Generation of a library of eGFP labelled bacterial clones for monitoring growth

Abstract

Environmental and climatic stress are problematic issues in today’s society that have multiple effects on various scenarios, including microbial viability and proliferation. The presence of this stress results in the expression of certain genes in microorganisms, which help in protein stabilisation and protection of the organism from these conditions. In understanding the mechanism of this response, relations between the climate and microorganisms present can be made. The aim of this project was to generate a validated library of reporter strains building upon previous research where one strain was produced. These can be used to evaluate and tackle the question of what the effects of varying environmental conditions on microbial cell proliferation actually are. In this project, an IPTG-inducible eGFP plasmid was inserted into an Escherichia coli strain and its isogenic deletion mutants in the rpoS, dnaK, gadB, gadC and gadD genes through chemical transformation after making the cells chemically competent. These mutants assessed the effect of the gene targets involved in response to climatic stress through the production of successful growth kinetics and expression of eGFP. The FTTD was used to estimate the μmax parameter for each clone at each condition used. At a temperature of 27°C/0.04% CO2, the rpoS gene was observed to affect the μmax, while at 37°C and 42°C, the gabD and dnaK genes had a significant effect. Results also highlighted the fundamentality of the genes involved in the GAD system as well as the nutrient depletion response in E. coli. The information obtained through this project should also be applicable to other similar microorganisms in order to provide information on the response of the organism to environmental stress.