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Check out our Project <a href="https://2020.igem.org/Team:IISER-Tirupati_India/Inspiration" target="_blank">Inspiration</a> page to know more!</p> | Check out our Project <a href="https://2020.igem.org/Team:IISER-Tirupati_India/Inspiration" target="_blank">Inspiration</a> page to know more!</p> | ||
<h2 id="imagine">Imagine</h2><br> | <h2 id="imagine">Imagine</h2><br> | ||
− | <p style="font-family:comfort;font-size:16px;">Once we had the project idea in place, we wanted to incorporate different independent modules in the project that would account for the different objectives our bacteria is set to achieve. We searched for sulfonamide degrading genes that would degrade sulfonamides into simpler molecules and came across the two-component flavin dependent monooxygenase system found naturally in <i>Microbacterium</i> sp. CJ77 [4]. This involves the genes <i>sulX</i> and <i>sulR</i> and work together to destroy sulfonamides. To prevent our engineered bacteria (being a GMO) from undergoing Horizontal Gene Transfer (HGT) we planned to incorporate the surface exclusion genes <i>traS</i> and <i>traT</i> which help reduce conjugation significantly [5]. The death of our GMO would ensure high biosafety and to ensure that we planned to use the <i> | + | <p style="font-family:comfort;font-size:16px;">Once we had the project idea in place, we wanted to incorporate different independent modules in the project that would account for the different objectives our bacteria is set to achieve. We searched for sulfonamide degrading genes that would degrade sulfonamides into simpler molecules and came across the two-component flavin dependent monooxygenase system found naturally in <i>Microbacterium</i> sp. CJ77 [4]. This involves the genes <i>sulX</i> and <i>sulR</i> and work together to destroy sulfonamides. To prevent our engineered bacteria (being a GMO) from undergoing Horizontal Gene Transfer (HGT) we planned to incorporate the surface exclusion genes <i>traS</i> and <i>traT</i> which help reduce conjugation significantly [5]. The death of our GMO would ensure high biosafety and to ensure that we planned to use the <i>DNaseI</i> gene, a highly efficient endonuclease [6], downstream to a user modulatable promoter to induce bacterial cell death and release the proteins. |
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Check out our Project <a href="https://2020.igem.org/Team:IISER-Tirupati_India/Description" target="_blank">Description</a> page to know more!</p> | Check out our Project <a href="https://2020.igem.org/Team:IISER-Tirupati_India/Description" target="_blank">Description</a> page to know more!</p> | ||
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<div class="humanpratice-content"> | <div class="humanpratice-content"> | ||
<div class="main-text"><h2 id="design">Design</h2><br> | <div class="main-text"><h2 id="design">Design</h2><br> | ||
− | <p style="font-family:comfort;font-size:16px;">After the search for genes came the part of designing the genetic circuit. <b>Having several teams already used the J23118 promoter for their experiments we chose to use this medium strength promoter along with the strong RBS (B0034) and call it the medium strength expression system.</b> This system was designed to be used along with all the genes in the system except <i> | + | <p style="font-family:comfort;font-size:16px;">After the search for genes came the part of designing the genetic circuit. <b>Having several teams already used the J23118 promoter for their experiments we chose to use this medium strength promoter along with the strong RBS (B0034) and call it the medium strength expression system.</b> This system was designed to be used along with all the genes in the system except <i>DNaseI</i> which was designed downstream to the araBAD promoter [7][8], which is an arabinose inducible promoter. <b>All these systems were mathematically modelled and simulated for their theoretical expression as we predicted their behaviour.</b> |
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Check out our Project <a href="https://2020.igem.org/Team:IISER-Tirupati_India/Design" target="_blank">Design</a> and <a href="https://2020.igem.org/Team:IISER-Tirupati_India/Model" target="_blank">Model</a> page to know more!</p><br> | Check out our Project <a href="https://2020.igem.org/Team:IISER-Tirupati_India/Design" target="_blank">Design</a> and <a href="https://2020.igem.org/Team:IISER-Tirupati_India/Model" target="_blank">Model</a> page to know more!</p><br> | ||
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<p><b>Gene Cloning</b></p> | <p><b>Gene Cloning</b></p> | ||
− | <p style="font-family:comfort;font-size:16px;">The genes <i>sulX</i> and <i>sulR</i> will be cloned into the vectors pSB1C3 and pSB3K3 respectively and will be transformed into calcium chloride competent DH5α cells individually in the presence of respective antibiotics. The colonies will be screened for positive clones, thereafter the colonies will be cultured followed by plasmid isolation. The recombinant plasmids will then be co-transformed into competent BL21 cells. Similar cloning strategy will be followed to clone <i>traS</i>, <i>traT</i> and <i> | + | <p style="font-family:comfort;font-size:16px;">The genes <i>sulX</i> and <i>sulR</i> will be cloned into the vectors pSB1C3 and pSB3K3 respectively and will be transformed into calcium chloride competent DH5α cells individually in the presence of respective antibiotics. The colonies will be screened for positive clones, thereafter the colonies will be cultured followed by plasmid isolation. The recombinant plasmids will then be co-transformed into competent BL21 cells. Similar cloning strategy will be followed to clone <i>traS</i>, <i>traT</i> and <i>DNaseI</i> into the same bacterium.</p><br> |
<p><b>Action of sulfa drugs on the engineered bacteria</b></p> | <p><b>Action of sulfa drugs on the engineered bacteria</b></p> | ||
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<p><b>Inducible expression of bpDNaseI and its purification</b></p> | <p><b>Inducible expression of bpDNaseI and its purification</b></p> | ||
− | <p style="font-family:comfort;font-size:16px;"><i> | + | <p style="font-family:comfort;font-size:16px;"><i>DNaseI</i> is used here as a death-inducing gene that cleaves DNA into smaller fragments thus inducing cellular death. This gene will be cloned downstream to the tightly regulated araBAD promoter into both the vectors. This will be transformed into BL21 cells. These cells will be stimulated with the appropriate arabinose concentration and cell death will be measured by monitoring the OD of the culture. The culture will be plated at regular intervals to determine the CFU. Also, DNA from such cultures will be isolated and run on an agarose gel to check for any intact DNA prevailing. The bpDNaseI will also be overexpressed and will be purified for in-vitro characterization to determine its DNA degrading efficiency.</p><br> |
</p></div> | </p></div> | ||
</div> | </div> |
Revision as of 11:53, 27 October 2020