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| </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><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> |
− | <h4>The final PCR product was tried to clone at AatII and ApaI site of pZS21MCS. But we did not get positive results for cloning. Therefore, we were not able to clone the construct1. Similarly, for cloning construct 2, PT7 - RBS2 - Chromoprotein II - RBS3 - tetR - terminator3. Three PCR reactions were performed.</h4> | + | <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> |
| <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><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> |
− | <h4>First PCR reaction was performed with primer 5 and 6 using the template K1343022 to amplify the PT7-RBS2. The size of 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. |
− | Second PCR reaction was performed with primer 7 and 8 using the template K1033910 to amplify the chromoprotein II. The size of 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. |
− | Third PCR was performed using the primers 9 and 10 (for PCR conditions and reaction mixture, refer notebook).
| + | The third PCR was performed using the primers 9 and 10 (for PCR conditions and reaction mixture, refer notebook). |
− | Then SOE1 PCR was performed using primer 5 and 8 to get 891 bp amplified fragment.
| + | Next, SOE1 PCR was performed using primers 5 and 8 to get an 891 bp amplified fragment. |
− | Then SOE2 PCR was performed using primer 5, 10 and the template SOE1 (891bp) fragment to amplify 1783 bp fragment. | + | Then, SOE2 PCR was performed using primers 5, primer 10 and the template SOE1 (891bp) fragment to amplify the 1783 bp fragment. |
− | This SOE2 PCR fragment was tried to clone in low copy number plasmid pZS21MCS, at SalI and BamHI site. But due to problems with digestion of vector, we were not able to clone in pZS21MCS plasmid.
| + | The cloning of this SOE2 PCR fragment was attempted in the low copy number plasmid pZS21MCS, at SalI and BamHI sites, but due to problems with digestion of the vector, we were not able to clone in pZS21MCS plasmid. |
− | Therefore, construct 2 was finally cloned at SalI and BamHI site of pRC10 (modified ptrc99a) which is intermediate copy number plasmid. | + | Therefore, construct 2 was finally cloned at SalI and BamHI sites of pRC10 (modified ptrc99a) which is an intermediate copy number plasmid. |
− | The <i>E. coli</i> transformants were screened for positive clones of construct 2 by restriction digestion and obtained positive clone 9 as shown in agraose gel 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><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> |
− | <h3>2.Cloning of module 2:</h3> | + | <h3>2. Cloning of module 2:</h3> |
− | <h4>Construct 1 of module II --> Pmar - RBS4 - ToxR - terminator 4. | + | <h4>Construct 1--> Pmar - RBS4 - ToxR - terminator 4.</h4> |
− | First PCR reaction was performed with primer 11 and 12 using the template <i>E. coli</i> genome to amplify the pmar - RBS4. The size of PCR product is 524 bp.
| + | <h4>The first PCR was performed with primers 11 and 12 using the template <i>E. coli</i> genome to amplify the pmar - RBS4. The size of the PCR product is 524 bp. |
− | Second PCR reaction was performed with primer 13 and 14 using the template K641009 to amplify ToxR. The size of PCR product is 1441 bp.
| + | The second PCR was performed with primers 13 and 14 using the template K641009 to amplify ToxR. The size of this PCR product is 1441 bp. |
− | Third PCR was performed using the primers 15 and 16 using the template pVenus to amplify terminator (271 bp). For PCR conditions and reaction mixture, refer notebook.
| + | The third PCR was performed using the primers 15 and 16 using the template pVenus to amplify terminator (271 bp). For PCR conditions and reaction mixture, refer notebook. |
− | Then, SOE1 PCR was performed using primer 11 and 14 to get 1965 bp amplified fragment.
| + | Next, SOE1 PCR was performed using primers 11 and 14 to get a 1965 bp amplified fragment. |
− | Then, SOE2 PCR was performed using primer 11, 16 and the template SOE1 (1965bp). Third PCR amplified fragment (271 bp) to amplify 2236 bp fragment. | + | Then, SOE2 PCR was performed using primer 11, primer 16, template SOE1 (1965bp) and third PCR amplified fragment (271 bp) to amplify the 2236 bp fragment. |
− | This SOE2 PCR fragment was tried to clone in low copy number plasmid pACYC177, at XhoI and XmaI site (high copy number plasmid). | + | This SOE2 PCR fragment was attempted to be cloned in low copy number plasmid pACYC177, at XhoI and XmaI sites (high copy number plasmid). |
| </h4> | | </h4> |
− | <h4>The <i>E. coli</i> transformants were screened for positive clones of construct 2 by restriction digestion and obtained positive clone in lane 2,3,4,6,7,8,9,10 and 11 are shown in agraose gel below.</h4> | + | <h4>The <i>E. coli</i> transformants were screened for positive clones of construct 2 by restriction |
| + | 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><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><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><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> |
− | <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><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> |
− | <h4>Two PCR reactions were performed. | + | <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. |
− | First PCR reaction was performed with primer 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. |
− | Second PCR reaction was performed with primer 19 and 20 using the template K1343022 to amplify the RBS5 - chromoprotein I - RBS6 - TetR - terminator5. The size of PCR product is 1645 bp.
| + | Thirdly, SOE PCR was performed using the primers 17 and 20 to amplify 1825 bp (for PCR conditions and reaction mixture, refer notebook). |
− | Third SOE PCR was performed using the primers 17 and 20 to amplify 1825 bp (for PCR conditions and reaction mixture, refer notebook).
| + | Finally, construct II of module II was cloned at BamHI and AatII site of pACYC177 plasmid. |
− | Final construct II of module II was cloned at BamHI and AatII site of pACYC177.
| + | 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 agraose gel 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><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> |
− | <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 <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><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> |
− | <h4>The reporter strain was grown to exponential phase (0.5 OD 600) and aliquoted 200 µl into three wells each of 96-well, clear bottom, black plate. Desired amount of salicylate was added from a stock into each well. LB media with and without salicylate were used as blank. The plates were incubated at 37°C with shaking and fluorescence was measured after 40 minutes of incubation 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 induction of fluorescence is obtained. Thus, the sensitivity of the promoter is limited to mM concentration of salicylate. To increase the sensitivity of the reporter a different strong promoter or mutations in the MarR binding site can be done. | + | 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 also investigated the time kinetics of the reporter, by measuring the fluorescence of the reporter strain induced with various concentrations of the salicylate. The 200 µl aliquots of the exponentially growing reporter strain cells were induced with desired concentrations of salicylate and fluorescence was measured after every 20 minutes. As seen in the figure below, the <i>mar</i> promoter responds very quickly to the salicylate. As 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><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> |
− | <h4>With higher concentrations of salicylate, the response was again very quick but with greater magnitude. Saturation of fluorescence signal could not be studied with higher concentrations as the fluorescence crossed the upper-limit of the instrument. Thus, we observed that the <i>mar</i> promoter responds within 20 minutes to the 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> |
| <h4>We selected five different chromoproteins for studying their kinetics to evaluate their utility in our circuit.</h4> | | <h4>We selected five different chromoproteins for studying their kinetics to evaluate their utility in our circuit.</h4> |