Demonstrate
1. Cloning of module 1:
For cloning of construct 1, Ptet- RBS1- T7 RNA polymerase –terminator 1-terminator 2 Two PCR reactions were performed. First PCR reaction is performed with primer 1 and 2 using the template R0040 to amplify the Ptet. The size of PCR product is 77 bp. Second PCR reaction is performed with primer 3 and 4 using the template K1450004 to amplify the RBS1- T7 RNA polymerase -terminator 1-terminator 2. The size of PCR product is 2338 bp. Third SOE PCR was performed using the primers 1 and 4 (for PCR conditions and reaction mixture, refer notebook).
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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, were not able to clone the construct1. Similarly, for cloning construct 2, PT7-RBS2- Chromoprotein II- RBS3-tetR-terminator3. Three PCR reactions were performed.
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First PCR reaction is performed with primer 5 and 6 using the template K1343022 to amplify the PT7-RBS2. The size of PCR product is 132 bp. Second PCR reaction is performed with primer 7 and 8 using the template K1033910 to amplify the chromoprotein II. The size of PCR product is 759 bp. 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 amiplified fragment. Then SOE2 PCR was performed using using primer 5 and 10 using the template SOE1 (891bp) fragment to amplify 1783 bp fragment. This SOE2 PCR frament 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 vector. Therefore, construct 2 was finally cloned at SalI and BamHI site of pRC10 (modified ptrc99a) which is intermediate copy number plasmid. The E. coli transformants were screened for positive clones of construct 2 by restriction digestion and obtained positive clone 9 and shown in agraose gel below.
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2.Cloning of module 2:
For cloning of construct 1 of module II, Pmar-RBS4- ToxR-terminator 4 First PCR reaction is performed with primer 11 and 12 using the template E. coli genome to amplify the pmar-RBS4. The size of PCR product is 524 bp. Second PCR reaction is performed with primer 13 and 14 using the template K641009 to amplify ToxR. The size of 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). Then SOE1 PCR was performed using primer 11 and 14 to get 1965 bp amiplified fragment. Then SOE2 PCR was performed using using primer 11 and 16 using the template SOE1 (1965bp) and third pcr amplified fragment (271 bp) to amplify 2236 bp fragment. This SOE2 PCR frament was tried to clone in low copy number plasmid pACYC177, at XhoI and XmaI site (high copy number plasmid).
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For cloning of construct 2, Pctx-RBS5- chromoprotein I- RBS6-TetR- terminator5.
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Two PCR reactions were performed. First PCR reaction is performed with primer 17 and 18 to amplify the Pctx. The size of PCR product is 180 bp. Second PCR reaction is 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. Third SOE PCR was performed using the primers 17 and 20 to amplify 1825 bp (for PCR conditions and reaction mixture, refer notebook). Final construct II of module II was cloned at BamHI and AatII site of pACYC177. The E. coli 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.
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To check the sensitivity of the mar promoter, we used an E. coli strain (taken from C.R. Rao’s lab), which contains mar cis-element cloned upstream of the Venus reporter gene (a fast folding variant of yellow fluorescent protein) integrated at the attλ site. We monitored the induction of fluorescence in the reporter strain with increasing amount of salicylate as shown in the figure below.
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The reporter strain was grown to exponential phase (0.5 OD600) 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 λexcitation = 515 nm and λemission= 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 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 mar 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.
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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 observe that the mar promoter responds within 20 minutes to the salicylate.
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Cloning of chromoprotein for selection and part submission for registry :
We select five different chromoproteins for studying their kinetics to evaluate their utility in our circuit.
We have observed that out of these five different chromoproteins, chromoprotein sequence no. 1-3 develop color very fast (24-30 hours). But chromoproteins sequence no. 4-5 develop color vey late. Hence can not be suitable for utilization in our circuit. Therefore, we select chromoprotein amilCP Blue chromoprotein and fwYellow chromoprotein for utilization circuit. Further all these chromoproteins are cloned with RBS and LacI promoter by 3A assembly and submitted in depository with accession no. (from BBa_K2320001- K2320005).
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