified our PCR protocol to adjust for the homemade Taq polymerase we were using. Template DNA was boiled for 10 minutes on its own to replace the initialization step. This is because this polymerase could not tolerate the 95 degree heat during the regular initialization step. Another modification we made was to boil our primers for 5 minutes prior to adding them to the master mix. This was done to discourage the formation of primer-dimers. Finally we also attempted a touch-down PCR which started at an annealing temperature greater than the Tm of our primers and gradually worked its way down to below the Tm of the primers. This was done to increase the specificity of our reaction and also eliminate the formation of primer-dimers. Unfortunately these techniques still saw the formation of dimers, preventing us from performing a PCR purification. Again, the gel purification procedure did not lead to successful purification of our target gene. Due to the problems we have had so far our goal for next week is to perform an assay experiment to try and determine which of the <i>Pseudomonas putida</i> strains we have access to can actually degrade carbazole and therefore contain the pCAR1 plasmid. </p><br />
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<h2>Week 10 (July 2-July 6)</h2><br />
<p>The goal for this week was to grow different strains of<i> Pseudomonas</i> in media containing B-N media + glucose with only carbazole as a source of nitrogen. In theory only those strains which contained the pCAR1 plasmid would be able to use carbazole as a nitrogen source and thrive in this media. The strains we tested from glycerol stock included <i>P. putida</i> LD1, <i>P.putida</i> environmental strains 28-30, and <i>P. fluorescens</i> PF5. Each of these strains had two cultures grown; one with carbazole and one without it. <i>E. coli</i> grown with carbazole was used as a negative control as it does not contain the pCAR plasmid. At timepoints of 22, 46, and 117 hours of growth samples were taken to do an OD600 reading to measure bacterial growth and had an ammonia assay performed on it. The detection of ammonia acted as a proxy for detecting carbazole degradation as it is an end-product of the pathway. With this in mind, an additional control strain of LD1 was grown in media containing glucose and ammonia. This is to detect the possibility that <i>Pseudomonas</i> can use ammonia as a source of nitrogen after it has degraded carbazole which would be problematic as far as using ammonia levels to determine carbazole degradation. So far we have been having difficulties reading the results of the ammonia assay with a plate reader and may resort to testing our samples using a different assay protocol next week. The OD600 readings were initially very high at 22 hours and then dropped at 46 hours. </p><br />
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<h2>Week 11 (July 9-July 13)</h2><br />
<p>This week we took our last readings from the experiment that we started last week. The OD600 readings taken at 117 hours showed minimal growth over the weekend for all strains except for environmental strain #29 (with carbazole) which increased from 0.086 to 0.313. No other strain showed an increase of 0.100 or greater. This shows that only strain #29 was able to thrive in the carbazole media, possibly indicating that it contains the pCAR plasmid enabling it to use carbazole as a nitrogen source. To verify this we used a new ammonia assay protocol to measure the production of ammonia in the different cultures and theoretically how much carbazole was degraded. Unlike the previous assay we were able to produce a standard curve with a consistent slope based on samples with known amounts of ammonia. However, the only samples that showed any elevation of ammonia levels above the blank were the LD1 samples that were cultured with ammonia chloride to control for the possibility of pseudomonas using ammonia as a nitrogen source. The ammonia levels in this sample were relatively constant which would indicate that the LD1 was not using ammonia as a nitrogen source. Unfortunately the fact that none of the test samples had elevated ammonia levels did not give us any evidence that these strains contain the pCAR plasmid. </p><br />
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</html>[[File: Standard_curve_for_ammonia_assay_UCalgary2012_denitrification_group.png|500px|thumb|Standard curve for ammonia assay using OD635 absorbance.|center|]]<html><br />
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<h2>Week 12 (July 16 -July 20)</h2><br />
<p>We received a new strain of Pseudomonas, LD2, this week that had previously been used to study the pCAR1 plasmid. It was grown up in PCA media and had its colonies used for colony PCR. We also received our new primers this week, further increasing the chances of successful PCR. Using the new strain and the new primers <i>CarAa</i>, <i>CarAc</i>, and <i>CarAd</i> all showed at least some bands of close to the correct size. After PCR purification we obtained DNA concentrations of 21.3 ng/μL for <i>CarAa</i>, 41.9 ng/ μL for <i>CarAc</i>, and 32.8 ng/μL for<i> CarAd</i>. These products were then restriction digested using EcoRI + PstI sites. </p><br />
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</html>[[File:07.20.2012 CarAa and CarAc.jpg|500px|thumb|Gel of colony PCR from Pseudomonas LD2 for CarAa and CarAc genes. CarAa expected size is 1155 bp and CarAc is 323 bp.|center]]<br />
[[File:07.20.2012 CarAd colony PCR from LD2.jpg|500px|thumb|Gel of colony PCR from Pseudomonas LD2 for CarAd gene. CarAd expected size is 989 bp.|center]] <html><br />
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<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The restriction digested <i>Car</i> genes were ligated into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> cells. White colonies grew for<i> CarAa</i> and <i>CarAc</i> suggesting that ligation and transformation were successful, however no colonies grew for <i>CarAd</i> suggesting that the transformation did not work. Colony PCR was performed to confirm the presence of these genes within the vectors, however only <i>CarAc</i> was positive. The colonies containing the <i>CarAc</i> plasmid were then mini prepped and restriction digested to isolate the plasmid and send for sequencing. Further attempts at transforming<i> CarAa</i> and <i>CarAd</i> were not successful. Another goal was to perform colony PCR on the <i>P. putida</i> to isolate the<i> AntABC</i> genes using both the primers for the entire operon and those for the individual genes. No attempt showed any bands, although salt and temperature gradients were used as well as different types of polymerase. LD2 cultures were grown up to be plasmid prepped and genomic prepped in order to have a higher concentration of template DNA in the PCR master mix and more PCR will be attempted on these genes next week. Mutagenesis primers were also designed this week to remove the illegal restriction cut sites in the<i> CarAd</i> and <i>AntB</i> genes (NotI and 2xEcoRI respectively). Only 1 mutagenesis primer was designed for <i>AntB</i> as one of its EcoRI sites was very close to the beginning of the gene and could be removed using a new primer. During this process it was also discovered that the original Ant primers were designed for the wrong genes and therefore do not match the intended sequence. Although the new <i>AntABC</i> primers were designed using the correct gene the PCR may not be working due to the length of the operon so the design of new individual primers may be necessary. Finally, we also aimed to grow up the freeze dried culture of <i>Rhodococcus erythropolis</i> (source of the<i> AmdA</i> gene) that we received from DSMZ. The reactivation media was produced and autoclaved to begin the process next week.</p><br />
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</html>[[File:07.23.2012.Restriction Digest of Car genes.jpg|500px|thumb|Gel of restriction digest of Car genes. A refers to CarAa, C to CarAc, and D to CarAd. Ladder shows that after purification and digestion DNA is still the correct size.|center]]<br />
[[File:CarAc biobrick confirmation PCR.jpg|500px|thumb|Gel of confirmation PCR done on competent Top 10 E. coli cells in which a PSB1C3 vector containing the CarAc gene had been inserted. Red X marks the 500 bp mark on the weak ladder showing the product to be the expected size (323 bp gene + 2*100 bp biobrick sites on each end).|center]] <html><br />
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<h2>Week 14 (July 30 - August 3)</h2><br />
<p>We received the sequencing results this week for <i>CarAc</i> which came back as a 100% match to the expected nucleotide sequence. We also successfully ligated both<i> CarAa</i> and<i> CarAd</i> into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into top 10 <i>E. coli</i> cells. Colony PCR verified the presence of the biobrick plasmid in these colonies. These were both miniprepped, digested and sent for sequencing by the end of the week. Using a salt gradient, addition of DMSO, and the KAPA buffer specifically designed for GC-rich amplicons we were able to amplify the Ant operon from a genomic prep of LD2. However, all attempts at ligating and transforming this sequence were met with the unfortunate presence of red colonies (indicating failed insertion into the plasmid). Next week we will recut a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector with Xba and Pst to attempt to mitigate this problem. We also reactivated the <i>R. erythropolis</i> using DSMZ media #1 and grew it on plates made from DSMZ media #220 at 28 degrees. Colonies grew very quickly and were used for colony PCR and to make overnight cultures for downstream applications. The colony PCR using the amidase primers was unsuccessful, so a genomic prep was performed on the overnight culture. Unfortunately we lacked some of the required equipment and the genomic prep did not work, but with a few procedural modifications and the right equipment we are confident it will work next week. A glycerol stock of the<i> R. erythropoplis</i> was also made from another overnight culture. We also started construction on the <i>CarAc </i>part this week by inserting a ribosome binding site (B0034) in front of the gene. Finally, since the<i> Ant</i> operon was successfully amplified by PCR we designed mutagenesis primers for the 2nd EcoRI site in the operon rather than making new individual primers. We also designed a mutagenesis primer to remove the SpeI site in <i>AmdA</i>. Now that we have conclusively proven that the LD2 strain contains the pCAR1 plasmid we aim to demonstrate its ability to degrade carbazole and other nitrogen containing heterocycles using an assay experiment similar to the one performed in Week 10.</p><br />
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</html>[[File:07.30.2012. CarAa, CarAd.jpg|500px|thumb|CarAa and CarAd amplified from genomic prep of Pseudomonas LD2. Amplicons are larger than expected, potentially the gel ran diagonally. The 6th lane for each gene was a negative control.|center]]<br />
[[File:07.31.2012.Ant operon.jpg|500px|thumb|Ant operon amplified from genomic prep of Pseudomonas LD2 at the expected size of 3000 bp.|center]]<br />
[[File:08.01.2012 CarAdBB.jpg|500px|thumb|Lanes 2,4,5, and 11 contain CarAd genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent E.Coli.|center]]<br />
[[File:08.01.2012 CarAaBB.jpg|500px|thumb|Lane 9 contains CarAa genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent E.Coli. Although larger than expected, the positive control RFP generator in lane 10 is also larger than its expected size (~1200 bp including biobrick bookends) indicating that the gel may have a slant in it.|center]]<html><br />
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<h2>Week 15 (August 7 - August 10)</h2><br />
<p>This week we received the sequencing results for <i>CarAa</i> and <i>CarAd</i>. <i>CarAd</i> was a match, however <i>CarAa</i> did not match at all and had misplaced biobrick restriction enzyme sites. Due to this we decided to restart the biobricking process for <i>CarAa</i> all the way back to PCR from the plasmid. By the end of the week we had progressed to the point of transforming it into competent <i>E. coli</i> cells, however a thick lawn of bacteria had grown overnight making it impossible to pick colonies. A streak plate was made from this plate to attempt to get less dense growth. Since the sequencing came back positive for <i>CarAd</i> the next step was to perform site directed mutagenesis on its NotI restriction enzyme site using the primers that were designed previously (for site-directed mutagenesis optimization, please refer to Desulfurization Journal Week 14). The PCR was successful, showing very bright bands at 3000 bp, and the PCR product was digested with DpnI to remove parental DNA. Transformation of this product will indicate whether the mutagenesis was successful. We were also able to successfully perform a genomic prep of <i>Rhodococcus erythropolis</i> and amplify the <i>AmdA</i> gene this week. It was ligated into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> by Friday, and a confirmation colony PCR was run on Friday evening to verify that the gene was inserted. These results will be available by Monday. After many attempts the <i>Ant</i> operon was finally ligated and transformed as well this week. Confirmation PCR showed bands of about 3000 bp which is consistent with the size of the operon. By Friday the successful colonies had been grown up and miniprepped, ready to send for sequencing early next week. The <i>CarAc</i>/B0034 construct was also successfully transformed this week and verified by running a PCR using the B0034 forward primer. It was also miniprepped and is ready to send for sequencing next week. </p><br />
<p>In addition to this work constructing biobricks we also started an experiment to demonstrate the function of the pCAR plasmid in LD2. Strains of LD2 were grown up in LB and then washed and placed in B-N media + glucose and various experimental additions. These additions included carbazole, 4-PBAH (a nitrogen containing naphthenic acid), ammonia chloride, and a negative control with no nitrogen source. Strains of <i>E. coli</i> were also grown under the same conditions to compare growth and ammonia production of LD2 to a strain that does not contain the pCAR plasmid. These results should act as a preview of what we expect our final biobrick containing <i>E. col</i>i species to produce as well as allowing us to establish LD2 as a positive control for this experiment. </p><br />
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</html>[[File:08.07.2012 amdA pcr from genomic prep.jpg|500px|thumb|AmdA amplified from the genomic prep of R. Erythropolis shows bands just greater than 1500 base pairs in lanes 2,4-6,9, and 12-13. Lane 14 was a negative control. The expected size of AmdA is 1566 base pairs.|center]]<br />
[[File:08.09.2012 confirmation pcr of ant operon.jpg|500px|thumb|The two labelled lanes show amplification at 3000 base pairs from a confirmation colony PCR using the biobrick primers. The expected size of the Ant operon with the biobrick bookends is about 3100 base pairs.|center]]<br />
[[File:08.10.2012 carad notI mutagenesis.jpg|500px|thumb|Strong amplification at just above 3000 base pairs from the CarAd PCR using mutagenic primers. Since PSB1C3 is 2070 base pairs and CarAd is 989 base pairs the amplification should run at about 3059 base pairs. 3 different amounts of template DNA were tested in lanes 2-7 as indicated and a positive control showed weaker amplification in lanes 8-9 at about 4000 base pairs.|center]]<br />
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<h2>Week 16 (August 13 - August 17)</h2><br />
<p>The sequencing for the <i>CarAc</i>/B0034 construct came back with only <i>CarAc</i> and no evidence of a <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> site preceding it. The confirmation PCR that was run last week using the <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> forward primer seemed to indicate that it was inserted, however since <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a> is only 12 base pairs the primer may not have been specific enough. The construction on this part was restarted this week. <i>CarAa</i> colony PCR on last week's transformation showed two positive colonies, however upon culturing, miniprepping, and digesting these colonies the plasmid appeared to be too large (3000 base pairs as opposed to the expected 2000). This may be due to a problem of a mislabeled plasmid tube that has been experienced by other team members, causing the gene to be inserted into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a> rather than <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. To test this, a streak plate was made from these colonies and grown on an ampicillin resistant plate. Mutagenized <i>CarAd</i> was successfully transformed into Top 10 <i>E. coli</i> cells, cultured, and miniprepped. The miniprep product was digested with NotI to test whether the NotI site was successfully removed. These results were good, as the plasmid ran at 2000 base pairs and the insert at just under 1000 base pairs in the mutagenized <i>CarAd</i> whereas the non-mutagenized <i>CarAd</i> had its insert cut into 2 bands. This will be sent for sequencing next week to confirm the mutation. Unfortunately the sequencing results came back negative for the <i>Ant</i> operon meaning we will need to restart genomic PCR on it next week. Multiple transformations and confirmation PCRs were run on <i>AmdA</i> this week, however none displayed a band size of 1700-1800 base pairs as expected. We have continued to try different digested plasmids as other team members also had difficulties this week with ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, possibly indicating a problem with our Antarctic Phosphatase stock.</p><br />
<p>The other news from this week was the final results from our carbazole degradation experiment attempting to demonstrate LD2's ability to degrade carbazole and hopefully other nitrogen containing compounds. Unfortunately, these results were very difficult to make sense of as the LD2 that were cultured with carbazole grew, but did not show elevated ammonia levels which would be indicative of carbazole degradation. One of our controls was LD2 cultured with ammonium chloride to test whether LD2 was capable of using ammonia as a nitrogen source. This would pose a problem for us as we were planning to use an increase in ammonia levels as a proxy for carbazole degradation rather than measuring carbazole degradation directly using GC-MS. However, the LD2 showed significantly less ammonia after 44 hours when compared to 26 hours indicating that it may be degraded, either by the LD2 or by an unaccounted for abiotic process. This has lead us to decide to use GC-MS and directly measure carbazole levels in our sample the next time we do this experiment.</p><br />
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<h2>Week 17 (August 20 - August 24)</h2><br />
<p>The <i>CarAa</i> was re-streaked onto ampicillin plates and grew, probably showing that it was ligated into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a>, not <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, which explained the irregularity of our confirmation digest last week. It was sent for sequencing, but in case it was incorrect we also started a new <i>CarAa</i> pipeline by PCRing it from the <i>Pseudomonas</i> genome again and redigesting and ligating. It was transformed into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> over the weekend. <i>CarAc</i> did not see a lot of progress this week as we spent much of the time attempting to construct a RBS (B0034) in front of it, but were unsuccessful. <i>CarAd</i> was also subjected to this construction with B0034 and we were able to send it for sequencing by the end of the week. Several new constructs were also started for <i>CarAd</i> in case the sequencing is negative. The <i>Ant</i> operon was successfully transformed and confirmed by colony PCR this week, after which it was grown up and sent for sequencing. We also began to attempt mutagenesis on one of the EcoRI sites in the operon, but had no amplification of the plasmid. We also re-digested some PCR purified product to start another ligation in case sequencing failed. After many attempts we were also able to finally successfully transform an <i>AmdA</i>/PSB1C3 plasmid into Top 10 cells this week. After a miniprep they showed bands of about 1800 base pairs on the confirmation digest gel, so they were also sent for sequencing this week and had mutagenesis started on the illegal SpeI site. Unfortunately, the sequencing sent early in the week came back with ambiguous results. The forward reaction for<i> AntABC</i> was a match, but its reverse reaction matched to <i>CarAa</i>. Both <i>CarAa</i> reactions matched nothing. Since these two genes are over 20 000 base pairs apart on the template plasmid it is virtually impossible that they were actually combined into one plasmid (PCR extension time would not have been long enough and even if it was the product would have run much above the 3000 base pair mark). This is possibly a mistake on Eurofins end, so we are not 100% sure if we have 1 good gene, both of them, or neither. Also the sequence for mutagenized <i>CarAd</i> showed only about the first 100 base pairs, which were correct however not long enough meaning it was a poor reaction and inconclusive. Finally, we also re-started our carbazole degradation assay using the GC-MS at biological sciences. This process will take 2 weeks to demonstrate LD2's ability to degrade carbazole. We are forcing it to use carbazole as a carbon and a nitrogen source in one sample and as only a nitrogen source in another sample (this sample has had glucose added to it).</p><br />
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<h2>Week 18 (August 27 - August 31)</h2><br />
<p>The new stock of <i>CarAa</i> that we had PCRed from LD2 was sent for sequencing this week and came back as a match, so we have begun constructing a RBS site and a TetR promoter (J13002) in front of it. At this point all the <i>Car</i> genes have been successfully amplified, inserted into a biobrick plasmid, and sequence verified. We also continued to attempt to construct <i>CarAc</i> with an RBS site (B0034) in front of it, but had no success. On a better note, we confirmed this week that<i> CarAd</i> was mutated properly and we were also able to insert an RBS site in front of it. The next step for this gene will be to attempt a plasmid switch as it is currently in <a href="http://partsregistry.org/Part:BBa_K590025">pSB1A3</a> and needs to be in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> for submission to the parts registry. After sending a new batch of the Ant operon for sequencing we got the same confusing results as we did last week. We therefore hypothesized that our primers were annealing to a homologous region that is similar to the<i> Ant</i> genes and very close to <i>CarAa</i>. This means that we need to restart PCR on this gene as all of our current product is incorrect. The sequencing results for <i>AmdA</i> that came back this week were also somewhat confusing as they did not quite match the sequence we expected. However, they were a 100% match for the <i>AmdA</i> gene in a different strain of <i>Rhodococcus erythropolis</i>. This means that DSMZ probably sent us the wrong strain of bacteria and that we were very lucky that the primers we designed based off of the expected template actually worked to amplify a slightly different gene in another strain. The good news is that this new sequence contains no illegal cut sites, which explains why mutagenesis was not working (there was nothing to mutate) and allows us to immediately begin construction of <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> in front of <i>AmdA</i> this week. </p><br />
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<h2>Week 19 (September 3- September 7)</h2><br />
<p><i>CarAd</i>/B0034 was successfully plasmid switched into PSB1C3, making it ready for submission. Construction of J13002 and <i>CarAa</i> may have been successful as the digest of the miniprep looked promising. It will be sent for sequencing next week. Unfortunately there was no progress made on the constructions of <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> or <i>CarAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. These will be redigested and religated for next week. We could not amplify the <i>Ant</i> operon despite trying many variations of PCR using high fidelity KAPA polymerase. This is probably due to the multiple, large regions of homology on the operon that the reverse primer binds to making amplification difficult. Because the function of this gene can theoretically be replaced by <i>AmdA</i>, we may scale back efforts dedicated to it, as the chances of successful amplification appear slim. </p><br />
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<h2>Week 20 (September 10 - September 14)</h2><br />
<p>Constructs that continued this week included <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, <i>CarAa</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, and <i>CarAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. Some sequencing was sent, but no positive results were obtained. A new approach to biobrick the <i>Ant</i> operon was implemented this week. A new forward primer was designed that would anneal about 1500 base pairs upstream of the <i>Ant</i> operon, avoiding the region of homology that is repeated at various points on the plasmid. Once this region is successfully amplified we could use this amplified DNA as a template for another PCR reaction which would use the normal <i>Ant</i> primers. Since the template would no longer include regions of homology these primers should be free to work properly and amplify the <i>Ant</i> operon. The PCR using the new primers was started this week, and although amplification at 4500 base pairs was observed as expected, there was also a lot of non-specific amplification. This means that to purify the amplified DNA that we want and avoid the other products we will have to perform a dreaded gel extraction next week. </p><br />
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<h2>Week 21 (September 17-21)</h2><br />
<p>Nothing worked this week, please see above </p><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DenitrogenationTeam:Calgary/Notebook/Denitrogenation2012-10-03T03:42:45Z<p>Achawdhry: </p>
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<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Denitrogenation Journal|<br />
CONTENT=<br />
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<h2>Week 1 (May 1-4)</h2><br />
<p>The following section covers wetlab aspect of our overall project focusing on microbial conversion of naphthenic acids into economically-valuable hydrocarbons. The approach taken in this endeavour will be from four strategic starting points - ring cleavage, decarboxylation, denitrogenation, and desulfurization. Overall, the 'hydrocarbons' aspect of the project is a critical one to our overall design and construction of a bioreactor capable of not only detecting but also converting naphthenic acids and other toxins to useful or at least innocuous compounds. </p><br />
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<h2>Week 2-3 (May 7-18)</h2><br />
<p>In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the <i>car</i> operon, <i>CarA</i> (<i>CarAaAcAd</i>), <i>CarB</i> (<i>CarBaBb</i>), and <i>CarC</i>. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the <i>ant</i> operon, <i>antA, B, and C</i>, yielding cathecol while releasing CO2 and NH3. The <i>car</i> and <i>ant</i> operons are both regulated by the P<sup>ant</sup> promoter which is induced by the protein, antR. <i>CarAa</i> also has its own promoter which is not induced by <i>antR</i> (Diaz & Garcia, 2010). We have also investigated an alternative pathway using <i>CarA</i> combined with an amidase (<i>amdA</i>) that selectively cleaves NH2 from an intermediate of the car pathway (Xu et al, 2006). This could bypass much of the car/ant pathway and is possibly more efficient.</p><br />
<p>We have decided to use <i>Pseudomonas resinovorans</i> and <i>Rhodococcus erythropolis</i> to derive these genes from. <i>CarABC</i> and <i>AntABC</i> from<i> P. resinovorans</i> has been shown to have a wide range of nitrogen containing substrate specificity. <i>R. erythropolis</i> contains the <i>amdA</i> gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its <i>CarABC</i> pathway.</p><br />
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<h2>Week 4 (May 22-25)</h2><br />
<p>This week we reviewed the primers listed in the database and also designed some new ones. Primers for <i>CarAa</i>,<i> CarAc</i>, and <i>CarAd</i> were designed individually, while primers for <i>CarBaBbC</i> and <i>AntABC</i> were designed to encompass multiple genes in a sequence. These primers were designed to be used on <i>Pseudomonas putida</i> which we decided to use as our gene source since it was available to us as opposed to ordering <i>Pseudomonas resinovorans</i>. We also designed a primer for the<i> AmdA</i> gene from <i>Rhodococcus erthyroplois</i>. In addition to ordering these primers we also ordered the nitrogen containing compounds that we will need to test these enzymes on. We decided on using carbazole to make sure the enzymes can perform their natural function as well as pyrrolidine to test them on a similar ring structure. We also ordered cyclohexamine in order to independently test the function of the alternative <i>AmdA</i> pathway. Finally, we decided to eventually order 4-Piperidine butyric acid hydrochloride to test how the enzymes will work on nitrogen containing naphthenic acids. However, we decided since it is a very expensive compound we would wait to make sure the enzyme's work on more simple compounds before ordering it.</p><br />
<p>We also started our work in the wet lab by plating colonies and making an overnight culture of -80 freezer stock <i>Psuedomonas putida</i> on Thursday. We also resuspended the primers for <i>CarAa</i>, <i>CarAc</i>, <i>CarAd</i>,<i> AntA</i>, <i>AntB</i>, and <i>Ant C</i> that were already in the database. We were able to use the colonies that grew on the streak plates to start a colony PCR to attempt to isolate each of these genes on Friday.</p><br />
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<h2>Week 5 (May 28 - June 1)</h2><br />
<p>On Monday and Tuesday we used the database primers to attempt to isolate <i>CarAa</i>, <i>CarAc</i>, <i>CarAd</i>, <i>AntA</i>, <i>AntB</i>, and <i>AntC</i> from the <i>Psuedomonas putida</i> we plated last week. <i>CarAd</i>, <i>AntB</i>, and <i>AntC</i> were all put in the gradient PCR machine to account for the wide range of their primers’ melting points, while the others were done via regular PCR. Unfortunately, no positive results were obtained from these reactions. The PCR on <i>CarAc</i> and <i>AntA</i> was repeated on Wednesday, still not giving positive results. Finally, we attempted to use a salt concentration gradient on the PCR reaction for <i>CarAc</i> and <i>AntA</i>, using concentrations that ranged from 1.0 microlitres/tube to 2.0 microlitres/tube in increments of 0.2 microlitres/tube. This helped as <i>CarAc</i> showed bands in samples that had concentrations of 1.2 and 1.4 microlitres/tube. <i> AntA</i> also showed bands, however they were not the correct size, indicating contamination and/or non-specific annealing of the primer. The positive control also showed bands of the correct size. Earlier in the week we also made overnight cultures of 3 environmental strains (28, 29, 30) of <i>Psuedomonas putida</i> from -80 glycerol stock. On Friday we performed a genomic prep on these cultures, and plan on attempting to amplify genes from the isolated DNA next week. </p><br />
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<h2>Week 6 (June 4 - June 8)</h2><br />
<p> We started off this week by determining the DNA concentration of our genomic prep samples from last week using the nanodrop. DNA concentration for all three<i> P. putida</i> strains was at least 1000 ng/microlitre, well above what was needed for PCR. 1/2 and 1/3 dilutions were prepared for all three strains so as not to have an excess of template DNA in PCR reactions. PCR was performed on all 3 strains using primers for <i>CarAc</i>, <i>CarAd</i>, <i>AntA</i>, <i> AntB</i>, and <i>AntC</i> using 6 replicates per gene. The only successful amplification appeared to be <i>AntB</i> and <i>CarAc</i>, both from strain 28 (with weaker bands in strain 29). We then performed another PCR, just on those two genes with an increased amount of Taq polymerase to hopefully get enough amplified DNA to move forward with. This resulted in strong bands for both at the expected size. We then performed PCR purification using the Qiaquick kit and obtained samples containing 33.5 ng/microlitre of <i>AntB</i> DNA and 129 ng/microlitre of <i>CarAc </i>DNA. These concentrations were both sufficient to begin a restriction digest and ligation of these parts into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Next week we hope to verify the results of the restriction digest, continue to amplify <i>CarAc</i> and <i>AntB</i> from strain 28, and hopefully submit a biobrick for sequencing.</p><br />
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<h2>Week 7 (June 11 - June 15)</h2><br />
<p>The purified PCR products for <i>CarAc</i> and <i>AntB</i> were digested with restriction enzymes EcoRI+SpeI, EcoRI+PstI, and XbaI+PstI. However, only <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmids that had been digested with EcoRI+SpeI and EcoRI+PstI were available to attempt ligation. Gel results were inconclusive on the restriction digest product as the plasmid size appeared much larger than expected. However, transformation was still attempted on the ligation products for both genes (both using EcoRI+SpeI ligation into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid). The transformation products were plated onto chloroamphenicol (chlor) plates to select for colonies that had the chloro resistance gene on the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> plasmid. Also this week another round of PCR was performed for the <i> Car</i> and <i> Ant</i> genes, however all, but <i>CarAc</i> showed bands in the negative control lane, possibly indicating contamination or the formation of primer dimers. The <i>CarAc</i> bands were fairly weak and a PCR purification resulted in very low DNA concentration, insufficient to move onto restriction digest. Next week's plan hinges heavily on the result of the transformation from this Friday. </p><br />
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<h2>Week 8 (June 18 - June 22)</h2><br />
<p>The results of our transformation on <i>CarAc</i> and <i>AntB</i> from last week showed mostly red colonies indicating that the ligation was unsuccessful and that the PSB1C3 plasmid simply closed on itself with no insert. However, there were three colonies for <i>CarAc</i> that were white (cut with both X+P and E+P). Colony PCR was performed on these three colonies using biobrick primer sets to verify that the part had been inserted in these colonies, but unfortunately all PCR results were negative indicating an unsuccessful ligation.</p><br />
<p>We also performed a miniprep on <i>Pseudomonas putida</i> strain #28 this week using a home-made protocol rather than the Qiagen kit. Miniprep A had a DNA concentration of 1539.8 ng/μL and Miniprep B had DNA concentration of 1001.2 ng/μL possibly indicating that there may have been a lot of genomic DNA contamination. However miniprep A product was still used as a PCR template for a reaction attempting to isolate <i>CarAc</i>, <i>CarAd</i>, <i>AntA</i>, <i>AntB</i>, and <i>AntC</i>. Special conditions for this PCR included replacing 60 μL of water with 60 μL of betaine heated to 37 degrees Celsius and running a Mg gradient ranging from 0.5 μL - 2.5 μL per tube for each gene. Of these only <i>AntB</i> had bands of the right size in 3 of the lanes (Mg concentrations of 1.5, 2, and 2.5 μL). However, all of the <i>AntB</i> bands (including the negative control) contained a contamination band at around 100 bases, probably due to the formation of primer dimers. This forced us to do a gel extraction rather than a PCR purification. This gel extraction did not work as the nanodrop results indicated high amounts of agarose contamination. Another PCR was performed using the same conditions, but using miniprep B instead as there was no more miniprep A product remaining. Unfortunately there were no bands for <i>AntB</i>, only the 100 base pair contamination bands. </p><br />
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<h2>Week 9 (June 25 - June 29)</h2><br />
<p>This week we modified our PCR protocol to adjust for the homemade Taq polymerase we were using. Template DNA was boiled for 10 minutes on its own to replace the initialization step. This is because this polymerase could not tolerate the 95 degree heat during the regular initialization step. Another modification we made was to boil our primers for 5 minutes prior to adding them to the master mix. This was done to discourage the formation of primer-dimers. Finally we also attempted a touch-down PCR which started at an annealing temperature greater than the Tm of our primers and gradually worked its way down to below the Tm of the primers. This was done to increase the specificity of our reaction and also eliminate the formation of primer-dimers. Unfortunately these techniques still saw the formation of dimers, preventing us from performing a PCR purification. Again, the gel purification procedure did not lead to successful purification of our target gene. Due to the problems we have had so far our goal for next week is to perform an assay experiment to try and determine which of the <i>Pseudomonas putida</i> strains we have access to can actually degrade carbazole and therefore contain the pCAR1 plasmid. </p><br />
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<h2>Week 10 (July 2-July 6)</h2><br />
<p>The goal for this week was to grow different strains of<i> Pseudomonas</i> in media containing B-N media + glucose with only carbazole as a source of nitrogen. In theory only those strains which contained the pCAR1 plasmid would be able to use carbazole as a nitrogen source and thrive in this media. The strains we tested from glycerol stock included <i>P. putida</i> LD1, <i>P.putida</i> environmental strains 28-30, and <i>P. fluorescens</i> PF5. Each of these strains had two cultures grown; one with carbazole and one without it. <i>E. coli</i> grown with carbazole was used as a negative control as it does not contain the pCAR plasmid. At timepoints of 22, 46, and 117 hours of growth samples were taken to do an OD600 reading to measure bacterial growth and had an ammonia assay performed on it. The detection of ammonia acted as a proxy for detecting carbazole degradation as it is an end-product of the pathway. With this in mind, an additional control strain of LD1 was grown in media containing glucose and ammonia. This is to detect the possibility that <i>Pseudomonas</i> can use ammonia as a source of nitrogen after it has degraded carbazole which would be problematic as far as using ammonia levels to determine carbazole degradation. So far we have been having difficulties reading the results of the ammonia assay with a plate reader and may resort to testing our samples using a different assay protocol next week. The OD600 readings were initially very high at 22 hours and then dropped at 46 hours. </p><br />
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<h2>Week 11 (July 9-July 13)</h2><br />
<p>This week we took our last readings from the experiment that we started last week. The OD600 readings taken at 117 hours showed minimal growth over the weekend for all strains except for environmental strain #29 (with carbazole) which increased from 0.086 to 0.313. No other strain showed an increase of 0.100 or greater. This shows that only strain #29 was able to thrive in the carbazole media, possibly indicating that it contains the pCAR plasmid enabling it to use carbazole as a nitrogen source. To verify this we used a new ammonia assay protocol to measure the production of ammonia in the different cultures and theoretically how much carbazole was degraded. Unlike the previous assay we were able to produce a standard curve with a consistent slope based on samples with known amounts of ammonia. However, the only samples that showed any elevation of ammonia levels above the blank were the LD1 samples that were cultured with ammonia chloride to control for the possibility of pseudomonas using ammonia as a nitrogen source. The ammonia levels in this sample were relatively constant which would indicate that the LD1 was not using ammonia as a nitrogen source. Unfortunately the fact that none of the test samples had elevated ammonia levels did not give us any evidence that these strains contain the pCAR plasmid. </p><br />
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</html>[[File: Standard_curve_for_ammonia_assay_UCalgary2012_denitrification_group.png|500px|thumb|Standard curve for ammonia assay using OD635 absorbance.|center|]]<html><br />
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<h2>Week 12 (July 16 -July 20)</h2><br />
<p>We received a new strain of Pseudomonas, LD2, this week that had previously been used to study the pCAR1 plasmid. It was grown up in PCA media and had its colonies used for colony PCR. We also received our new primers this week, further increasing the chances of successful PCR. Using the new strain and the new primers <i>CarAa</i>, <i>CarAc</i>, and <i>CarAd</i> all showed at least some bands of close to the correct size. After PCR purification we obtained DNA concentrations of 21.3 ng/μL for <i>CarAa</i>, 41.9 ng/ μL for <i>CarAc</i>, and 32.8 ng/μL for<i> CarAd</i>. These products were then restriction digested using EcoRI + PstI sites. </p><br />
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</html>[[File:07.20.2012 CarAa and CarAc.jpg|500px|thumb|Gel of colony PCR from Pseudomonas LD2 for CarAa and CarAc genes. CarAa expected size is 1155 bp and CarAc is 323 bp.|center]]<br />
[[File:07.20.2012 CarAd colony PCR from LD2.jpg|500px|thumb|Gel of colony PCR from Pseudomonas LD2 for CarAd gene. CarAd expected size is 989 bp.|center]] <html><br />
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<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The restriction digested <i>Car</i> genes were ligated into vector <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> cells. White colonies grew for<i> CarAa</i> and <i>CarAc</i> suggesting that ligation and transformation were successful, however no colonies grew for <i>CarAd</i> suggesting that the transformation did not work. Colony PCR was performed to confirm the presence of these genes within the vectors, however only <i>CarAc</i> was positive. The colonies containing the <i>CarAc</i> plasmid were then mini prepped and restriction digested to isolate the plasmid and send for sequencing. Further attempts at transforming<i> CarAa</i> and <i>CarAd</i> were not successful. Another goal was to perform colony PCR on the <i>P. putida</i> to isolate the<i> AntABC</i> genes using both the primers for the entire operon and those for the individual genes. No attempt showed any bands, although salt and temperature gradients were used as well as different types of polymerase. LD2 cultures were grown up to be plasmid prepped and genomic prepped in order to have a higher concentration of template DNA in the PCR master mix and more PCR will be attempted on these genes next week. Mutagenesis primers were also designed this week to remove the illegal restriction cut sites in the<i> CarAd</i> and <i>AntB</i> genes (NotI and 2xEcoRI respectively). Only 1 mutagenesis primer was designed for <i>AntB</i> as one of its EcoRI sites was very close to the beginning of the gene and could be removed using a new primer. During this process it was also discovered that the original Ant primers were designed for the wrong genes and therefore do not match the intended sequence. Although the new <i>AntABC</i> primers were designed using the correct gene the PCR may not be working due to the length of the operon so the design of new individual primers may be necessary. Finally, we also aimed to grow up the freeze dried culture of <i>Rhodococcus erythropolis</i> (source of the<i> AmdA</i> gene) that we received from DSMZ. The reactivation media was produced and autoclaved to begin the process next week.</p><br />
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</html>[[File:07.23.2012.Restriction Digest of Car genes.jpg|500px|thumb|Gel of restriction digest of Car genes. A refers to CarAa, C to CarAc, and D to CarAd. Ladder shows that after purification and digestion DNA is still the correct size.|center]]<br />
[[File:CarAc biobrick confirmation PCR.jpg|500px|thumb|Gel of confirmation PCR done on competent Top 10 E. coli cells in which a PSB1C3 vector containing the CarAc gene had been inserted. Red X marks the 500 bp mark on the weak ladder showing the product to be the expected size (323 bp gene + 2*100 bp biobrick sites on each end).|center]] <html><br />
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<h2>Week 14 (July 30 - August 3)</h2><br />
<p>We received the sequencing results this week for <i>CarAc</i> which came back as a 100% match to the expected nucleotide sequence. We also successfully ligated both<i> CarAa</i> and<i> CarAd</i> into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into top 10 <i>E. coli</i> cells. Colony PCR verified the presence of the biobrick plasmid in these colonies. These were both miniprepped, digested and sent for sequencing by the end of the week. Using a salt gradient, addition of DMSO, and the KAPA buffer specifically designed for GC-rich amplicons we were able to amplify the Ant operon from a genomic prep of LD2. However, all attempts at ligating and transforming this sequence were met with the unfortunate presence of red colonies (indicating failed insertion into the plasmid). Next week we will recut a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector with Xba and Pst to attempt to mitigate this problem. We also reactivated the <i>R. erythropolis</i> using DSMZ media #1 and grew it on plates made from DSMZ media #220 at 28 degrees. Colonies grew very quickly and were used for colony PCR and to make overnight cultures for downstream applications. The colony PCR using the amidase primers was unsuccessful, so a genomic prep was performed on the overnight culture. Unfortunately we lacked some of the required equipment and the genomic prep did not work, but with a few procedural modifications and the right equipment we are confident it will work next week. A glycerol stock of the<i> R. erythropoplis</i> was also made from another overnight culture. We also started construction on the <i>CarAc </i>part this week by inserting a ribosome binding site (B0034) in front of the gene. Finally, since the<i> Ant</i> operon was successfully amplified by PCR we designed mutagenesis primers for the 2nd EcoRI site in the operon rather than making new individual primers. We also designed a mutagenesis primer to remove the SpeI site in <i>AmdA</i>. Now that we have conclusively proven that the LD2 strain contains the pCAR1 plasmid we aim to demonstrate its ability to degrade carbazole and other nitrogen containing heterocycles using an assay experiment similar to the one performed in Week 10.</p><br />
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</html>[[File:07.30.2012. CarAa, CarAd.jpg|500px|thumb|CarAa and CarAd amplified from genomic prep of Pseudomonas LD2. Amplicons are larger than expected, potentially the gel ran diagonally. The 6th lane for each gene was a negative control.|center]]<br />
[[File:07.31.2012.Ant operon.jpg|500px|thumb|Ant operon amplified from genomic prep of Pseudomonas LD2 at the expected size of 3000 bp.|center]]<br />
[[File:08.01.2012 CarAdBB.jpg|500px|thumb|Lanes 2,4,5, and 11 contain CarAd genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent E.Coli.|center]]<br />
[[File:08.01.2012 CarAaBB.jpg|500px|thumb|Lane 9 contains CarAa genes amplified from colony PCR (using biobrick primers) of the PCR product that had been ligated into PSB1C3 and transformed into competent E.Coli. Although larger than expected, the positive control RFP generator in lane 10 is also larger than its expected size (~1200 bp including biobrick bookends) indicating that the gel may have a slant in it.|center]]<html><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p>This week we received the sequencing results for <i>CarAa</i> and <i>CarAd</i>. <i>CarAd</i> was a match, however <i>CarAa</i> did not match at all and had misplaced biobrick restriction enzyme sites. Due to this we decided to restart the biobricking process for <i>CarAa</i> all the way back to PCR from the plasmid. By the end of the week we had progressed to the point of transforming it into competent <i>E. coli</i> cells, however a thick lawn of bacteria had grown overnight making it impossible to pick colonies. A streak plate was made from this plate to attempt to get less dense growth. Since the sequencing came back positive for <i>CarAd</i> the next step was to perform site directed mutagenesis on its NotI restriction enzyme site using the primers that were designed previously (for site-directed mutagenesis optimization, please refer to Desulfurization Journal Week 14). The PCR was successful, showing very bright bands at 3000 bp, and the PCR product was digested with DpnI to remove parental DNA. Transformation of this product will indicate whether the mutagenesis was successful. We were also able to successfully perform a genomic prep of <i>Rhodococcus erythropolis</i> and amplify the <i>AmdA</i> gene this week. It was ligated into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> and transformed into competent <i>E. coli</i> by Friday, and a confirmation colony PCR was run on Friday evening to verify that the gene was inserted. These results will be available by Monday. After many attempts the <i>Ant</i> operon was finally ligated and transformed as well this week. Confirmation PCR showed bands of about 3000 bp which is consistent with the size of the operon. By Friday the successful colonies had been grown up and miniprepped, ready to send for sequencing early next week. The <i>CarAc</i>/B0034 construct was also successfully transformed this week and verified by running a PCR using the B0034 forward primer. It was also miniprepped and is ready to send for sequencing next week. </p><br />
<p>In addition to this work constructing biobricks we also started an experiment to demonstrate the function of the pCAR plasmid in LD2. Strains of LD2 were grown up in LB and then washed and placed in B-N media + glucose and various experimental additions. These additions included carbazole, 4-PBAH (a nitrogen containing naphthenic acid), ammonia chloride, and a negative control with no nitrogen source. Strains of <i>E. coli</i> were also grown under the same conditions to compare growth and ammonia production of LD2 to a strain that does not contain the pCAR plasmid. These results should act as a preview of what we expect our final biobrick containing <i>E. col</i>i species to produce as well as allowing us to establish LD2 as a positive control for this experiment. </p><br />
<br />
<br />
</html>[[File:08.07.2012 amdA pcr from genomic prep.jpg|500px|thumb|AmdA amplified from the genomic prep of R. Erythropolis shows bands just greater than 1500 base pairs in lanes 2,4-6,9, and 12-13. Lane 14 was a negative control. The expected size of AmdA is 1566 base pairs.|center]]<br />
[[File:08.09.2012 confirmation pcr of ant operon.jpg|500px|thumb|The two labelled lanes show amplification at 3000 base pairs from a confirmation colony PCR using the biobrick primers. The expected size of the Ant operon with the biobrick bookends is about 3100 base pairs.|center]]<br />
[[File:08.10.2012 carad notI mutagenesis.jpg|500px|thumb|Strong amplification at just above 3000 base pairs from the CarAd PCR using mutagenic primers. Since PSB1C3 is 2070 base pairs and CarAd is 989 base pairs the amplification should run at about 3059 base pairs. 3 different amounts of template DNA were tested in lanes 2-7 as indicated and a positive control showed weaker amplification in lanes 8-9 at about 4000 base pairs.|center]]<br />
<html><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>The sequencing for the <i>CarAc</i>/B0034 construct came back with only <i>CarAc</i> and no evidence of a B0034 site preceding it. The confirmation PCR that was run last week using the B0034 forward primer seemed to indicate that it was inserted, however since B0034 is only 12 base pairs the primer may not have been specific enough. The construction on this part was restarted this week. <i>CarAa</i> colony PCR on last week's transformation showed two positive colonies, however upon culturing, miniprepping, and digesting these colonies the plasmid appeared to be too large (3000 base pairs as opposed to the expected 2000). This may be due to a problem of a mislabeled plasmid tube that has been experienced by other team members, causing the gene to be inserted into PSB1AC3 rather than PSB1C3. To test this, a streak plate was made from these colonies and grown on an ampicillin resistant plate. Mutagenized <i>CarAd</i> was successfully transformed into Top 10 <i>E. coli</i> cells, cultured, and miniprepped. The miniprep product was digested with NotI to test whether the NotI site was successfully removed. These results were good, as the plasmid ran at 2000 base pairs and the insert at just under 1000 base pairs in the mutagenized <i>CarAd</i> whereas the non-mutagenized <i>CarAd</i> had its insert cut into 2 bands. This will be sent for sequencing next week to confirm the mutation. Unfortunately the sequencing results came back negative for the <i>Ant</i> operon meaning we will need to restart genomic PCR on it next week. Multiple transformations and confirmation PCRs were run on <i>AmdA</i> this week, however none displayed a band size of 1700-1800 base pairs as expected. We have continued to try different digested plasmids as other team members also had difficulties this week with ligation into PSB1C3, possibly indicating a problem with our Antarctic Phosphatase stock.</p><br />
<p>The other news from this week was the final results from our carbazole degradation experiment attempting to demonstrate LD2's ability to degrade carbazole and hopefully other nitrogen containing compounds. Unfortunately, these results were very difficult to make sense of as the LD2 that were cultured with carbazole grew, but did not show elevated ammonia levels which would be indicative of carbazole degradation. One of our controls was LD2 cultured with ammonium chloride to test whether LD2 was capable of using ammonia as a nitrogen source. This would pose a problem for us as we were planning to use an increase in ammonia levels as a proxy for carbazole degradation rather than measuring carbazole degradation directly using GC-MS. However, the LD2 showed significantly less ammonia after 44 hours when compared to 26 hours indicating that it may be degraded, either by the LD2 or by an unaccounted for abiotic process. This has lead us to decide to use GC-MS and directly measure carbazole levels in our sample the next time we do this experiment.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<p>The <i>CarAa</i> was re-streaked onto ampicillin plates and grew, probably showing that it was ligated into <a href="http://partsregistry.org/Part:pSB1AC3">pSB1AC3</a>, not <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, which explained the irregularity of our confirmation digest last week. It was sent for sequencing, but in case it was incorrect we also started a new <i>CarAa</i> pipeline by PCRing it from the <i>Pseudomonas</i> genome again and redigesting and ligating. It was transformed into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> over the weekend. <i>CarAc</i> did not see a lot of progress this week as we spent much of the time attempting to construct a RBS (B0034) in front of it, but were unsuccessful. <i>CarAd</i> was also subjected to this construction with B0034 and we were able to send it for sequencing by the end of the week. Several new constructs were also started for <i>CarAd</i> in case the sequencing is negative. The <i>Ant</i> operon was successfully transformed and confirmed by colony PCR this week, after which it was grown up and sent for sequencing. We also began to attempt mutagenesis on one of the EcoRI sites in the operon, but had no amplification of the plasmid. We also re-digested some PCR purified product to start another ligation in case sequencing failed. After many attempts we were also able to finally successfully transform an <i>AmdA</i>/PSB1C3 plasmid into Top 10 cells this week. After a miniprep they showed bands of about 1800 base pairs on the confirmation digest gel, so they were also sent for sequencing this week and had mutagenesis started on the illegal SpeI site. Unfortunately, the sequencing sent early in the week came back with ambiguous results. The forward reaction for<i> AntABC</i> was a match, but its reverse reaction matched to <i>CarAa</i>. Both <i>CarAa</i> reactions matched nothing. Since these two genes are over 20 000 base pairs apart on the template plasmid it is virtually impossible that they were actually combined into one plasmid (PCR extension time would not have been long enough and even if it was the product would have run much above the 3000 base pair mark). This is possibly a mistake on Eurofins end, so we are not 100% sure if we have 1 good gene, both of them, or neither. Also the sequence for mutagenized <i>CarAd</i> showed only about the first 100 base pairs, which were correct however not long enough meaning it was a poor reaction and inconclusive. Finally, we also re-started our carbazole degradation assay using the GC-MS at biological sciences. This process will take 2 weeks to demonstrate LD2's ability to degrade carbazole. We are forcing it to use carbazole as a carbon and a nitrogen source in one sample and as only a nitrogen source in another sample (this sample has had glucose added to it).</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<p>The new stock of <i>CarAa</i> that we had PCRed from LD2 was sent for sequencing this week and came back as a match, so we have begun constructing a RBS site and a TetR promoter (J13002) in front of it. At this point all the <i>Car</i> genes have been successfully amplified, inserted into a biobrick plasmid, and sequence verified. We also continued to attempt to construct <i>CarAc</i> with an RBS site (B0034) in front of it, but had no success. On a better note, we confirmed this week that<i> CarAd</i> was mutated properly and we were also able to insert an RBS site in front of it. The next step for this gene will be to attempt a plasmid switch as it is currently in <a href="http://partsregistry.org/Part:BBa_K590025">pSB1A3</a> and needs to be in <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> for submission to the parts registry. After sending a new batch of the Ant operon for sequencing we got the same confusing results as we did last week. We therefore hypothesized that our primers were annealing to a homologous region that is similar to the<i> Ant</i> genes and very close to <i>CarAa</i>. This means that we need to restart PCR on this gene as all of our current product is incorrect. The sequencing results for <i>AmdA</i> that came back this week were also somewhat confusing as they did not quite match the sequence we expected. However, they were a 100% match for the <i>AmdA</i> gene in a different strain of <i>Rhodococcus erythropolis</i>. This means that DSMZ probably sent us the wrong strain of bacteria and that we were very lucky that the primers we designed based off of the expected template actually worked to amplify a slightly different gene in another strain. The good news is that this new sequence contains no illegal cut sites, which explains why mutagenesis was not working (there was nothing to mutate) and allows us to immediately begin construction of J13002 in front of <i>AmdA</i> this week. </p><br />
<br />
<h2>Week 19 (September 3- September 7)</h2><br />
<p><i>CarAd</i>/B0034 was successfully plasmid switched into PSB1C3, making it ready for submission. Construction of J13002 and <i>CarAa</i> may have been successful as the digest of the miniprep looked promising. It will be sent for sequencing next week. Unfortunately there was no progress made on the constructions of <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a> or <i>CarAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. These will be redigested and religated for next week. We could not amplify the <i>Ant</i> operon despite trying many variations of PCR using high fidelity KAPA polymerase. This is probably due to the multiple, large regions of homology on the operon that the reverse primer binds to making amplification difficult. Because the function of this gene can theoretically be replaced by <i>AmdA</i>, we may scale back efforts dedicated to it, as the chances of successful amplification appear slim. </p><br />
<br />
<h2>Week 20 (September 10 - September 14)</h2><br />
<p>Constructs that continued this week included <i>AmdA</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, <i>CarAa</i> with <a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>, and <i>CarAc</i> with <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>. Some sequencing was sent, but no positive results were obtained. A new approach to biobrick the <i>Ant</i> operon was implemented this week. A new forward primer was designed that would anneal about 1500 base pairs upstream of the <i>Ant</i> operon, avoiding the region of homology that is repeated at various points on the plasmid. Once this region is successfully amplified we could use this amplified DNA as a template for another PCR reaction which would use the normal <i>Ant</i> primers. Since the template would no longer include regions of homology these primers should be free to work properly and amplify the <i>Ant</i> operon. The PCR using the new primers was started this week, and although amplification at 4500 base pairs was observed as expected, there was also a lot of non-specific amplification. This means that to purify the amplified DNA that we want and avoid the other products we will have to perform a dreaded gel extraction next week. </p><br />
<br />
<h2>Week 21 (September 17-21)</h2><br />
<p>Nothing worked this week, please see above </p><br />
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</html><br />
}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T05:54:05Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
<br />
CONTENT=<html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">"PetroBrick"</a> (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) and aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). We have planned to verify the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from <a href="http://www.dsmz.de">DSMZ</a>. In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
</html><br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
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<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the PetroBrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html><br />
<br />
<br />
<br />
<p> These results suggest that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> sequencing results with a complementarity of 87%, as compared to the theoretical <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">General Production Protocol</a> that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed eight overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> has worked on <a href="https://2011.igem.org/Team:Washington/Alkanes/Future/Vector">system optimization</a>, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> that is preventing effective operation. We may need to design inner primers to sequence the entire <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "<a href="https://2011.igem.org/Team:Washington/Alkanes/Future/Vector">system optimization</a>" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) was missing. This explained why our sequencing results for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) gene and no aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). Either the parts registry did not have the proper <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> (containing both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>. It was determined that the latter was true. As such, the transformation for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> (submitted by the University of Washington). This involved transforming the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">production media protocol</a> in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">production media protocol</a> to attempt to produce hydrocarbons because we are not certain as to whether the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> sent to us in the 2012 kit plate does not contain a proper functional <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, b) transformation and isolation of the aldehyde decarbonylate (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) and c) culturing the Nocardia strain NRRL 5646. With regards to the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). The <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a> was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) being ligated with aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). Upon successfully forming this biobrick (ideally the same as a functional <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
</html><br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/<a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a> gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> gene to produce a <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>. The Washington iGEM team agreed to send us a plasmid stock of their <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with twelve colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. Eight overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>, and NPT (X/P) with <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
</html>[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with eight different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the<a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>vector. Following standard <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> would be unable to degrade naphthenic acids, we found that bacteria containing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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</html><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T05:47:44Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
<br />
CONTENT=<html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">"PetroBrick"</a> (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) and aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). We have planned to verify the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from <a href="http://www.dsmz.de">DSMZ</a>. In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
</html><br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the PetroBrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html><br />
<br />
<br />
<br />
<p> These results suggest that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> sequencing results with a complementarity of 87%, as compared to the theoretical <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">General Production Protocol</a> that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed eight overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> has worked on <a href="https://2011.igem.org/Team:Washington/Alkanes/Future/Vector">system optimization</a>, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> that is preventing effective operation. We may need to design inner primers to sequence the entire <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "<a href="https://2011.igem.org/Team:Washington/Alkanes/Future/Vector">system optimization</a>" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) was missing. This explained why our sequencing results for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) gene and no aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). Either the parts registry did not have the proper <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> (containing both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>. It was determined that the latter was true. As such, the transformation for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> (submitted by the University of Washington). This involved transforming the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">production media protocol</a> in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">production media protocol</a> to attempt to produce hydrocarbons because we are not certain as to whether the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> sent to us in the 2012 kit plate does not contain a proper functional <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, b) transformation and isolation of the aldehyde decarbonylate (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) and c) culturing the Nocardia strain NRRL 5646. With regards to the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). The <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a> was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) being ligated with aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). Upon successfully forming this biobrick (ideally the same as a functional <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
</html><br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a> gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> gene to produce a <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>. The Washington iGEM team agreed to send us a plasmid stock of their <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with twelve colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. Eight overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
</html>[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with eight different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the<a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> would be unable to degrade naphthenic acids, we found that bacteria containing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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</html><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T05:34:39Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
<br />
CONTENT=<html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">"PetroBrick"</a> (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) and aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). We have planned to verify the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from <a href="http://www.dsmz.de">DSMZ</a>. In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
</html><br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the PetroBrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html><br />
<br />
<br />
<br />
<p> These results suggest that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our PetroBrick sequencing results with a complementarity of 87%, as compared to the theoretical <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">General Production Protocol</a> that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed eight overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> has worked on <a href="https://2011.igem.org/Team:Washington/Alkanes/Future/Vector">system optimization</a>, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> that is preventing effective operation. We may need to design inner primers to sequence the entire <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "<a href="https://2011.igem.org/Team:Washington/Alkanes/Future/Vector">system optimization</a>" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) was missing. This explained why our sequencing results for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) gene and no aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). Either the parts registry did not have the proper <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> (containing both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>. It was determined that the latter was true. As such, the transformation for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> (submitted by the University of Washington). This involved transforming the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">production media protocol</a> in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">production media protocol</a> to attempt to produce hydrocarbons because we are not certain as to whether the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> sent to us in the 2012 kit plate does not contain a proper functional <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, b) transformation and isolation of the aldehyde decarbonylate (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) and c) culturing the Nocardia strain NRRL 5646. With regards to the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a>, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). The <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a> was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) being ligated with aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). Upon successfully forming this biobrick (ideally the same as a functional petrobrick), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional petrobrick that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
</html><br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a> gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> gene to produce a PetroBrick. The Washington iGEM team agreed to send us a plasmid stock of their <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with twelve colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. Eight overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
</html>[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with eight different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the PetroBrick stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the PetroBrick as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard PetroBrick assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> would be unable to degrade naphthenic acids, we found that bacteria containing the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T05:07:34Z<p>Achawdhry: </p>
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<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
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CONTENT=<html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">"Petrobrick"</a> (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) and aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). We have planned to verify the PetroBrick in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from <a href="http://www.dsmz.de">DSMZ</a>. In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
</html><br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a>, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the petrobrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html><br />
<br />
<br />
<br />
<p> These results suggest that the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our PetroBrick sequencing results with a complementarity of 87%, as compared to the theoretical <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">General Production Protocol</a> that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed eight overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> has worked on <a href="https://2011.igem.org/Team:Washington/Alkanes/Future/Vector">system optimization</a>, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> that is preventing effective operation. We may need to design inner primers to sequence the entire <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a>, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "<a href="https://2011.igem.org/Team:Washington/Alkanes/Future/Vector">system optimization</a>" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) was missing. This explained why our sequencing results for the petrobrick yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) gene and no aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). Either the parts registry did not have the proper petrobrick (containing both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a>. It was determined that the latter was true. As such, the transformation for the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> for the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> (submitted by the University of Washington). This involved transforming the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">production media protocol</a> in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">production media protocol</a> to attempt to produce hydrocarbons because we are not certain as to whether the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> sent to us in the 2012 kit plate does not contain a proper functional petrobrick. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the petrobrick, b) transformation and isolation of the aldehyde decarbonylate (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) and c) culturing the Nocardia strain NRRL 5646. With regards to the petrobrick, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the <a href="http://partsregistry.org/Part:BBa_K590025">PetroBrick</a> by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). The <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a> was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) being ligated with aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). Upon successfully forming this biobrick (ideally the same as a functional petrobrick), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional petrobrick that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
</html><br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the ADC gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our AAR gene to produce a PetroBrick. The Washington iGEM team agreed to send us a plasmid stock of their PetroBrick and ADC. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with 12 colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. 8 overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
</html>[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with 8 different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the PetroBrick stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp PetroBrick was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the PetroBrick as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard PetroBrick assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the PetroBrick would be unable to degrade naphthenic acids, we found that bacteria containing the PetroBrick were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T04:46:55Z<p>Achawdhry: </p>
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<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
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CONTENT=<html><br />
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<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">"Petrobrick"</a> (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) and aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). We have planned to verify the PetroBrick in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from <a href="http://www.dsmz.de">DSMZ</a>. In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
</html><br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a>, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the petrobrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html><br />
<br />
<br />
<br />
<p> These results suggest that the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our PetroBrick sequencing results with a complementarity of 87%, as compared to the theoretical <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">General Production Protocol</a> that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed eight overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> has worked on system optimization, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> that is preventing effective operation. We may need to design inner primers to sequence the entire <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a>, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "system optimization" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) was missing. This explained why our sequencing results for the petrobrick yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) gene and no aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). Either the parts registry did not have the proper petrobrick (containing both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the petrobrick. It was determined that the latter was true. As such, the transformation for the petrobrick had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with AAR for the petrobrick (submitted by the University of Washington). This involved transforming the petrobrick into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the production media protocol in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the petrobrick did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the production media protocol to attempt to produce hydrocarbons because we are not certain as to whether the petrobrick part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the petrobrick sent to us in the 2012 kit plate does not contain a proper functional petrobrick. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the petrobrick, b) transformation and isolation of the aldehyde decarbonylate (ADC) and c) culturing the Nocardia strain NRRL 5646. With regards to the petrobrick, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the petrobrick they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the petrobrick by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (ADC). The ADC was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for ADC, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (AAR) being ligated with aldehyde decarbonylase (ADC). Upon successfully forming this biobrick (ideally the same as a functional petrobrick), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional petrobrick that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
</html><br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the ADC gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our AAR gene to produce a PetroBrick. The Washington iGEM team agreed to send us a plasmid stock of their PetroBrick and ADC. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with 12 colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. 8 overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
</html>[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with 8 different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the PetroBrick stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp PetroBrick was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the PetroBrick as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard PetroBrick assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the PetroBrick would be unable to degrade naphthenic acids, we found that bacteria containing the PetroBrick were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T04:40:24Z<p>Achawdhry: </p>
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<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
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CONTENT=<html><br />
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<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">"Petrobrick"</a> (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) and aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). We have planned to verify the PetroBrick in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from <a href="http://www.dsmz.de">DSMZ</a>. In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
</html><br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a>, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the petrobrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html><br />
<br />
<br />
<br />
<p> These results suggest that the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our PetroBrick sequencing results with a complementarity of 87%, as compared to the theoretical <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our <a href=" https://2011.igem.org/Team:Washington/alkanebiosynthesis ">General Production Protocol</a> that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed eight overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the petrobrick) has worked on system optimization, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our PetroBrick that is preventing effective operation. We may need to design inner primers to sequence the entire <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a>, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "system optimization" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (ADC) was missing. This explained why our sequencing results for the petrobrick yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (AAR)gene and no aldehyde decarbonylase (ADC). Either the parts registry did not have the proper petrobrick (containing both AAR and ADC) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the petrobrick. It was determined that the latter was true. As such, the transformation for the petrobrick had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with AAR for the petrobrick (submitted by the University of Washington). This involved transforming the petrobrick into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the production media protocol in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the petrobrick did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the production media protocol to attempt to produce hydrocarbons because we are not certain as to whether the petrobrick part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the petrobrick sent to us in the 2012 kit plate does not contain a proper functional petrobrick. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the petrobrick, b) transformation and isolation of the aldehyde decarbonylate (ADC) and c) culturing the Nocardia strain NRRL 5646. With regards to the petrobrick, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the petrobrick they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the petrobrick by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (ADC). The ADC was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for ADC, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (AAR) being ligated with aldehyde decarbonylase (ADC). Upon successfully forming this biobrick (ideally the same as a functional petrobrick), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional petrobrick that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
</html><br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the ADC gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our AAR gene to produce a PetroBrick. The Washington iGEM team agreed to send us a plasmid stock of their PetroBrick and ADC. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with 12 colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. 8 overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
</html>[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
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<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with 8 different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the PetroBrick stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp PetroBrick was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the PetroBrick as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard PetroBrick assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the PetroBrick would be unable to degrade naphthenic acids, we found that bacteria containing the PetroBrick were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
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</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
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</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T04:25:03Z<p>Achawdhry: </p>
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<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
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CONTENT=<html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">"Petrobrick"</a> (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) and aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). We have planned to verify the PetroBrick in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from <a href="http://www.dsmz.de">DSMZ</a>. In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
</html><br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a>, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the petrobrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html><br />
<br />
<br />
<br />
<p> These results suggest that the <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our PetroBrick sequencing results with a complementarity of 87%, as compared to the theoretical <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our General Production Protocol that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed 8 overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the petrobrick) has worked on system optimization, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our PetroBrick that is preventing effective operation. We may need to design inner primers to sequence the entire PetroBrick, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "system optimization" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (ADC) was missing. This explained why our sequencing results for the petrobrick yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (AAR)gene and no aldehyde decarbonylase (ADC). Either the parts registry did not have the proper petrobrick (containing both AAR and ADC) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the petrobrick. It was determined that the latter was true. As such, the transformation for the petrobrick had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with AAR for the petrobrick (submitted by the University of Washington). This involved transforming the petrobrick into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the production media protocol in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the petrobrick did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the production media protocol to attempt to produce hydrocarbons because we are not certain as to whether the petrobrick part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the petrobrick sent to us in the 2012 kit plate does not contain a proper functional petrobrick. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the petrobrick, b) transformation and isolation of the aldehyde decarbonylate (ADC) and c) culturing the Nocardia strain NRRL 5646. With regards to the petrobrick, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the petrobrick they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the petrobrick by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (ADC). The ADC was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for ADC, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (AAR) being ligated with aldehyde decarbonylase (ADC). Upon successfully forming this biobrick (ideally the same as a functional petrobrick), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional petrobrick that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
</html><br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the ADC gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our AAR gene to produce a PetroBrick. The Washington iGEM team agreed to send us a plasmid stock of their PetroBrick and ADC. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with 12 colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. 8 overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
</html>[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with 8 different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the PetroBrick stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp PetroBrick was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the PetroBrick as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard PetroBrick assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the PetroBrick would be unable to degrade naphthenic acids, we found that bacteria containing the PetroBrick were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T04:14:36Z<p>Achawdhry: </p>
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<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
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CONTENT=<html><br />
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<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">"Petrobrick"</a> (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) and aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). We have planned to verify the PetroBrick in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from <a href="http://www.dsmz.de">DSMZ</a>. In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
</html><br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the PetroBrick on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the PetroBrick, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the petrobrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html><br />
<br />
<br />
<br />
<p> These results suggest that the PetroBrick plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our PetroBrick plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed Petrobrick E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our PetroBrick sequencing results with a complementarity of 87%, as compared to the theoretical PetroBrick in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our General Production Protocol that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed 8 overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the petrobrick) has worked on system optimization, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our PetroBrick that is preventing effective operation. We may need to design inner primers to sequence the entire PetroBrick, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "system optimization" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (ADC) was missing. This explained why our sequencing results for the petrobrick yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (AAR)gene and no aldehyde decarbonylase (ADC). Either the parts registry did not have the proper petrobrick (containing both AAR and ADC) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the petrobrick. It was determined that the latter was true. As such, the transformation for the petrobrick had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with AAR for the petrobrick (submitted by the University of Washington). This involved transforming the petrobrick into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the production media protocol in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the petrobrick did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the production media protocol to attempt to produce hydrocarbons because we are not certain as to whether the petrobrick part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the petrobrick sent to us in the 2012 kit plate does not contain a proper functional petrobrick. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the petrobrick, b) transformation and isolation of the aldehyde decarbonylate (ADC) and c) culturing the Nocardia strain NRRL 5646. With regards to the petrobrick, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the petrobrick they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the petrobrick by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (ADC). The ADC was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for ADC, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (AAR) being ligated with aldehyde decarbonylase (ADC). Upon successfully forming this biobrick (ideally the same as a functional petrobrick), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional petrobrick that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
</html><br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the ADC gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our AAR gene to produce a PetroBrick. The Washington iGEM team agreed to send us a plasmid stock of their PetroBrick and ADC. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with 12 colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. 8 overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
</html>[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with 8 different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the PetroBrick stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp PetroBrick was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the PetroBrick as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard PetroBrick assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the PetroBrick would be unable to degrade naphthenic acids, we found that bacteria containing the PetroBrick were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T04:06:29Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
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CONTENT=<html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">"Petrobrick"</a> (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) and aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). We have planned to verify the PetroBrick in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from DSMZ (http://www.dsmz.de). In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
<br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the PetroBrick on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the PetroBrick, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the petrobrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html></html><br />
<br />
<br />
<br />
<p> These results suggest that the PetroBrick plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our PetroBrick plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed Petrobrick E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our PetroBrick sequencing results with a complementarity of 87%, as compared to the theoretical PetroBrick in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our General Production Protocol that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed 8 overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the petrobrick) has worked on system optimization, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our PetroBrick that is preventing effective operation. We may need to design inner primers to sequence the entire PetroBrick, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "system optimization" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (ADC) was missing. This explained why our sequencing results for the petrobrick yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (AAR)gene and no aldehyde decarbonylase (ADC). Either the parts registry did not have the proper petrobrick (containing both AAR and ADC) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the petrobrick. It was determined that the latter was true. As such, the transformation for the petrobrick had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with AAR for the petrobrick (submitted by the University of Washington). This involved transforming the petrobrick into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the production media protocol in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the petrobrick did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the production media protocol to attempt to produce hydrocarbons because we are not certain as to whether the petrobrick part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the petrobrick sent to us in the 2012 kit plate does not contain a proper functional petrobrick. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the petrobrick, b) transformation and isolation of the aldehyde decarbonylate (ADC) and c) culturing the Nocardia strain NRRL 5646. With regards to the petrobrick, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the petrobrick they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the petrobrick by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (ADC). The ADC was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for ADC, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (AAR) being ligated with aldehyde decarbonylase (ADC). Upon successfully forming this biobrick (ideally the same as a functional petrobrick), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional petrobrick that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
<br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html></html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the ADC gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our AAR gene to produce a PetroBrick. The Washington iGEM team agreed to send us a plasmid stock of their PetroBrick and ADC. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with 12 colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. 8 overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html></html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with 8 different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the PetroBrick stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp PetroBrick was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the PetroBrick as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard PetroBrick assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the PetroBrick would be unable to degrade naphthenic acids, we found that bacteria containing the PetroBrick were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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</html><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T04:05:41Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
<br />
CONTENT=<html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032">AAR</a>) and aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). We have planned to verify the PetroBrick in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from DSMZ (http://www.dsmz.de). In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
<br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the PetroBrick on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the PetroBrick, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the petrobrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html></html><br />
<br />
<br />
<br />
<p> These results suggest that the PetroBrick plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our PetroBrick plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed Petrobrick E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our PetroBrick sequencing results with a complementarity of 87%, as compared to the theoretical PetroBrick in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our General Production Protocol that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed 8 overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the petrobrick) has worked on system optimization, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our PetroBrick that is preventing effective operation. We may need to design inner primers to sequence the entire PetroBrick, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "system optimization" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (ADC) was missing. This explained why our sequencing results for the petrobrick yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (AAR)gene and no aldehyde decarbonylase (ADC). Either the parts registry did not have the proper petrobrick (containing both AAR and ADC) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the petrobrick. It was determined that the latter was true. As such, the transformation for the petrobrick had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with AAR for the petrobrick (submitted by the University of Washington). This involved transforming the petrobrick into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the production media protocol in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the petrobrick did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the production media protocol to attempt to produce hydrocarbons because we are not certain as to whether the petrobrick part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the petrobrick sent to us in the 2012 kit plate does not contain a proper functional petrobrick. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the petrobrick, b) transformation and isolation of the aldehyde decarbonylate (ADC) and c) culturing the Nocardia strain NRRL 5646. With regards to the petrobrick, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the petrobrick they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the petrobrick by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (ADC). The ADC was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for ADC, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (AAR) being ligated with aldehyde decarbonylase (ADC). Upon successfully forming this biobrick (ideally the same as a functional petrobrick), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional petrobrick that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
<br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html></html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the ADC gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our AAR gene to produce a PetroBrick. The Washington iGEM team agreed to send us a plasmid stock of their PetroBrick and ADC. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with 12 colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. 8 overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html></html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with 8 different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the PetroBrick stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp PetroBrick was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the PetroBrick as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard PetroBrick assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the PetroBrick would be unable to degrade naphthenic acids, we found that bacteria containing the PetroBrick were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
<br />
<br />
<br />
</html><br />
<br />
}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T04:03:59Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
<br />
CONTENT=<html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's <a href="http://partsregistry.org/Part:BBa_K590025">Petrobrick</a> (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (AAR) and aldehyde decarbonylase (ADC). We have planned to verify the PetroBrick in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from DSMZ (http://www.dsmz.de). In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
<br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the PetroBrick on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the PetroBrick, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the petrobrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html></html><br />
<br />
<br />
<br />
<p> These results suggest that the PetroBrick plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our PetroBrick plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed Petrobrick E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our PetroBrick sequencing results with a complementarity of 87%, as compared to the theoretical PetroBrick in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our General Production Protocol that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed 8 overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the petrobrick) has worked on system optimization, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our PetroBrick that is preventing effective operation. We may need to design inner primers to sequence the entire PetroBrick, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "system optimization" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (ADC) was missing. This explained why our sequencing results for the petrobrick yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (AAR)gene and no aldehyde decarbonylase (ADC). Either the parts registry did not have the proper petrobrick (containing both AAR and ADC) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the petrobrick. It was determined that the latter was true. As such, the transformation for the petrobrick had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with AAR for the petrobrick (submitted by the University of Washington). This involved transforming the petrobrick into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the production media protocol in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the petrobrick did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the production media protocol to attempt to produce hydrocarbons because we are not certain as to whether the petrobrick part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the petrobrick sent to us in the 2012 kit plate does not contain a proper functional petrobrick. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the petrobrick, b) transformation and isolation of the aldehyde decarbonylate (ADC) and c) culturing the Nocardia strain NRRL 5646. With regards to the petrobrick, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the petrobrick they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the petrobrick by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (ADC). The ADC was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for ADC, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (AAR) being ligated with aldehyde decarbonylase (ADC). Upon successfully forming this biobrick (ideally the same as a functional petrobrick), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional petrobrick that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
<br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html></html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the ADC gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our AAR gene to produce a PetroBrick. The Washington iGEM team agreed to send us a plasmid stock of their PetroBrick and ADC. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with 12 colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. 8 overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html></html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with 8 different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the PetroBrick stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp PetroBrick was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the PetroBrick as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard PetroBrick assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the PetroBrick would be unable to degrade naphthenic acids, we found that bacteria containing the PetroBrick were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
<br />
<br />
<br />
</html><br />
<br />
}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T04:00:31Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
<br />
CONTENT=<html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's "Petrobrick" (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (AAR) and aldehyde decarbonylase (ADC). We have planned to verify the PetroBrick in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from DSMZ (http://www.dsmz.de). In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
<br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the PetroBrick on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the PetroBrick, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the petrobrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html></html><br />
<br />
<br />
<br />
<p> These results suggest that the PetroBrick plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our PetroBrick plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed Petrobrick E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our PetroBrick sequencing results with a complementarity of 87%, as compared to the theoretical PetroBrick in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our General Production Protocol that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed 8 overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the petrobrick) has worked on system optimization, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our PetroBrick that is preventing effective operation. We may need to design inner primers to sequence the entire PetroBrick, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "system optimization" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (ADC) was missing. This explained why our sequencing results for the petrobrick yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (AAR)gene and no aldehyde decarbonylase (ADC). Either the parts registry did not have the proper petrobrick (containing both AAR and ADC) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the petrobrick. It was determined that the latter was true. As such, the transformation for the petrobrick had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with AAR for the petrobrick (submitted by the University of Washington). This involved transforming the petrobrick into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the production media protocol in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the petrobrick did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the production media protocol to attempt to produce hydrocarbons because we are not certain as to whether the petrobrick part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the petrobrick sent to us in the 2012 kit plate does not contain a proper functional petrobrick. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the petrobrick, b) transformation and isolation of the aldehyde decarbonylate (ADC) and c) culturing the Nocardia strain NRRL 5646. With regards to the petrobrick, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the petrobrick they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the petrobrick by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (ADC). The ADC was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for ADC, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (AAR) being ligated with aldehyde decarbonylase (ADC). Upon successfully forming this biobrick (ideally the same as a functional petrobrick), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional petrobrick that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
<br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html></html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the ADC gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our AAR gene to produce a PetroBrick. The Washington iGEM team agreed to send us a plasmid stock of their PetroBrick and ADC. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with 12 colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. 8 overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html></html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with 8 different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the PetroBrick stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp PetroBrick was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the PetroBrick as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard PetroBrick assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the PetroBrick would be unable to degrade naphthenic acids, we found that bacteria containing the PetroBrick were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
<br />
<br />
<br />
</html><br />
<br />
}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T03:59:40Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
<br />
CONTENT=<html></html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's "Petrobrick" (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (AAR) and aldehyde decarbonylase (ADC). We have planned to verify the PetroBrick in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from DSMZ (http://www.dsmz.de). In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
<br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the PetroBrick on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the PetroBrick, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the petrobrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html></html><br />
<br />
<br />
<br />
<p> These results suggest that the PetroBrick plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our PetroBrick plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed Petrobrick E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our PetroBrick sequencing results with a complementarity of 87%, as compared to the theoretical PetroBrick in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our General Production Protocol that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed 8 overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the petrobrick) has worked on system optimization, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our PetroBrick that is preventing effective operation. We may need to design inner primers to sequence the entire PetroBrick, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "system optimization" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (ADC) was missing. This explained why our sequencing results for the petrobrick yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (AAR)gene and no aldehyde decarbonylase (ADC). Either the parts registry did not have the proper petrobrick (containing both AAR and ADC) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the petrobrick. It was determined that the latter was true. As such, the transformation for the petrobrick had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with AAR for the petrobrick (submitted by the University of Washington). This involved transforming the petrobrick into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the production media protocol in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the petrobrick did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the production media protocol to attempt to produce hydrocarbons because we are not certain as to whether the petrobrick part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the petrobrick sent to us in the 2012 kit plate does not contain a proper functional petrobrick. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the petrobrick, b) transformation and isolation of the aldehyde decarbonylate (ADC) and c) culturing the Nocardia strain NRRL 5646. With regards to the petrobrick, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the petrobrick they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the petrobrick by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (ADC). The ADC was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for ADC, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (AAR) being ligated with aldehyde decarbonylase (ADC). Upon successfully forming this biobrick (ideally the same as a functional petrobrick), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional petrobrick that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
<br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html></html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the ADC gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our AAR gene to produce a PetroBrick. The Washington iGEM team agreed to send us a plasmid stock of their PetroBrick and ADC. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with 12 colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. 8 overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html></html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with 8 different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the PetroBrick stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp PetroBrick was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the PetroBrick as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard PetroBrick assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the PetroBrick would be unable to degrade naphthenic acids, we found that bacteria containing the PetroBrick were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
<br />
<br />
<br />
</html><br />
<br />
}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T03:52:19Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
<br />
CONTENT=<html></html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's "PetroBrick" (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (AAR) and aldehyde decarbonylase (ADC). We have planned to verify the PetroBrick in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from DSMZ (http://www.dsmz.de). In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
<br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the PetroBrick on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the PetroBrick, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the petrobrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html></html><br />
<br />
<br />
<br />
<p> These results suggest that the PetroBrick plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our PetroBrick plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed Petrobrick E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our PetroBrick sequencing results with a complementarity of 87%, as compared to the theoretical PetroBrick in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our General Production Protocol that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed 8 overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the petrobrick) has worked on system optimization, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our PetroBrick that is preventing effective operation. We may need to design inner primers to sequence the entire PetroBrick, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "system optimization" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (ADC) was missing. This explained why our sequencing results for the petrobrick yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (AAR)gene and no aldehyde decarbonylase (ADC). Either the parts registry did not have the proper petrobrick (containing both AAR and ADC) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the petrobrick. It was determined that the latter was true. As such, the transformation for the petrobrick had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with AAR for the petrobrick (submitted by the University of Washington). This involved transforming the petrobrick into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the production media protocol in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the petrobrick did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the production media protocol to attempt to produce hydrocarbons because we are not certain as to whether the petrobrick part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the petrobrick sent to us in the 2012 kit plate does not contain a proper functional petrobrick. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the petrobrick, b) transformation and isolation of the aldehyde decarbonylate (ADC) and c) culturing the Nocardia strain NRRL 5646. With regards to the petrobrick, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the petrobrick they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the petrobrick by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (ADC). The ADC was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for ADC, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (AAR) being ligated with aldehyde decarbonylase (ADC). Upon successfully forming this biobrick (ideally the same as a functional petrobrick), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional petrobrick that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
<br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html></html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the ADC gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our AAR gene to produce a PetroBrick. The Washington iGEM team agreed to send us a plasmid stock of their PetroBrick and ADC. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with 12 colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. 8 overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html></html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with 8 different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the PetroBrick stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp PetroBrick was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the PetroBrick as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard PetroBrick assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the PetroBrick would be unable to degrade naphthenic acids, we found that bacteria containing the PetroBrick were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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</html><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T03:51:46Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
<br />
CONTENT=<html></html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's "PetroBrick" (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (AAR) and aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">Petrobrick</a>). We have planned to verify the PetroBrick in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from DSMZ (http://www.dsmz.de). In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
<br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the PetroBrick on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the PetroBrick, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the petrobrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html></html><br />
<br />
<br />
<br />
<p> These results suggest that the PetroBrick plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our PetroBrick plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed Petrobrick E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our PetroBrick sequencing results with a complementarity of 87%, as compared to the theoretical PetroBrick in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our General Production Protocol that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed 8 overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the petrobrick) has worked on system optimization, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our PetroBrick that is preventing effective operation. We may need to design inner primers to sequence the entire PetroBrick, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
<br />
<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "system optimization" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (ADC) was missing. This explained why our sequencing results for the petrobrick yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (AAR)gene and no aldehyde decarbonylase (ADC). Either the parts registry did not have the proper petrobrick (containing both AAR and ADC) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the petrobrick. It was determined that the latter was true. As such, the transformation for the petrobrick had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
<br />
<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with AAR for the petrobrick (submitted by the University of Washington). This involved transforming the petrobrick into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the production media protocol in an attempt to produce hydrocarbons. </p><br />
<br />
<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the petrobrick did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the production media protocol to attempt to produce hydrocarbons because we are not certain as to whether the petrobrick part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the petrobrick sent to us in the 2012 kit plate does not contain a proper functional petrobrick. </p><br />
<br />
<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the petrobrick, b) transformation and isolation of the aldehyde decarbonylate (ADC) and c) culturing the Nocardia strain NRRL 5646. With regards to the petrobrick, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the petrobrick they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the petrobrick by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (ADC). The ADC was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
<br />
<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for ADC, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (AAR) being ligated with aldehyde decarbonylase (ADC). Upon successfully forming this biobrick (ideally the same as a functional petrobrick), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional petrobrick that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
<br />
<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
<br />
[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html></html><br />
<br />
<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the ADC gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our AAR gene to produce a PetroBrick. The Washington iGEM team agreed to send us a plasmid stock of their PetroBrick and ADC. </p><br />
<br />
<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with 12 colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. 8 overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
<br />
[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html></html><br />
<br />
<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
<br />
<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
<br />
<br />
</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
<br />
<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with 8 different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
<br />
<h2>Week 17 (August 20 - August 24)</h2><br />
<br />
<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the PetroBrick stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp PetroBrick was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
<br />
<h2>Week 18 (August 27 - August 31)</h2><br />
<br />
<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
<br />
<h2> Week 19 (September 3 - September 7) </h2><br />
<br />
<p>The team focussed primarily on assays this week, including that of the PetroBrick as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard PetroBrick assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the PetroBrick would be unable to degrade naphthenic acids, we found that bacteria containing the PetroBrick were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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</html><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Notebook/DecarboxylationTeam:Calgary/Notebook/Decarboxylation2012-10-02T03:51:11Z<p>Achawdhry: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookBlue|<br />
TITLE=Decarboxylation Journal|<br />
<br />
CONTENT=<html></html><br />
<br />
<h3> Decarboxylation </h3><br />
<br />
<h2>Week 2 (May 7-11)</h2><br />
<p>For the decarboxylation sub-project, the second week was entirely focused on literature research and the practice of basic laboratory techniques. 8 potential pathways were identified as potential candidates for naphthenic acid decarboxylation. The first of these would utilize only the University of Washington's "PetroBrick" (from iGEM 2011), consisting of the genes encoding the enzymes acyl-ACP reductase (AAR) and aldehyde decarbonylase (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031">ADC</a>). We have planned to verify the PetroBrick in the distribution plates and test its efficacy on naphthenic acids in the coming weeks. If this proves to be unsuccessful, we will begin investigating the alternative approaches, beginning with replacing AAR with carboxylic acid reductase (CAR) from Nocardia iowensis, a very unspecific reductase shown to work on structures resembling naphthenic acids. Failing this, the remaining pathways will be examined; however, the disadvantage in these pathways is their direct reliance on the success of the other steps, as the naphthenic acids must be degraded to the point of resembling branched-chain fatty acids (since all remaining pathways are related to fatty acid metabolism).</p><br />
<br />
<h2>Week 3 (May 14-18)</h2><br />
<p>The third week included some additional literature investigation in the first two days. The iGEM distributions arrived this week, and verification began on May 17th by transformation into E. coli, followed by colony PCR on May 18th (using standard protocols on the wiki). Additionally, primers were designed for CAR in N. iowensis, along with primers for Nocardia posphopantetheine transferase (NPT), a second enzyme required for optimal function in the former, and a short list of contacts were acquired to request the donation of the required strain (called NRRL 5646) from researchers who have worked with it previously. A sort of form email was drafted for this purpose, and should this be unsuccessful, we will be purchasing the strain from DSMZ (http://www.dsmz.de). In the following week, we will begin with development of overnight cultures and gel preparation.</p><br />
<br />
<h2>Week 4 (May 22-25)</h2><br />
<p> This week, a gel was prepared for the colony PCR prepared last week, and the gel was run, yielding results that were very difficult to see. The PCR and gel electrophoresis process were repeated, yielding the following gel: </p><br />
<br />
[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 1.PNG|thumb|Results indicate transformation with the PetroBrick. Lane 1 contains the 1 kb plus ladder, while Lanes 2-11 contain the results of colony PCR. Each of these bands correspond to the PetroBrick vector, which is 2070bp, indicating that the transformation was successful. Lane 12 contains a positive control, which varies distinctly from the colony PCR results. The negative control lane is empty as expected.|500px|center]]<html><br />
<br />
<br />
<p> Overnight cultures were grown from the colonies were prepared this week. Sigma compounds - for the purpose of testing the PetroBrick on naphthenic acid analogues - were selected from the list provided. The compounds to be used include cyclohexanepentanoic acid, cycohexane-1,1-dicarboxylic acid, and benzo[b]thiophene-3-acetic acid. A mini-prep was completed on the overnight cultures prepared earlier in the week, according to protocol obtained from the wiki. The DNA concentration in the resulting samples was measured by nanodrop to confirm successful plasmid extraction, and the resulting DNA concentrations were as follows: </p><br />
<br />
<ul><br />
<li> Tube 1: 44.9 ng/mL </li><br />
<li> Tube 2: 54.2 ng/mL </li><br />
<li> Tube 3: 54.4 ng/mL </li><br />
<li> Tube 4: 41.6 ng/mL </li><br />
<li> Tube 5: 34.3 ng/mL </li><br />
</ul><br />
<br />
<p> Because tube 3 (prepared from Colony 3, Plate 3) had the highest concentration, it was to be used for the eventual sequencing of the plasmid. The restriction digest was completed also, but it was not run on a gel this week; this was left for Week 5. </p><br />
<br />
<br />
<h2>Week 5 (May 28 - June 1)</h2> <br />
<p> On Tuesday (May 29), a gel was run on the restriction digests of the extracted plasmids from Week 4, which appeared to confirm the successful transformation of the PetroBrick, showing clear bands at the pertinent locations. The gel is as follows: </p> <br />
<br />
</html>[[File:UCalgary igem PCRverfication Hydrocarbon PetroBrick Gel 2.PNG|thumb|Lane 1 contained the 1 kb plus ladder, while lanes 2-6 contained the restriction digest results, which had used the five different miniprep tubes with plasmids isolated from five different colonies. As can be observed in the gel, the upper row of bands corresponds to 2392bp, indicating the petrobrick part. The lower set of bands corresponds to the PetroBrick pSB1C3 vector at 2070 bp.|500px|center]]<html></html><br />
<br />
<br />
<br />
<p> These results suggest that the PetroBrick plasmid had been successfully purified. The black circle indicates undigested plasmid in lane 3. There simply means that not all of the plasmid was digested into two separate DNA strands. It indicates the entire size of our PetroBrick plasmid. </p><br />
<br />
<p> Based on the apparent success of the gel, the products were sent away for sequencing. Long-term stocks were prepared for the alkane production medium outlined in University of Washington's PetroBrick protocols from 2011 (see https://2011.igem.org/Team:Washington/alkanebiosynthesis). These stocks are as follows: </p><br />
<br />
<ul><br />
<li> 1 L of 1M Tris (pH = 7.25) </li><br />
<li> 10 mL of 1 mg/mL Thiamine </li><br />
<li> 10 mL of 10% Triton x-100 </li><br />
<li> 1 M MgSO4 </li><br />
<li> 0.1 M FeCl3 (anhydrous) </li><br />
</ul><br />
<br />
<p> The medium itself is to be prepared in Week 6 once the results of sequencing are (hopefully) acquired. </p><br />
<h2>Week 6 (June 4 - June 8)</h2><br />
<p> This week, we first made a streak plate of our confirmed Petrobrick E.coli Colony 3, Plate 2, as well as a long-term glycerol stock for storage. In accordance with the Washington team’s protocols, we prepared our M9 minGlucose Media (Production Media) with the following reagents: </p><br />
<br />
<ul><br />
<li> 75mL ddH2O </li> <br />
<li> 0.6g Na2HPO4 </li><br />
<li> 0.3g KH2PO4 </li><br />
<li> 0.05g NaCl (855.6mL 1M solution) </li><br />
<li> 0.2g NH4Cl </li><br />
<li> 20mL of 1M Tris (pH = 7.25) </li><br />
<li> 1mL of 10% Triton </li><br />
<li> 100mL of 1mg/mL thiamine hydrochloride </li><br />
<li> 10mL of FeCl3 (anhydrous) </li><br />
<li> 100mL of MgSO4 </li><br />
</ul><br />
<br />
<p> **3.0g glucose needed to be added later after the mixture was autoclaved, as glucose is known to caramelize under the conditions of the autoclave. </p><br />
<br />
<p> We also prepared an overnight stock containing 5mL of LB broth inoculated with E.coli from our verified colony. The following day we measured its optical density, demonstrating that our OD was 1.311, suitable for our purposes. We obtained our PetroBrick sequencing results with a complementarity of 87%, as compared to the theoretical PetroBrick in the PartsRegistry, which was deemed by iGEM team leaders to be high enough to continue in our protocol. We also performed glucose filtration, transferring 3g of glucose into our Production Media solution after it had been autoclaved in order to ensure sterile transfer. On the final day of the week, we were able to start our General Production Protocol that would be followed by a 48 hour incubation. It was in this time that hydrocarbon production was reportedly supposed to occur. Use of the GC would occur in the following week to test for alkane production. </p><br />
<h2>Week 7 (June 11 - June 15)</h2><br />
<p> After preparing our production media 48 hour incubation sample for gas chromatography and extracting with ethyl acetate, we left our sample for use in the GC. Our results indicated a small hydrocarbon peak, indicating a possibility that hydrocarbon production had been a success, but it was not distinct enough to make this conclusion. The procedure would be repeated, this time creating sigma compound solutions as well. These sigma compounds would resemble naphthenic acids. In order to ensure that hydrocarbon peaks would be produced from these compounds and not from glucose, we prepared a new production media lacking glucose, in hopes that incubated bacteria could survive for long enough without a food source to decarboxylate a sigma compound to some extent. Since the sigma compounds we initially selected for our purposes had not arrived as of yet, we opted for others: Cyclohexanepentanoic acid (CHPA), cyclohexanecarboxylic acid, and 1,4-cyclohexanedicarboxylic acid. We performed 8 overnight cultures and prepared sigma compound solutions. Despite our many attempts, we were only able to acquire an OD of about 0.6 for these 8 tubes. We then performed our production protocol with the tubes, preparing them with the following reagents and inoculating them with the contents of our broth cultures: </p><br />
<ul><br />
<li> Tube 1: Glucose </li><br />
<li> Tube 2: Glucose </li><br />
<li> Tube 3: Glucose and CHPA </li><br />
<li> Tube 4: Non-glucose and CHPA </li><br />
<li> Tube 5: Glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 6: Non-glucose and cyclohexanecarboxylic acid </li><br />
<li> Tube 7: Glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
<li> Tube 8: Non-glucose and 1,4-cyclohexanedicarboxylic acid </li><br />
</ul><br />
<p> In the above tubes, “glucose” indicated that glucose-containing production media was used, whereas “non-glucose” indicated that the production media lacking glucose was used. In each case where a sigma compound was added, it was present at a concentration of 25mg/L. Each production tube was then left for a 48 hour incubation over the weekend, to perform gas chromatography tests the following week. </p><br />
<h2>Week 8 (June 18 - June 22)</h2><br />
<p>This week, we received the results of our gas chromatography of the eight tubes created last week, and unfortunately no hydrocarbons had been produced in any of them. It was thus very important for us to spend time researching why this was unsuccessful. We have found a number of things to consider. Firstly, the Washington iGEM team (creator of the petrobrick) has worked on system optimization, and has come up with an number of ways to increase alkane production. Information regarding this can be found here: https://2011.igem.org/Team:Washington/Alkanes/Future/Vector. There are many other considerations as well. We have received feedback on our sequencing results, and have been told that 87% is not high enough; we should be obtaining results of 100%. This is a problem because it presents the possibility that there is a mutation somewhere in our PetroBrick that is preventing effective operation. We may need to design inner primers to sequence the entire PetroBrick, as it is 2392bp and this may have prevented 100% sequencing results. We have also been using plastic falcon tubes, while hydrocarbon production has been tested in glass tubes. Incubation time and initial amount of cells to be innoculated are also concerns, as we have been having many problems with cell growth. The Washington Team often had an OD600 of 10 in their production media, whereas we have been tracking the OD of our initial broth only, so it is possible that the OD600 of our broth has been very low. As demonstrated by iGEM Washington, the OD600 is of utmost importance when it comes to hydrocarbon production, and is something critical that we need to improve. We are also considering extracting alkanes from our cells using HCl rather than ethyl acetate, as we many not be removing hydrocarbons effectively from the cells. We will consider each of these things when we attempt hydrocarbon production again.</p><br />
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<h2>Week 9 (June 25 - June 29)</h2><br />
<p>The week began initially with planning for continued attempts to produce hydrocarbons (Based on information that was learned using the University of Washington's "system optimization" techniques. We also backtracked to determine any additional sources of error within our procedures for the production media protocol. During the week, the sequencing results were examined in further detail, and it was determined that the entire gene sequence for aldehyde decarbonylase (ADC) was missing. This explained why our sequencing results for the petrobrick yielded only 87% complementarity. Based on this, there were two possible justifications for only having the acyl-acp reductase (AAR)gene and no aldehyde decarbonylase (ADC). Either the parts registry did not have the proper petrobrick (containing both AAR and ADC) in the assigned well in the 2012 kit plate sent to us, or we had extracted from the wrong well in the kit plate, assuming that it was the well containing the petrobrick. It was determined that the latter was true. As such, the transformation for the petrobrick had to be redone, and if gene sequencing yielded positive results (high complementarity), we would try the production media protocol once more in an attempt to get hydrocarbon production. </p><br />
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<h2>Week 10 (July 2-July 6)</h2><br />
<p>The main goal this week was to repeat the transformation and verification steps that were done previously with AAR for the petrobrick (submitted by the University of Washington). This involved transforming the petrobrick into E.coli, colony PCR for verification of a successful transformation, followed by mini-preparation and restriction digest. Results were submitted for gene sequencing. If the sequencing results come back as being successful (showing a high rate of complementarity), we will proceed forward with the production media protocol in an attempt to produce hydrocarbons. </p><br />
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<h2>Week 11 (July 9-July 13)</h2><br />
<p>The sequencing results sent in for the petrobrick did not yield a high complementarity rate, therefore were unsuccessful. As such, we were not yet able to proceed with the production media protocol to attempt to produce hydrocarbons because we are not certain as to whether the petrobrick part has been isolated. It was then decided that the colony PCR would be redone with a new colony, followed again by a mini-preparation and restriction digest before gene sequencing occurs. If the sequencing results again yield a low complementarity rate, it can be deduced that the petrobrick sent to us in the 2012 kit plate does not contain a proper functional petrobrick. </p><br />
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<h2>Week 12 (July 16 -July 20)</h2><br />
<p>It was decided that the decarboxylation team would be split up into three individual components, a) to strictly work with the petrobrick, b) transformation and isolation of the aldehyde decarbonylate (ADC) and c) culturing the Nocardia strain NRRL 5646. With regards to the petrobrick, the sequencing results were once again unsuccessful and yielded a low complementarity rate. As such, it was decided that we would contact the University of Washington, and ask them to send us the petrobrick they composed and were working with in 2011. In addition, we also decided to produce our own biobrick identical to the petrobrick by using the acyl-acp reductase (which we had successfully transformed and isolated previously) with aldehyde decarbonylase (ADC). The ADC was transformed into the E.coli, and colony PCR was done in order to determine whether successful this was successful. This was followed by mini-preparation and restriction digest before gene sequencing occurred. </p><br />
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<h2>Week 13 (July 23 - July 27)</h2><br />
<p>The gene sequencing results for the aldehyde decarbonylase were unsuccessful. It was presumed that this occurred because the mini-preparation procedure being used may potentially be leading to a high contamination rate. As such, a colony PCR was done with a new colony for ADC, followed my mini-preparation using a different procedure, and then a restriction digest. The sequencing results now yielded a high complementarity rate and were considered successful. We then proceeded with making the biobrick consisting of the acyl-acp reductase (AAR) being ligated with aldehyde decarbonylase (ADC). Upon successfully forming this biobrick (ideally the same as a functional petrobrick), we will be able to test for hydrocarbon production using the general production media protocol. The University of Washington also responded, and said that they will be able to send us their functional petrobrick that was being used in 2011. <br />
With regards to culturing Nocardia, a “brain and heart infusion (BHI)” broth was used to inoculate the Nocardia in an attempt to get growth. Six pairs of mutagenic primers were also composed for carboxylic acid reductase (CAR), which is found in the Nocardia, as well as primers for the OleT enzyme. </p><br />
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<p>This week, we were pleased to find ample Nocardia iowensis growth on our BHI plate. We performed a PCR with our constructed CAR and NPT BioBrick primers to attempt to amplify these genes, so that we could insert them into BioBricks. This PCR utilized primers specific to our genes of interest. After running the gel, we found that we had ample amplification of both of these genes, as shown in the picture below:</p><br />
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[[File:CAR NPT PCR-Amplification.jpg|thumb|In the above gel, lanes 4-5, 7, and 9-12 represent successfully amplifed NPT (669 bp). Lanes 14-16 and 18-20 show that CAR (4690 bp) has been successfully amplifed.|500px|center]]<html></html><br />
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<p> Following this, we performed a Cycle-Pure Spin Protocol to purify the amplified CAR and NPT DNA. The resulting concentration of CAR was 171.6 ng/ul, while NPT was 20.8 ng/ul. Using a restriction digest, the CAR gene was cut with AscI and HindIII, while NPT was cut with both XbaI and PstI, as well as with EcorI and PstI. Using these same enzymes, the PET vector was cut to be ligated with CAR, and the PSB1C3 vector was cut to be ligated with both NPT digests. Following incubation, the inserts were ligated together with their vectors. Transformations were then performed, with the CAR/PET ligation transformed on a Kan plate, and the NPT/PSB1C3 E/P and X/P ligations transformed on chloramphenicol plates. Transformations were successful, but there were not a great deal of colonies on the CAR plate, and both NPT plates revealled a very high proportion of red colonies, indicating that our desired NPT gene had not been successfully ligated and transformed. Nonetheless, colony PCRs were run in order to test for successful transformations of the ligations the following week. Meanwhile, the results of the ADC gene sequencing came in, but unfortunately it was found that this gene did not correspond well to its theoretical sequence on PartsRegistry. It therefore could not be used with our AAR gene to produce a PetroBrick. The Washington iGEM team agreed to send us a plasmid stock of their PetroBrick and ADC. </p><br />
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<h2>Week 15 (August 7 - August 10)</h2><br />
<p> We ran the gel on the CAR and NPT colony PCRs from last week, but unfortunately these gels were blank. Another colony PCR was performed, with 12 colonies from each of the following plates: CAR/PET, NPT(X/P), NPT(E/P). Both NPT plates did not look very promising, as they were completely covered by red colonies, with only a few white ones. Many of the white colonies from the previous week had become red. 8 overnight cultures were also performed so that a restriction digest could be done on the CAR colonies, to see if the part was there. These overnight cultures were prepared with the Kan antibiotic. Another ligation was done with the three combinations of cut inserts and vectors: CAR with PET, NPT (E/P) with PSB1C3, and NPT (X/P) with PSB1C3. Restriction digests were performed on the CAR overnight colonies with AscI and HindIII, and when the gel was run, a single lane was shown to contain both the CAR gene and PET vector, as shown below:</p><br />
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[[File:CARinPET.jpg|thumb|As shown in the gel above, lane 3 indicates 2 bands, the uppermost at about 5.4kb, identifying as PET. This single lane also contains a clear band of CAR, a bit under the 5000bp mark, at 4690bp. The ladder used was 1 kb plus. |500px|center]]<html></html><br />
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<p>This successful result allowed us to proceed with mutagenesizing the 6 illegal cut sites of CAR, including: 2 PstI sites, 1 EcorI site, and 3 NotI sites, with the primers that we had designed. Two additional colony PCRs of NPT were performed, but unfortunately a band indicating NPT was not visible in any of the traisl, even when an additional plate of both the NPT (E/P) and NPT (X/P) restriction digests were ligated and transformed. Very few white colonies could be seen on any of the transformations, but we were determined to continue trying. We then attempted to mutagenesize the first illegal cut site of CAR, which would be a PstI site. The mutagenesis primers needed to be diluted and stock solutions were made. Then, we attempted to mutagenesize the PstI site of CAR with that site's specific primers, using various concentrations of template DNA (5ng, 20ng, 50ng). We used the KAPA system to perform this plasmid amplification, using the thermocycler settings of the Desulfurization team. The mutagenesis PCR would be run over the weekend to hopefully have successful results. </p><br />
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<h2>Week 16 (August 13 - August 17)</h2><br />
<p>This week, the attempted PstI mutagenesis of CAR ws run on a gel. Unfortunately, amplification was unsuccessful, but we had an idea for why this might have occurred. It was discovered that our plasmid of 9kb would need a 7.5 minute extention time, rather than the 1 minute extention time that we had originally programmed into the thermocycler. The mutagenesis experiment was repeated, this time with a 7.5 minute extention time. When these results were analyzed with gel electrophoresis, we had bands appearing to indicate success with the 50ng samples of CAR that we had run, as shown below.</p><br />
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</html>[[File:Band_with_CAR_success_50ng.JPG|500px|thumb|This gel shows 2 bands of CAR that indicate successful mutagenesis of the first PstI site in CAR. The CAR gene is contained in the PET vector. Lane 1 contains the 1 kb plus ladder, while lanes 2,3 contained samples in which 5ng of original CAR+PET plasmid were used, lanes 4,5 contained samples in which 20ng of original plasmid were used, and lanes 6,7 contained 50ng of plasmid sample and underwent mutagenesis by PCR amplification successfully. The bands are slightly over the 9kb band of the ladder, at about 10kb, which is roughly the size of the 10,090bp plasmid. |center]]<html><br />
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<p>After combining these two PCR tubes and incubating with DpnI, we performed a transformation with this DNA and allowed the plate to incubate overnight. Also, we continued to try and verify NPT, but were once again unsuccessful. When we viewed the plate that we had transformed the following day, it was found that we did not have any colonies. We presumed this was because the 9kb plasmid was very difficult to uptake with its large size. Several more transformations were attempted. We also attempted a new procedure called site-directed mutagenesis, which would use a single mutagenesis primer per site, rather than one for each strand, and supposedly had the capacity to mutagenize multiple illegal cut sites at once. We would try to mutate 3 sites with this protocol: the 2 PstI sites, and 1 EcorI site. The PCR was run overnight, with 8 different samples, at two different concentrations of DNA (50ng and 80ng), with four different annealing temperatures. The following day, each sample was incubated with DpnI for 6 hours, and then each was tranformed on a kan plate. A restriction digest of the NPT plasmid was once again performed, cutting with both (E/P) and (X/P) as before, this time using more DNA. An overnight culture of a single potentially successful NPT colony was made, to perform a restrictin digest to see if NPT was present - this would be done over the weekend. Finally, a transformation of the single mutagenesis KAPA PCR of PstI was performed, to hopefully yield more colonies.</p><br />
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<h2>Week 17 (August 20 - August 24)</h2><br />
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<p>This week, we carried forth with the second and third mutagenesis sites of the CAR gene. Our new plasmid with our first PstI site was PCR amplified with the Kapa system and our EcorI mutagenesis primers to carry out the second mutation, to remove the EcorI illegal cut site from our gene. After the PCR was complete, we ran our product on a gel to see if the amplification worked. Bands indicated success. Following this verification, we mixed our two seemingly successful PCR tubes with 1 uL of DpnI, and left them to incubate for one hour. The PCR product was then transformed into highly competent E.coli cells, and the plates were left overnight in the 37 degree incubator. Unfortunately a mistake prevented colonies from growing, but the process was repeated and our second attempt yielded many colonies. We made overnight cultures of 8 of these colonies, with 4 from each plate, with Kan as the antibiotic for selective growth. The next day, a miniprep was performed on each overnight culture to isolate the CAR+PET plasmids that had hopefully undergone their second mutation, of their single EcorI site. Once the plasmid isolation was complete, we performed a restriction digest using EcorI to cut, in order to verify that the illegal cut site had been removed and the plasmid could no longer be digested with this enzyme. Our success is demonstrated in the gel below. We immediately proceeded with the third mutagenesis, which would this time be performed on the second (and last) PstI site. Using our new plasmid now with two illegal cut sites removed, we performed an amplification PCR, using our PstI(2) primers and the Kapa system. After running this on a gel, we determined bands of success, as shown below. We then once again treated our PCR amplified tubes with DpnI for one hour, and then transformed into highly competent E.coli. The plates were left overnight to allow colony growth. Our second accomplishment of the week was successfully amplifying the OleT gene from Jeotgalicoccus, by using the Kapa system. This was very successful, as shown in the gel below. We then performed the cycle-pure spin protocol to remove contamination from our PCR product, and then proceeded to perform a restriction digest on the product with HindIII and AscI so that we could ligate it into the PET vector. After this ligation was complete, it was transformed with E.coli on a Kan plate, and left overnight to incubate. Unfortunately we do not see colonies yet, but the incubation is not complete yet, and might still be successful. Thirdly, we obtained the PetroBrick stock from the Washington team on Tuesday, and immediately proceeded to transform it. We obtained a great deal of colonies, and then performed colony PCR on 12 of these colonies to ensure that our ~2400bp PetroBrick was present in these cells. Last but not least, we have still been working on a successful purification of NPT from a transformed plate. A colony PCR indicated that NPT was potentially in a few of these colonies, so we made overnight cultures of these four colonies, and performed a miniprep the next morning, and then a restriction digest to verify that the gene was present.</p><br />
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<h2>Week 18 (August 27 - August 31)</h2><br />
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<p>This week, the primary objectives from last week were continued. Another restriction digest of OleT and then a ligation was performed, in order to see if we could obtain more colonies following transformation. After preparing overnight cultures and minipreps of eight of these colonies, we attempted to cut the OleT/PET vector with EcorI to verify that the gene had successfully inserted into the vector. Unfortunately, we did not have any luck, even after several attempts. Our attempts to verify this will continue. We also performed ligations with NPT (cut with XbaI and PstI) with JI3002 (cut with SpeI and PstI), so that we could produce a biobrick with JI3002 before NPT to act as a promoter and ribosome binding site. We decided on how we will build our two primary constructions, and where each part will be. Also, we attempted to amplify the CAR gene in the PET vector (with 3 mutagenesis sites replaced) with biobrick primers, so that we could get it inserted into a biobrick plasmid. After several tries, this amplification was successful.</p><br />
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<h2> Week 19 (September 3 - September 7) </h2><br />
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<p>The team focussed primarily on assays this week, including that of the PetroBrick as well as OleT. Procedures were also continued with CAR and NPT, and each were sent for sequencing, both shown to be successfully isolated when the results came in. It was decided that we would use CAR that had been not fully mutagenesized, still with three NotI sites, as a piece in our construction with NPT so that we could perform assays. Mutagenesis would continue later, once we were able to obtain actual assay results. We continued to attempt to ligate JI3002 in front of NPT in a PSB1C3 vector. Following standard PetroBrick assay procedures (see earlier weeks), we were finally able to have successful assay results with proper graphical data. Instead of performing the assay with glucose, however, we used a standard mixture of naphthenic acids. Despite our earlier hypothesis that the PetroBrick would be unable to degrade naphthenic acids, we found that bacteria containing the PetroBrick were able to degrade these naphthenic acids into both alkanes and alkenes. These successful graphs are shown below.</p><br />
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</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 1: The relative intensity of alkane production over a retention time in both E.coli that contain the PetroBrick, and in E.coli that are lacking the PetroBrick, as measured with GC-MS. Naphthenic acids were used as a substrate. A naphthenic acid standard was required to compare peaks.|700px]]<html><br />
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</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 2: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from E.coli containing the PetroBrick as in Figure 1, using naphthenic acids as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
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}}</div>Achawdhryhttp://2012.igem.org/Team:Calgary/Project/FRED/ReportingTeam:Calgary/Project/FRED/Reporting2012-10-02T03:44:23Z<p>Achawdhry: </p>
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<div>{{Team:Calgary/TemplateProjectGreen|<br />
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<p>For FRED to be able to tell us about the toxins he's sensing we needed a good reporter system that could function in a wide array of environments. Unfortunately the traditional fluorescent or luminescent reporters have significant drawbacks that prevent them from being useful in a tailings environment that is murky and potentially anaerobic. Due to these limitations we decided to improve upon <a href="https://2011.igem.org/Team:Calgary">last year's electrochemical sensor</a> using the <i>lacZ</i> gene to cleave a substrate into an easily detectable analyte. Our team has developed a system that utilizes three separate reporter genes to provide a triple-output biosensor. This system overcomes traditional reporters in that it is fast, accurate, and can function in turbid environments and even in the absence of oxygen!</p><br />
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<h2>How Does it Work?</h2><br />
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<p>The enzymes encoded by our reporter genes are specific sugar hydrolases. This means that they target one kind of sugar and remove it from whatever compound they are attached to. We have chosen to use the sugars glucose, glucuronide, and galactose for our system. The genes reposnsible for their respective hydrolases are <i>bglX</i>, <i>uidA</i>, and <i>lacZ</i>. By having our electrochemical analyte conjugated to this sugar, when the hydrolase is expressed the sugar is cleaved from the analyte, allowing for it's electrochemical detection. A diagrammatic representation of this system is shown below in figure 2.</p><br />
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[[File:Calgary2012 EchemWikiFig1.jpg|thumb|600px|center|Figure 2: Representation of cleavage of the sugar-analyte substrate by a hydrolase enzyme.]]<br />
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<p>After the analyte is released we need to detect it. Electrochemistry is an excellent approach for this because of it's fast and quantitative nature. A voltage is applied between two electrodes compared to a reference electrode and the resulting current is measured. By changing the applied voltage to that of the oxidation voltage of one of our analytes, the increase in current due to it's oxidation when compared to an analyte free baseline is proportional to the amount of analyte present in the solution. This process happens so quickly that you can have an output value in a matter of seconds.</p><br />
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<p>We used two different electrochemical techniques in our testing depending on what question the experiment was trying to answer. When we were characterizing the voltages at which our products oxidized we used cyclic voltammetry, which is where you apply a voltage and then slowly increase and decrease it over a designated sweep range. Any bumps in the graph are due to a reaction and can be standardized against baseline measurements. After the oxidation potential has been localized we can speed up our experiments by using potentiostatic runs. In this case instead of sweeping the voltage we apply to the solution we hold it steady at the voltage that will oxidize our compound the moment it is released into the solution. Both of these techniques require the three electrodes in an electrolyte solution such as phosphate buffered saline and can routinely detect nanomolar concentrations of electrochemical analytes.</p><br />
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<h2>Genes, Chemicals, and Circuits</h2><br />
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<p>For our system to have a triplex outplut we need three seperate genetic circuits with three analytes possessing unique oxidation potentials. If one chemical overlaps with another we could get false-positives of one chemical due to oxidation of another. To this end we have chosen to use chlorophenol red (CPR), para-diphenol (PDP), and para-nitrophenol (PNP). These compounds are conjugated with their sugars to form CPR-&beta;-D-galactopyranoside (CPRG), PDP-&beta;-D-glucopyranoside (PDPG), and PNP-&beta;-D-glucuronide (PNPG). An easy way to tell the analytes from their sugar conjugates is the addition of the letter G to the acronym. These chemicals are summarized below in figure 3 along with the reporter genes used with each one.</p><br />
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[[File:Calgary2012 ECHEMWikiFig2.png|thumb|700px|center|Figure 3: Analyte/sugar combinations as well as the reporter genes responsible for the detection of each compound.]]<br />
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<p>Out of the three sugar conjugates the only one that exhibits any electrochemical activity is PDPG, with it's oxidation potential at 0.6V vs the reduction of hydrogen reference electrode (RHE). The three analytes have potentials at 0.825V for PDP, 1.325V for CPR, and 1.6V for PNP vs RHE. As none of these peaks overlap and no sugar conjugates interfere with their signals the three chemicals can be detected in the same solution. This is shown below in figure 4.<br />
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[[File:Calgary2012 FRED triple.png|thumb|700px|center|Figure 4: Cyclic voltammogram of the three electrochemical analytes vs RHE. PDP has a peak at 0.825V, while CPR is at 1.325V and PNP is at 1.6V. The concentration of all analytes was 40&micro;M]]<br />
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<p>With the chemicals finalized we now needed to construct our circuits. The <i>lacZ</i> gene under the control of the <i>lacI</i> promoter in the registry has a frameshift mutation, rendering the enzyme nonfunctional. As such we have reconstructed this circuit and submitted it to the registry (BBa_K902008???). The <i>bglX</i> and <i>uidA</i> genes were amplified from the <i>E. coli</i> genome using PCR and biobricked as <a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a> and <a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a> respectively. These genes were then also constructed under the <a href="http://partsregistry.org/Part:BBa_R0010"><i>lacI</i> promoter</a> to allow for comparison testing.</p><br />
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<h2>Does it Work?</h2><br />
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<p>Yes! We have been able to show that when induced with IPTG we can observe the cleavage of our compounds into the detectable analyte. Shown below in figure 5 is the detection of PNP by the cleave of PNPG when <i>uidA</i> was induced by IPTG or not. This also shows that there was a lot of background expression of our promoter when it was not induced.</p><br />
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[[File:Calgary2012 ECHEM PNP.png|thumb|600px|center|Figure 5: Potentiostatic detection of PNP at 1.6V vs RHE by either an IPTG induced <i>uidA</i> gene or leaky expression of an uninduced <i>uidA</i> gene.]]<br />
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<p>More data coming soon for the other two enzymes! (Provided we can actually build the bloody circuits...)</p><br />
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<h2>What Next?</h2><br />
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<p>With our electrochemical system functioning properly we can now hook up our reporter genes to promoters found in the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon library</a> for a final detection system. We have also created a <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">hardware and software platform</a> for a field-ready biosensor. Our system has also been <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">mathematically modeled</a> in MATLAB to aid us in planning time courses for the experiments and the final prototype. When combined with the mechanical and biological containment mechanisms used in our system these genes create a novel and safe approach to biosensing in the oil sands and in many other potential applications.</p><br />
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