Team:IISER-Pune-India/Results/regulatory
Regulatory System
The regulatory system has not been completely developed. However, an intermediate part the negative feedback loop has been made and experimentally characterised (BBa_K3285666). The system consists of the cI-lam promoter upstream of a mRFP gene and cI gene. The cI gene, hence, represses its own promoter, thus forming a negative feedback loop.
The following results have been obtained:-
Interval Fluorescence results
After 1, 2, 3, 4 and 10 hours of inoculation, OD (Absorbance at 600nm) and Fluorescence intensity (Excitation:- 584nm; Emission:- 607nm) measurements were taken. Baseline correction was done w.r.t the reading taken at the zeroth time point i.e after inoculation
This shows that the fluorescence is rather comparable to that of constitutively expressing mRFP; but is lower in expression due to the negative feedback loop as expected. The saturation occurs in both cases; but as the model will soon show the difference in saturation values is expected behavior of the part.
recA mediated interference results
After 1 hour of incubation, Hydroxy-Urea was added to one of the test cultures. The cultures were grown for another 1 hour. The cells were then pelleted, washed and resuspended in 1x PBS buffer. The solution were then all diluted to OD (Absorbance @600nm) value 1.0. This solution were loaded into black bottom plates with 3 technical replicates and fluorescence readings were taken (Excitation:- 584nm; Emission:- 607nm). The following was obtained.
The other set of experiment done by inoculating at 1/6 dilution and adding Hydroxy-Urea(HU) at the beginning yielded the following result
The increase in mRFP readout due to HU induction shows that CI repressor in the system has been blocked. Hence, due to such a halt of negative feedback loop, one can expect to see such a increase in mRFP readout. The increase being higher than constitutive mRFP shows that disruption of feedback loop has turned it to a constitutively expressing system. Thus, the low mRFP readout of non treated system shows effective lowering of saturation point by the negative feedback.
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