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Improved Monitoring of Low-Level Transcription in Escherichia coli by a β-Galactosidase α-Complementation System.

ABSTRACT: Genetically encoded reporter proteins are important and widely used tools for the identification and capture of a promoter, tracking the dynamic behavior of transcription, and the quantification of promoter activity. The sensitivity of the reporter gene is a critical factor for an ideal reporter system because weak transcriptional signal has usually failed to be detected using classical reporters. In this study, we present a novel reporter system for improved monitoring of transcription in E. coli based on β-galactosidase α-complementation. In this reporter system, the β-galactosidase activity resulting from the assembly of a reporter lacZα and an existing α-acceptor in advance serves as a measure of transcriptional activity in vivo. To validate the potential of the lacZα-derived reporter system, a series of artificial operons were constructed, and the moderately strong lac promoter, ara promoter, and weak pbr promoter were chosen as the detection promoters. The response profiles of lacZα was similar to that of wild type lacZ in artificial lac operons. Due to its small size and efficient expression profile, the detection sensitivity of a lacZα-derived reporter system was significantly higher than that of the traditional full-length β-galactosidase and the fluorescent protein mCherry reporter system in artificial ara operons. As expected, the response sensitivity of the lacZα-derived reporter system was also demonstrated to be significantly higher than that of the β-galactosidase and mCherry reporter systems in lead-sensitive artificial pbr operons. The lacZα-derived reporter system may prove to be a valuable tool for detecting promoter activity, especially low-level transcription in vivo.

SUBMITTER: Guo Y

PROVIDER: S-EPMC6607957 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:1. Repression by glucose of beta-galactosidase synthesis is spontaneously reversible in all strains of Escherichia coli examined long before the glucose has all been consumed. The extent of recovery and the time necessary for reversal differ among various strains. Other inducible enzymes show similar effects. 2. This transient effect of glucose repression is observed in constitutive (i(-)) and permease-less (y(-)) cells as well as in the corresponding i(+) and y(+) strains. 3. Repression is exerted by several rapidly metabolizable substrates (galactose, ribose and ribonucleosides) but not by non-metabolized or poorly metabolized compounds (2-deoxyglucose, 2-deoxyribose, phenyl thio-beta-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of beta-galactosidase synthesis (e.g. isopropyl thio-beta-galactoside) but not with a weak inducer (lactose); in the latter instance glucose repression is permanent. Diauxic growth on glucose plus lactose can be abolished by including isopropyl thio-beta-galactoside in the medium. 5. In some strains phosphate starvation increases catabolite repression; in others it relieves it. Adenine starvation in an adenine-requiring mutant also relieves catabolite repression by glycerol but not that by glucose. Restoration of phosphate or adenine to cells starved of these nutrients causes a pronounced temporary repression. Alkaline-phosphatase synthesis is not affected by the availability of adenine. 6. During periods of transient repression of induced enzyme synthesis the differential rate of RNA synthesis, measured by labelled uracil incorporation in 2min. pulses, shows a temporary rise. 7. The differential rate of uracil incorporation into RNA falls during exponential growth of batch cultures of E. coli. This is equally true for uracil-requiring and non-requiring strains. The fall in the rate of incorporation has been shown to be due to a real fall in the rate of RNA synthesis. The significance of the changes in the rate of RNA synthesis is discussed. 8. A partial model of catabolite repression is presented with suggestions for determining the chemical identification of the catabolite co-repressor itself.

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Improved Monitoring of Low-Level Transcription in Escherichia coli by a β-Galactosidase α-Complementation System.

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