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| <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><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 it's exponential phase (0.5 OD600) and 200 µl was aliquoted 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 observe 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> |
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| <center> | | <center> |
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− | <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 to study their kinetics and to evaluate their utility in our circuit.</h4> |
− | <h4>We have observed that out of these five different chromoproteins, chromoprotein sequence number 1-3 developed color relatively fast (24-30 hours). But chromoproteins sequence number 4-5 developed color relatively slow. Hence, 4-5 can not be suitable for utilization in our circuit. | + | <h4>We have observed that out of these five different chromoproteins, chromoprotein sequence numbers 1-3 developed color relatively fast (24-30 hours) but chromoprotein sequence numbers 4-5 developed color relatively slow. Hence, 4 and 5 are not suitable for our circuit. |
− | Therefore, we select chromoprotein amilCP Blue chromoprotein and fwYellow chromoprotein for using in our circuit. | + | Therefore, we selected chromoproteins: amilCP Blue chromoprotein and fwYellow chromoprotein for use in our circuit. |
− | Further, all these chromoproteins were cloned with RBS and LacI promoter by 3A assembly and submitted in depository with accession no. (from BBa_K2320001- K2320005). | + | Further, all these chromoproteins were cloned with RBS and LacI promoter by 3A assembly and submitted in the depository (accession number from BBa_K2320001- K2320005). |
| </h4> | | </h4> |
| <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/a/a4/T--IISER-Mohali-INDIA--dish.jpg" alt="Dishes"></td></tr></table></center> | | <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/a/a4/T--IISER-Mohali-INDIA--dish.jpg" alt="Dishes"></td></tr></table></center> |
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| <div class="page-title"><h6>Software</h6> | | <div class="page-title"><h6>Software</h6> |
| </div> | | </div> |
− | <section><h3>We seek to automate the process of reading gel doc slides and give reliable band sizes as outputs in a comprehensible format with an easy-to-use software. A manual mode hass also been developed for graphical analysis to ease the detection of fainter bands that are too close to each other to differentiate using one's eyes. The software requires minimum resources: a few Python modules, and can be adjusted to suit any gel doc machine and a variety of standard ladders. The main difficulty in differentiating very closely spaced bands, due to the inefficiency of human eyes, is done away with in this module. Furthermore, we are also working on introducing machine learning in the software to give better accuracy in this regard and also to detect extremely faint bands. The software will help students and researchers to automate the mundane and routine task of reading gel doc slides.</h3> | + | <section><h3>We seek to automate the process of reading gel doc slides and give reliable band sizes as outputs in a comprehensible format with an easy-to-use software. A manual mode has also been developed for graphical analysis to ease the detection of fainter bands that are too close to each other to distinguish using one's eyes. The software requires minimal resources: a few Python modules, any gel doc machine and a variety of standard ladders. The main difficulty in differentiating very closely spaced bands (due to the inefficiency of human eyes), is done away with in this module. Furthermore, we are working on introducing machine learning in the software to give better accuracy and to detect extremely faint bands. The software will help students and researchers to automate the mundane and routine task of reading gel doc slides.</h3> |
| </section> | | </section> |
| <section><center><table> | | <section><center><table> |