Difference between revisions of "Team:IIT Kharagpur/Modeling"

As a rookie team, we faced a lot of challenges and at the end, due to lack of time, we were not able to make the construct we had set out to. However, we learned a great deal from all the failures we had(after all, iGEM is all about the learning experience). We hope that this summary, of the issues we faced and solutions we tried to overcome them, would help other teams.

As mentioned, we were working with three parts namely : BBa_K274100(CrtEBI), BBa_R0062(pLuxR) and BBa_K546003(LuxR). Our primary aim was to ligate the pLuxR promoter in front of the CrtEBI gene. And then ligate the LuxR gene downstream of this. This was done to ensure that the reporter system(pLuxR + CrtEBI) and the detection system(LuxR) would remain in separate constructs and could be co-transformed.

We first tried this to do this using the Standard Protocol. We could go about doing this in two ways :

• Restriction digest the CrtEBI plasmid using the enzymes, XbaI and PstI. Run the digest on agarose gel and extract the CrtEBI gene, using a gel extraction kit. Open up the pLuxR plasmid by digestion using the enzymes, SpeI and PstI. Ligate the two parts. Transform the colonies and plate on culture plates to select for the transformants.
  
  
• We were not successful in our attempt. We didn’t get any colony on the plates. This prompted us to conclude that the pLuxR plasmid was not getting digested properly. We tried to repeat the digestion reaction for longer durations but, that didn’t work.
• Restriction digest the pLuxR plasmid using the enzymes, EcoRI and SpeI. Run the digest on agarose gel and extract the pLuxR gene, using a gel extraction kit. Open up the pLuxR plasmid by digestion using the enzymes, EcoRI and . Ligate the two parts. Transform the colonies and plate on ---- plates to select for the transformants.

• However, we were not successful in this attempt also as we could not isolate the pLuxR gene. The failure can be attributed to the fact that the pLuxR gene is only 55 base pairs long. As such, it does not bind to the spin column used for DNA extraction from agarose gel. The kit available with us could be used only for DNA >100 bp long.

Next, we tried to make the constructs using 3A assembly.

• We first tried to ligate CrtEBI and pLuxR using the standard 3A assembly through the protocol prescribed by iGEM. As our 'two inserts were in Amp and Cam backbone, we decided to use kanamycin backbone for the final construct. We did the ligations with different ratios of the three parts(1:1:0.5 and 3:1:0.5 for pLuxR:CrtEBI:kana backbone.
  
  We got colonies on kanamycin plate. This was our first successful attempt. We got a construct that seemed to contain the ligated part of the correct size(for the 1:1:0.5 reaction). We went on to check the construct by double restriction digestion, first with EcoRI and PstI and then with XbaRI and SpeI. The expected bands were of size 2.2kb(backbone) and 3.4kb(CrtEBI + pLuxR) in both cases. For the first digestion, we got the expected bands but, with the second digestion, we got a band of 3.3kb and another band of around 4.4kb. Subsequent digestion with PstI resulted in two bands of size 2.2kb and 5.5kb. This suggested that there were 2 PstI restriction sites in the construct(Which is why we had got a 2.2kb band. They were actually 2 different fragments of size 2.2kb). We suspected backbone ligation of kanamycin and chloramphenicol backbones. The possible construct could have been [Amp + Cam + pLuxR + CrtEBI]. So, we went on to streak the colony on chloramphenicol plate. But, didn’t observe any growth.
  
  
• The size of the pLuxR gene is too small(in comparison with the size of CrtEBI) to see any retardation between CrtEBI and [CrtEBI + pLuxR]. So, we searched for restriction sites inside the CrtEBI gene to digest CrtEBI and check for retardation, because retardation can be observed better with smaller pieces. The results were inconclusive.
  
• To address the issue of backbone ligation, in our next 3A assembly attempt, we ran the CrtEBI digest on agarose gel and extracted the gene from the gel and used that gene alone(without the backbone) instead of the digestion mix. We did not get constructs of the right size.
  
• Finally, we attempted to ligate the CrtEBI gene downstream of the LuxR construct using the standard assembly . As mentioned, earlier the LuxR construct(BBa_K546003) has a pLuxR downstream of the LuxR gene. It can act as a promoter for the CrtEBI gene. The results were inconclusive, though.
  

  Conclusions
  
  In our system, the intensity of the red colour produced by lycopene would act as an indicator for the concentration of AHLs in the medium, and in turn, a measure of the degree of spoilage. The intensity can be measured by measuring the absorbance of the culture solution at “maximum wavelength(lambda max)” for lycopene. However, the dose response curve of AHL concentration vs. Absorbance will not show a simple linear relationship. For all practical purposes, the curve would be a step function, that is, the absorbance would stay more or less unchanged for initial rise in AHL concentration and would rise steeply after a threshold concentration is reached and would become constant again soon afterwards. The dose response curve would resemble the graph shown below.

  

  The exact parameters of the curve would depend on the following factors:

  • Rate of production of LuxR(determined by strength of the pLuxR promoter and concentration of AHL inside the cell)
  • Diffusivity of the cell wall for AHL(This will relate the concentration of AHL in the medium to the concentration inside the cell)
  • Degradation rate of LuxR(characteristic of the protein)
  
  As such, further analysis need to be done to correlate the threshold concentration of AHL with the concentration at which spoilage happens. We believe, we’ll need to tweak the system and try out different promoters so that steep rise in response happens at the critical concentration after which the food can be deemed spoiled.