Team:ICT-Mumbai/Parts

ICT-Mumbai 2017

Our project involves metabolism of ammonia by Escherichia coli to produce a blue-coloured dye, indigoidine. We had to choose between using a constitutive and an inducible promoter to drive expression of the genes that we wish to express in E. coli.

Expression from inducible promoters requires addition of an inducing molecule. However, as it would be cumbersome and tedious to add the inducer to the device that will house the engineered E. coli, and as it would also contribute to the cost, we decided to use a constitutive promoter to drive gene expression.

We reasoned that the constitutive promoter of choice should have the following two properties: (1) it should not be a very strong promoter, so as to not lead to any toxicity to the cell, and (2) it should be active in low-nutrient conditions. Based on these two considerations, the commonly used glycolytic promoters were ruled out as possible choices.

The ychH promoter has been described in literature to be active under low glucose conditions (Ref. 1). Crp acts as a positive transcription regulator of ychH, which means that this gene is expressed when glucose concentrations are low (Ref. 2; EcoCyc database). Moreover, it is not a very strong promoter, compared to those frequently employed to express recombinant proteins in E. coli (Ref. 3). Therefore, the ychH promoter became our promoter of choice.


Part Table

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References

  1. Hollands K, Busby SJ, Lloyd GS (2007). New targets for the cyclic AMP receptor protein in the Escherichia coli K-12 genome. FEMS Microbiol Lett 274:89-94. PMID: 17608696
  2. Keseler IM, Mackie A, Peralta-Gil M, Santos-Zavaleta A, Gama-Castro S, Bonavides-Martínez C, Fulcher C, Huerta AM, Kothari A, Krummenacker M, Latendresse M, Muñiz-Rascado L, Ong Q, Paley S, Schröder I, Shearer AG, Subhraveti P, Travers M, Weerasinghe D, Weiss V, Collado-Vides J, Gunsalus RP, Paulsen I, Karp PD (2013). EcoCyc: fusing model organism databases with systems biology. Nucleic Acids Res 41(Database issue):D605-612. PMID: 23143106
  3. Deb SS, Reshamwala SMS, Lali AM (2016). A series of template plasmids for Escherichia coli genome engineering. J Microbiol Methods 125:49-57. PMID: 27071533

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