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| Thirdly, SOE PCR was performed using the primers 1 and 4 (for PCR conditions and reaction mixture, refer notebook). | | Thirdly, SOE PCR was performed using the primers 1 and 4 (for PCR conditions and reaction mixture, refer notebook). |
| </h4> | | </h4> |
− | <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/8/8c/T--IISER-Mohali-INDIA--CS2.png" alt="Flow chart"></td></tr></table></center> | + | <center><div class="data-img"><img width="80%" src="https://static.igem.org/mediawiki/2017/8/8c/T--IISER-Mohali-INDIA--CS2.png" alt="Flow chart"></center> |
| <h4>The final PCR product was attempted to be cloned at AatII and ApaI sites of pZS21MCS. However, we did not get positive results for cloning, resulting in failure to clone the construct 1. | | <h4>The final PCR product was attempted to be cloned at AatII and ApaI sites of pZS21MCS. However, we did not get positive results for cloning, resulting in failure to clone the construct 1. |
| Similarly, cloning of construct 2 (PT7 - RBS2 - Chromoprotein II - RBS3 - tetR - terminator3), three PCR reactions were performed.</h4> | | Similarly, cloning of construct 2 (PT7 - RBS2 - Chromoprotein II - RBS3 - tetR - terminator3), three PCR reactions were performed.</h4> |
− | <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/b/b3/T--IISER-Mohali-INDIA--CS3.png" alt="Flow chart"></td></tr></table></center> | + | <center><div class="data-img"><img width="80%" src="https://static.igem.org/mediawiki/2017/b/b3/T--IISER-Mohali-INDIA--CS3.png" alt="Flow chart"></center> |
| <h4>The first PCR reaction was performed with primer 5 and 6 using the template K1343022 to amplify PT7-RBS2. The size of the PCR product is 132 bp. | | <h4>The first PCR reaction was performed with primer 5 and 6 using the template K1343022 to amplify PT7-RBS2. The size of the PCR product is 132 bp. |
| The second PCR reaction was performed with primer 7 and 8 using the template K1033910 to amplify chromoprotein II. The size of the PCR product is 759 bp. | | The second PCR reaction was performed with primer 7 and 8 using the template K1033910 to amplify chromoprotein II. The size of the PCR product is 759 bp. |
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| The <i>E. coli</i> transformants were screened for positive clones of construct 2 by restriction digestion and a positive clone 9 was obtained as shown in the image below. | | The <i>E. coli</i> transformants were screened for positive clones of construct 2 by restriction digestion and a positive clone 9 was obtained as shown in the image below. |
| </h4> | | </h4> |
− | <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/2/26/T--IISER-Mohali-INDIA--results1.jpg" alt="Screening of transformants containing construct 2 by resriction digestion with SalI and BamHI"></td></tr></table></center> | + | <center><div class="data-img"><img width="80%" src="https://static.igem.org/mediawiki/2017/2/26/T--IISER-Mohali-INDIA--results1.jpg" alt="Screening of transformants containing construct 2 by resriction digestion with SalI and BamHI"></center> |
| <h3>2. Cloning of module 2:</h3> | | <h3>2. Cloning of module 2:</h3> |
| <h4>Construct 1 --> Pmar - RBS4 - ToxR - terminator 4.</h4> | | <h4>Construct 1 --> Pmar - RBS4 - ToxR - terminator 4.</h4> |
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| digestion and positive clones were obtained in lane 2,3,4,6,7,8,9,10 and 11 as shown in image below:</h4> | | digestion and positive clones were obtained in lane 2,3,4,6,7,8,9,10 and 11 as shown in image below:</h4> |
| | | |
− | <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/7/72/T--IISER-Mohali-INDIA--rslstss.png" alt="Flow chart"></td></tr></table></center> | + | <center><div class="data-img"><img width="80%" src="https://static.igem.org/mediawiki/2017/7/72/T--IISER-Mohali-INDIA--rslstss.png" alt="Flow chart"></center> |
− | <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/3/31/T--IISER-Mohali-INDIA--CS4.png" alt="Flow chart"></td></tr></table></center> | + | <center><div class="data-img"><img width="80%" src="https://static.igem.org/mediawiki/2017/3/31/T--IISER-Mohali-INDIA--CS4.png" alt="Flow chart"></center> |
| <h4>Construct 2 --> Pctx - RBS5 - chromoprotein I - RBS6 - TetR - terminator5.</h4> | | <h4>Construct 2 --> Pctx - RBS5 - chromoprotein I - RBS6 - TetR - terminator5.</h4> |
− | <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/6/63/T--IISER-Mohali-INDIA--CS5.png" alt="Flow chart"></td></tr></table></center> | + | <center><div class="data-img"><img width="80%" src="https://static.igem.org/mediawiki/2017/6/63/T--IISER-Mohali-INDIA--CS5.png" alt="Flow chart"></center> |
| <h4>Two PCR reactions were performed. The first PCR reaction was performed with primers 17 and 18 to amplify the Pctx. The size of PCR product is 180 bp. | | <h4>Two PCR reactions were performed. The first PCR reaction was performed with primers 17 and 18 to amplify the Pctx. The size of PCR product is 180 bp. |
| The second PCR reaction was performed with primers 19 and 20 using the template K1343022 to amplify the RBS5 - chromoprotein I - RBS6 - TetR - terminator5. The size of PCR product is 1645 bp. | | The second PCR reaction was performed with primers 19 and 20 using the template K1343022 to amplify the RBS5 - chromoprotein I - RBS6 - TetR - terminator5. The size of PCR product is 1645 bp. |
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| Finally, construct II of module II was cloned at BamHI and AatII site of pACYC177 plasmid. | | Finally, construct II of module II was cloned at BamHI and AatII site of pACYC177 plasmid. |
| The <i>E. coli</i> transformants were screened for positive clones of construct 2 by PCR and obtained positive clone 4,6,7,8 and 10 shown in image below:</h4> | | The <i>E. coli</i> transformants were screened for positive clones of construct 2 by PCR and obtained positive clone 4,6,7,8 and 10 shown in image below:</h4> |
− | <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/a/a2/T--IISER-Mohali-INDIA--results2.jpg" alt="Flow chart"></td></tr></table></center> | + | <center><div class="data-img"><img width="80%" src="https://static.igem.org/mediawiki/2017/a/a2/T--IISER-Mohali-INDIA--results2.jpg" alt="Flow chart"></center> |
| <h4>To check the sensitivity of the <i>mar</i> promoter, we used an <i>E. coli</i> strain (taken from C.R. Rao’s lab), which contains a <i>mar</i> cis-element cloned upstream of the Venus reporter gene (a fast folding variant of yellow fluorescent protein) integrated at the <i>attλ</i> site. We monitored the amount of fluorescence in the reporter strain with increasing amount of salicylate as shown in the figure below:</h4> | | <h4>To check the sensitivity of the <i>mar</i> promoter, we used an <i>E. coli</i> strain (taken from C.R. Rao’s lab), which contains a <i>mar</i> cis-element cloned upstream of the Venus reporter gene (a fast folding variant of yellow fluorescent protein) integrated at the <i>attλ</i> site. We monitored the amount of fluorescence in the reporter strain with increasing amount of salicylate as shown in the figure below:</h4> |
− | <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/c/cf/T--IISER-Mohali-INDIA--results3.jpg" alt="Flow chart"></td></tr></table></center> | + | <center><div class="data-img"><img width="80%" src="https://static.igem.org/mediawiki/2017/c/cf/T--IISER-Mohali-INDIA--results3.jpg" alt="Flow chart"> </center> |
| <h4>The reporter strain was grown to it's exponential phase (0.5 OD600) and aliquoted 200 µl per well into three wells of 96-well, clear bottom, black plate. A desired amount of salicylate was added from a stock into each well. LB media with and without salicylate were used as blanks. The plates were incubated at 37°C with shaking, and fluorescence was measured after 40 minutes of incubation at λ<sub>excitation</sub> = 515 nm and λ<sub>emission</sub>= 547 nm. | | <h4>The reporter strain was grown to it's exponential phase (0.5 OD600) and aliquoted 200 µl per well into three wells of 96-well, clear bottom, black plate. A desired amount of salicylate was added from a stock into each well. LB media with and without salicylate were used as blanks. The plates were incubated at 37°C with shaking, and fluorescence was measured after 40 minutes of incubation at λ<sub>excitation</sub> = 515 nm and λ<sub>emission</sub>= 547 nm. |
| We observed that below 1.25 mM salicylate, very little fluorescence was obtained. Thus, the sensitivity of the promoter is limited to mM concentrations of salicylate. To increase the sensitivity of the reporter, a stronger promoter or mutations in the MarR binding site can be done.</h4> | | We observed that below 1.25 mM salicylate, very little fluorescence was obtained. Thus, the sensitivity of the promoter is limited to mM concentrations of salicylate. To increase the sensitivity of the reporter, a stronger promoter or mutations in the MarR binding site can be done.</h4> |
| <h4>We also investigated the time kinetics of the reporter, by measuring the fluorescence of the reporter strain induced with various concentrations of salicylate. The 200 µl aliquots of the exponentially growing reporter strain cells were induced with desired concentrations of salicylate and their fluorescence was measured after every 20 minutes. As seen in the figure below, the <i>mar</i> promoter responds very quickly to the salicylate - eg in case of 0.625 mM salicylate concentration, the minimal fluorescent signal reached saturation in 150 minutes.</h4> | | <h4>We also investigated the time kinetics of the reporter, by measuring the fluorescence of the reporter strain induced with various concentrations of salicylate. The 200 µl aliquots of the exponentially growing reporter strain cells were induced with desired concentrations of salicylate and their fluorescence was measured after every 20 minutes. As seen in the figure below, the <i>mar</i> promoter responds very quickly to the salicylate - eg in case of 0.625 mM salicylate concentration, the minimal fluorescent signal reached saturation in 150 minutes.</h4> |
− | <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/e/e7/T--IISER-Mohali-INDIA--results4.jpg" alt="Flow chart"></td></tr></table></center> | + | <center><div class="data-img"><img width="80%" src="https://static.igem.org/mediawiki/2017/e/e7/T--IISER-Mohali-INDIA--results4.jpg" alt="Flow chart"> </center> |
| <h4>With higher concentrations of salicylate, the response was as quick as before, but with greater magnitude. The saturation of the fluorescence signal could not be studied with higher concentrations as the fluorescence intensity crossed the upper-limit of the instrument. Thus, we observe that the <i>mar</i> promoter responds within 20 minutes of adding salicylate.</h4> | | <h4>With higher concentrations of salicylate, the response was as quick as before, but with greater magnitude. The saturation of the fluorescence signal could not be studied with higher concentrations as the fluorescence intensity crossed the upper-limit of the instrument. Thus, we observe that the <i>mar</i> promoter responds within 20 minutes of adding salicylate.</h4> |
| <h3>Cloning of chromoprotein for selection and part submission for registry:</h3> | | <h3>Cloning of chromoprotein for selection and part submission for registry:</h3> |