Influence of global gene regulatory networks on single cell heterogeneity of green fluorescent protein production in Bacillus subtilis

Microbial Cell Factories
Haojie Cao, Oscar P Kuipers

Abstract

Gram-positive bacterium Bacillus subtilis has been extensively studied as a microbial cell factory for high-level producing a wide range of interesting products. Green fluorescent protein (GFP) is commonly used as a marker for determining the strength of a given promoter or for the subcellular localization of a fusion protein. However, the inherent heterogeneity of GFP expression among individual cells that can arise from global regulation differences in the expression host, has not yet been systematically assessed. B. subtilis strains with single mutation(s) in the two major transcriptional regulators CcpA and/or CodY were earlier found to improve overall heterologous protein production levels. Here, we investigate the dynamic production performance of GFP in the reporter strains with chromosomally integrated Physpank-sfGFP(Sp). The mutation R214C in the DNA-binding domain of CodY effectively enhances GFP production at the population level relative to two other strains, i.e. wildtype (WT) and CcpAT19S. During the late stationary phase, the high- and low-level GFP-producing cells coexist in the WT population, while the CodYR214C population at the single-cell level shows higher phenotypic homogeneity of fluorescence signals. Exp...Continue Reading

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Methods Mentioned

BETA
fluorescence microscopy
Fluorescence
flow cytometry
PCR

Software Mentioned

Elements AR
ImageJ
Flowing
NIS

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