Team:IISER Kolkata/Contribution
Contributions
Characterization of BBa_K381001
Our model relies on the ability of the bacterial chassis to detect the various Nitric Oxide (NO) concentrations inside the macrophage and trigger the modified genetic circuit accordingly. Thus as proof of model we are characterizing the induction of PyeaR promoter in different concentrations. For experimental purposes, we are using Sodium Nitroprusside (SNP) as the NO donor. From literature, it is known that one molecule of SNP releases one molecule of NO in aqueous solution.
Aim
BBa_K381001 was used as a test circuit to quantify the PyeaR promoter activity in different concentrations of NO in the LB medium.
Method
E. coli DH5-α were transformed using re-suspended BBa_k381001 in pSB1C3 plasmids from iGEM 2019 distribution kit and selected using Chloramphenicol LB agar plates.
- Single positive colony of BBa_k381001 was added to 10ml of LB media and left overnight at 37° C on 150 rpm in an BOD incubator.
- 4 mL of overnight culture was added to 200 mL of LB media and incubated until the OD reached 0.4.
- The secondary culture was divided into five flasks each with 50mL and calculated stock solution of Sodium nitroprusside was added to give the following final concentration of Nitric oxide in the media - 0 M, 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M.
- Four hours after induction, 100uL of the culture was added to each well of 96 well plate.
- Varioskan LUX multimode reader was used to measure the GFP fluorescence: Excitation at 475 nm and emission at 545 nm.
Result
BBa_k381001 transformed E. coli shows high GFP expression in moderate nitric oxide concentration and decreased expression in both very low and very high NO concentration.
Conclusion
The bacterial promoter PyeaR is inducible depending on the concentration of NO available in the media. It is induced best in the range of 10-5 M to 10-4 M.
Characterization of BBa_I13522
In our project to combat Leishmaniasis using a genetically modified bacterium we planned to use bacterial promoter pTET as a upstream part of our coding region, but for it to act effectively we need to quantify that its promoter activity is comparable in both bacterial and mammalian culture medium.
Aim
BBa_I13522 was used as a test circuit to compare the activity of pTET bacterial promoter in bacterial medium Luria Broth and mammalian cell culture medium DMEM.
Method
E. coli DH5-α were transformed using re-suspended BBa_I13522 pSB1C3 plasmids from iGEM 2019 distribution kit and selected using Chloramphenicol LB agar plates.
- Single positive colony of BBa_I13522 was added to 10ml of LB media and left overnight at 37° C on 150 rpm in an incubator. Along with this, a positive control with non-transformed E coli cells was also kept in liquid culture in the similar conditions.
- 100 µL of overnight culture was added to 10ml of LB media and DMEM media in two different test tubes. For positive control secondary culture was added in 10ml of LB media and all the test tubes were left in incubator for 4 hours until the OD for the control reached 0.6
- 2 mL of liquid culture from each of the test tube was centrifuged, washed and re-suspended in 500 µL of Phosphate-Buffered Saline (PBS)
- 100uL of the re-suspended culture was added to each well of 96 well plate.
- Varioskan LUX multimode reader was used to measure the GFP fluorescence: Excitation at 494 nm and emission at 525 nm.
Result
BBa_I13522 transformed E. coli shows similar GFP expression in both bacterial and mammalian media.
Conclusion
The bacterial promoter pTET constitutively expresses comparable amount the downstream coding region (GFP in this case) irrespective of medium in which it is grown.
Characterization of BBa_K861173
BBa_K861173 has a mRFP controlled by PcstA which is a Glucose repressible promoter. It has been shown by previous teams that with the increased concentration of glucose there is a decrease in the activity of PcstA. Team iGEM IISER Kolkata is trying to characterize what is the effect of increasing concentration of other glucose analogues on the same system.
Aim
BBa_K861173 was used to characterize the promoter activity of PcstA in a range of concentration of glucose and its analogues in the growth media.
Method
E. coli DH5-α were transformed using re-suspended BBa_K861173 pSB1C3 plasmids from iGEM 2019 distribution kit and selected using Chloramphenicol LB agar plates.
- Single positive colony of BBa_I13522 was added to 10ml of LB media and left overnight at 37° C on 150 rpm in an incubator. Along with this, a positive control with non-transformed E. coli cells was also kept in liquid culture under similar conditions.
- 200 µL of the overnight culture was added to 10 mL of M9 minimal media with different concentrations of sugars in individual test tubes. One control was kept in LB media.
- It was kept in incubated for 8 hours at 37°C at 150 rpm.
- At the end of 8 hours of incubation in the incubator, 100uL of the culture was added to each well of 96 well plate. The culture in LB was pelleted down and resuspended in equivalent amount of M9 media.
- Varioskan LUX multimode reader was used to measure the GFP fluorescence: Excitation at 494 nm and emission at 525 nm.
- Additionally, Shimazadu UV1900 was used to measure the OD at 600 nm to quantify the bacterial growth.
Result
Conclusion
As expected, there is a linear increase in the O.D600 of the bacterial culture with the increase in the concentration of sugar (except maltose) as seen in figure 1. Moreover, we could also show a clear co-relation that with the increase in concentration of glucose, fructose and xylose there is a decrease in mRFP fluorescence. This signifies the fact that the PcstA promoter activity decreases with the increase in concentration of glucose, fructose and xylose.