Fluorescence Reporters turboGFP and turboRFP for Promoter Evaluation in Escherichia coli: Possibilities and Limitations

• Published in: Applied biochemistry and microbiology Vol. 59; no. 9; pp. 1168 - 1176
• Main Authors: Lavrov, K. V., Grechishnikova, E. G., Shemyakina, A. O., Mungarro, A. H. Bernal, Potapova, M. S., Derbikov, D. D., Yanenko, A. S.
• Format: Journal Article
• Language: English
• Published: Moscow Pleiades Publishing 01-12-2023; Springer Nature B.V
• Subjects: Accumulation; Biochemistry; Biology; Biomedical and Life Sciences; Biotechnology; Cassettes; Cell culture; Cloning; Cytotoxicity; E coli; Escherichia coli; Fluorescence; Gene Engineering; Genes; Life Sciences; Medical Microbiology; Microbiology; Producers; Proteins; Selection; Synthesis; Toxicity; cytofluorimetry; fluorescent protein; reporters; cytotoxicity; gene expression
• ISSN: 0003-6838; 1608-3024
• DOI: 10.1134/S0003683823090107

Fluorescent proteins are convenient reporters widely used in cell biology. However, there is insufficient information at present on the possibilities and limitations of their use for the development and optimization of expression cassettes in the design of bacterial producers for biotechnology. Herein, the features of overexpression of genes of green (turboGFP) and red (turboRFP) fluorescent proteins in
Escherichia coli
under the control of the IPTG-regulated phage promoter T5 were investigated. Both proteins were mainly synthesized in the soluble form and had a significant cytotoxicity. In the presence of IPTG in the culture, overexpression of the turboGFP or turboRFP led to a delay in culture growth and the accumulation of clones that did not synthesize these proteins. Under the same cultivation conditions, the accumulation of such clones was more pronounced for turboRFP than for turboGFP. At a reduced expression level (in the absence of IPTG), these effects diminished (for turboRFP) or did not appear at all (for turboGFP). Possible reasons for the cytotoxicity of the studied proteins for
E. coli
cells were discussed. Practical recommendations for using the genes of investigated proteins as reporters in the development of expression cassettes were proposed. In particular, the level of production of the investigated proteins at which no negative effects were observed was determined, and the possibilities and limitations of the cytofluorometric cell sorting method in the development of strains-producers were indicated.