Team:IISER-Pune-India/Design

Mutatis Mutandis

An En-“lightened” tool for directed evolution

Design

Mutator Plasmid

In this module, we aim to create the gene circuit that is activated with blue light to form the required protein product, MutD5 and is controlled with an introduced self-regulatory system.
The Const. Promoter constitutively expresses YF1 and FixJ. In the absence of blue light, the YF1 protein activates the FixJ protein by phosphorylation [1]. The activated FixJ protein activates the FixK2 promoter. This allows the expression of cI gene. cI is a lambda repressor protein which represses the promoter cI lam. This inhibits the expression of the genes downstream of cI lam, here, mutD5 and cI* (cI-ts2). In the presence of blue light, the YF1 protein is inhibited from activating the FixJ protein [1]. Therefore, cI is no longer expressed. The cI lam promoter is hence active and the mutD5 gene is expressed in the system. The cI* is also expressed. cI* (cI-ts2) is a modified version of cI which is temperature sensitive and has lower binding affinity to cI lam than cI at 37 degrees Celsius [7]. This cI* acts on the cI lam promoter in a negative feedback loop and thus creates an upper limit on the amount of mutD5 expressed in the system. This allows us to stop the mutation rates from getting too high which ensures that the evolutionary adaptation of the E.coli is not hindered as a result of very high mutation rates [2].

Biosensor system

This biosensor was previously developed by the 2015 Bielefeld iGEM team. It consists of sfGPF downstream of the pbrAP promoter and pbrR downstream of a constitutive promoter in the opposite direction. pbrR is an inhibitor of the pbrAP promoter but in the presence of lead, the pbrR binds to it hindering it from binding to the promoter thereby leading to gfp expression. More the lead concentration, more the gfp output will be.

Bioremediation system

This system is an extension of the biosensor having pbrD downstream of sfGFP and pbrT downstream of pbrR in the opposite direction. The constitutive promoter constitutively expresses both pbrR and pbrT. pbrT codes for the transporter protein allowing more lead to come inside. Once inside, the lead inhibits pbrR from binding to the promoter leading to gfp and pbrD expression. pbrD codes for the protein that dimerizes with lead leading to a heterodimer further sequestering the lead[3].

References

class="w3-justify">[1] Robert Ohlendorf, Roee R.Vidavski, Avigdor Eldar, Keith Moffat, and Andreas Möglich (2012). From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. J. Mol. Biol., 416(4), 534-542.

class="w3-justify">[2] Badran, A. H., & Liu, D. R. (2015). Development of potent in vivo mutagenesis plasmids with broad mutational spectra. Nature communications, 6, 8425.

class="w3-justify">[3]https://www.sciencedirect.com/science/article/pii/S104659281830603X

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