Difference between revisions of "Team:NCKU Tainan/Parts"
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| − | + | <!--Sidebar (Remember to change the subtitle context, but don't change the id)--> | |
| − | + | <aside class="sidenav d-none d-lg-flex flex-column justify-content-start align-items-center col-2"> | |
| − | + | <ul class="nav flex-column"> | |
| − | + | <li class="nav-item"> | |
| − | + | <a class="nav-link" href="#Subtitle1">Overview</a> | |
| − | + | </li> | |
| − | + | <li class="nav-item"> | |
| − | + | <a class="nav-link" href="#Subtitle2">Experimental Approach & Results</a> | |
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| − | + | <a class="nav-link" href="#Subtitle1">Reference</a> | |
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| − | + | <main class=""> | |
| − | + | <!--The Page Name--> | |
| − | + | <div class="Improve"> | |
| − | + | <h1>Improve</h1> | |
| + | <div class="divider"></div> | ||
| + | </div> | ||
| + | <hr> | ||
| + | <section> | ||
| + | <!--section name:Overview--> | ||
| + | <h2 id ="Subtitle1">Overview</h2> | ||
| + | <p> In our project, one of the major goals is to reduce the uremic toxin, p-Cresol. Clostridium bacteria inside the gut will ferment excess tyrosine into p-Cresol. With the help of <a href="https://2019.igem.org/Team:NCKU_Tainan/Parts">Tyrosine ammonia-lyase (TAL)</a> , we are able to convert this excess tyrosine into a harmless product, p-Coumaric acid instead of the toxic p-Cresol. As such, the concentration of p-Cresol (mainly produced by C. difficile) can be reduced.</p> | ||
| + | <p> For our construct design, we focused on <a href="http://parts.igem.org/Part:BBa_I742148">BioBrick BBa_I742148</a>from the <a href="https://2007.igem.org/Edinburgh">2007 iGEM Edinburgh team</a><sup>[<a href="#ref1">1</a>]</sup> in the iGEM BioBrick library. This enzyme has been shown to have the highest Km value compared to other TAL enzyme. </p> | ||
| + | <p> We hypothesized that the native ribosome binding site(NRBS) from Saccharothrix espanaensis will not have a high translation efficiency in E.coli Nissle, so we changed the NRBS in front of TAL into a strong ribosome binding site B0034. Also, we noticed that there was no spacer sequence between NRBS and the TAL coding sequence(CDS) in <a href="http://parts.igem.org/Part:BBa_I742148">BBa_I742148</a>. </p> | ||
| + | <p> According to research, the length of the spacer sequence between RBS and CDS strongly affects translation efficiency.<sup>[<a href="#ref2">2</a>]</sup> We decided to follow the standard iGEM BioBrick assembly rule to add 6bp of scar sequence between RBS and CDS. The schematic below depicts how we improved the BioBrick.</p> | ||
| + | </section> | ||
| + | <hr> | ||
| + | <section> | ||
| + | <!--section name:Experimental Approach & Results--> | ||
| + | <h2 id ="Subtitle2">Experimental Approach & Results</h2> | ||
| + | <p> To prove that TAL expression was improved with the change of RBS (<a href="http://parts.igem.org/Part:BBa_I742146">I742146</a> into B0034), we checked the protein expression level by SDS-PAGE and measured the amount of p-coumaric acid production normalized by O.D. value. </p> | ||
| + | <p> The result of SDS-PAGE is shown below whereby the expected protein size for TAL is 54kDa.</p> | ||
| + | <p> As seen in the results above, there’s no distinguishable band around the expected size. Since we cannot identify any significant protein bands on SDS gel, we performed an RT-PCR experiment to confirm that the constructed BioBrick is being transcribed. The result is shown below.</p> | ||
| + | <p> As shown in Fig.3(b), the cDNA for both TAL constructs are being detected by RT-PCR. Hence, it confirms that the TAL genes are actually being transcribed in E.coli Nissle.</p> | ||
| + | <p> Finally, to determine the protein activity of TAL, we performed a functional test using the n-octanol extraction method (hyperlink: TAL and TyrP functional test), which was previously proposed by the <a href="https://2013.igem.org/Team:Uppsala">2013 iGEM Uppsala team</a> <sup>[<a href="#ref3">3</a>]</sup>and has been verified by HPLC. The p-Coumaric acid concentration was measured through the absorbance value at 310nm wavelength under Nanodrop UV-Vis wavelength. </p> | ||
| + | <p> We compared the TAL constructs containing the native and B0034 ribosome binding sites, (BBa_K2997009 and BBa_K2997010) to determine if p-Coumaric Acid production is improved by changing the ribosome binding sites. From the results seen in Fig 6., BBa_K2997010 is able to produce a higher amount of p-Coumaric acid. Hence, we are able to prove that by changing the RBS (from Native to B0034), the conversion of tyrosine into p-Coumaric acid can increase by 1.73-fold. Therefore, we have shown that we have improved a previous BioBrick.</p> | ||
| + | <p> For more information, please visit our <a href="https://2019.igem.org/Team:NCKU_Tainan/Results">Results page.</a></p> | ||
| + | </section> | ||
| + | |||
| + | <section class="ref"> | ||
| + | <h2>References</h2> | ||
| + | <ol> | ||
| + | <!--id name must corespond --> | ||
| + | <li id="ref1">2007 iGEM Edinburgh Team </li> | ||
| + | <li id="ref2">Chen, H., Bjerknes, M., Kumar, R., & Jay, E. (1994, November 25). Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC523762/. </li> | ||
| + | <li id="ref3">2013 iGEM Uppsala Team</li> | ||
| + | </ol> | ||
| + | </section> | ||
| + | </main> | ||
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Revision as of 13:02, 19 October 2019
Improve
Overview
In our project, one of the major goals is to reduce the uremic toxin, p-Cresol. Clostridium bacteria inside the gut will ferment excess tyrosine into p-Cresol. With the help of Tyrosine ammonia-lyase (TAL) , we are able to convert this excess tyrosine into a harmless product, p-Coumaric acid instead of the toxic p-Cresol. As such, the concentration of p-Cresol (mainly produced by C. difficile) can be reduced.
For our construct design, we focused on BioBrick BBa_I742148from the 2007 iGEM Edinburgh team[1] in the iGEM BioBrick library. This enzyme has been shown to have the highest Km value compared to other TAL enzyme.
We hypothesized that the native ribosome binding site(NRBS) from Saccharothrix espanaensis will not have a high translation efficiency in E.coli Nissle, so we changed the NRBS in front of TAL into a strong ribosome binding site B0034. Also, we noticed that there was no spacer sequence between NRBS and the TAL coding sequence(CDS) in BBa_I742148.
According to research, the length of the spacer sequence between RBS and CDS strongly affects translation efficiency.[2] We decided to follow the standard iGEM BioBrick assembly rule to add 6bp of scar sequence between RBS and CDS. The schematic below depicts how we improved the BioBrick.
Experimental Approach & Results
To prove that TAL expression was improved with the change of RBS (I742146 into B0034), we checked the protein expression level by SDS-PAGE and measured the amount of p-coumaric acid production normalized by O.D. value.
The result of SDS-PAGE is shown below whereby the expected protein size for TAL is 54kDa.
As seen in the results above, there’s no distinguishable band around the expected size. Since we cannot identify any significant protein bands on SDS gel, we performed an RT-PCR experiment to confirm that the constructed BioBrick is being transcribed. The result is shown below.
As shown in Fig.3(b), the cDNA for both TAL constructs are being detected by RT-PCR. Hence, it confirms that the TAL genes are actually being transcribed in E.coli Nissle.
Finally, to determine the protein activity of TAL, we performed a functional test using the n-octanol extraction method (hyperlink: TAL and TyrP functional test), which was previously proposed by the 2013 iGEM Uppsala team [3]and has been verified by HPLC. The p-Coumaric acid concentration was measured through the absorbance value at 310nm wavelength under Nanodrop UV-Vis wavelength.
We compared the TAL constructs containing the native and B0034 ribosome binding sites, (BBa_K2997009 and BBa_K2997010) to determine if p-Coumaric Acid production is improved by changing the ribosome binding sites. From the results seen in Fig 6., BBa_K2997010 is able to produce a higher amount of p-Coumaric acid. Hence, we are able to prove that by changing the RBS (from Native to B0034), the conversion of tyrosine into p-Coumaric acid can increase by 1.73-fold. Therefore, we have shown that we have improved a previous BioBrick.
For more information, please visit our Results page.
References
- 2007 iGEM Edinburgh Team
- Chen, H., Bjerknes, M., Kumar, R., & Jay, E. (1994, November 25). Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC523762/.
- 2013 iGEM Uppsala Team