Part:BBa_K3285666
Negative feedback system with lamda promoter
This part consists of the lambda phage promoter which regulates the expression of mRFP and cI genes. CI protein is the phage repressor protein for the same phage promoter. Hence, it effectively along with mRFP expresses its own repressor and hence acts as a negative feedback loop. To use this part in a project, one can effectively replace the mRFP with gene(s) of interest. The CI protein is known to be degraded through a recA mediated pathway [1][2][3]. Hence, induction of recA would lead to loss of the negative feedback and higher the expression of mRFP. One such method to induce recA is through usage of Hydroxy-Urea [4][5].
===Sequence and Features===
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 630
Illegal AgeI site found at 742 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
The gene circuit is designed such that the gene(s) under cI-lam is constitutively expressed. However, this expression is limited by the negative feedback imposed due to the expression of cI gene. cI produces a phage protein that acts as a repressor to the phage promoter cI-lam. In this circuit, CI essentially represses its own promoter and hence, on average maintains a lower saturation point with instantaneous variations. The gene circuit can be used to limit expression of certain genes and the limiting amount can be set by changing incubation temperatures as CI has varying degradation rate at different temperatures. This is primarily due to the known thermally unstable structure of CI protein [6]. This variation has been shown in our model. The negative feedback can be killed through induction of recA. This is because recA mediates degradation and effectively stops activity of CI protein in the system. This recA mediation can be achieved through various ways, one of them being induction through 50mM Hydroxy-Urea. This was done in our experimental characterisation of the system.
Experimental Characterisation
Two experiments were carried out to characterise the designed part:-
1. Fluorescence measurements over time The transformed cells were grown in Minimal Media (M9) in flat black, clear bottom 96-well plates. 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. The following data was obtained.
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.
2. recA induced disbanding of feedback system Transformed cells were grown from a primary culture into a secondary culture 1/2 bottleneck in Nutrient Broth. 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.
Another set of experiment was done by inoculating at 1/6 dilution and Hydroxy-Urea(HU) was added at the beginning and cultured in for 2 hours. The processing later was same as the previous and these results were obtained.
This shows that induction of recA relieves the negative feedback loop and increases the effective mRFP expression of the system.
Model
Based on deterministic modelling on the basis of information present in Jana et al., the repression of cIlam promoter by cI using Hill function was modelled and the following graph for RFP(in number of molecules) versus time(in minutes) in both processes were plotted.
Also, since cI is temperature-sensitive, there should be a variation in the RFP expression at saturation versus temperature as:
References
[1] Pal A1, Chattopadhyaya R. (2009) RecA-mediated cleavage of lambda cI repressor accepts repressor dimers: probable role of prolyl cis-trans isomerization and catalytic involvement of H163, K177, and K232 of RecA. J Biomol Struct Dyn. 27(2):221-33.
[2] Ogawa H1, Ogawa T. (1990) Regulation in repressor inactivation by RecA protein. Adv Biophys. 26:33-49.
[3] Vitold E. Galkin,1 Xiong Yu,1 Jakub Bielnicki,1 Dieudonné Ndjonka,2,3 Charles E. Bell,2 and Edward H. Egelman (2010) Cleavage of Bacteriophage λ cI Repressor Involves the RecA C-terminal Domain. J Mol Biol. 2009 Jan 23; 385(3): 779–787.
[4] Barbe et al. (1987) Induction of the SOS response by hydroxyurea in Escherichia coli K12. Mutation Research Letters Volume 192, Issue 2, October 1987, Pages 105-108
[5] Gangan M.S. and Athale C.A. (2017) Threshold Effect of Growth Rate on Population Variability of Escherichia coli Cell Lengths Roy. Soc. open science DOI: 10.1098/rsos.160417 22-Feb-2017
[6] Little JW, Michalowski CB. 2010. Stability and instability in the lysogenic state of phage lambda. J Bacteriol 192:6064–6076. doi:10.1128/JB.00726-10.
===Sequence and Features===
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 630
Illegal AgeI site found at 742 - 1000COMPATIBLE WITH RFC[1000]
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