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Improving the stability of the TetR/Pip-OFF mycobacterial repressible promoter system.

ABSTRACT: Tightly regulated gene expression systems are powerful tools to study essential genes and characterize potential drug targets. In a past work we reported the construction of a very stringent and versatile repressible promoter system for Mycobacterium tuberculosis based on two different repressors (TetR/Pip-OFF system). This system, causing the repression of the target gene in response to anhydrotetracycline (ATc), has been successfully used in several laboratories to characterize essential genes in different mycobacterial species both in vitro and in vivo. One of the limits of this system was its instability, leading to the selection of mutants in which the expression of the target gene was no longer repressible. In this paper we demonstrated that the instability was mainly due either to the loss of the integrative plasmid carrying the genes encoding the two repressors, or to the selection of a frameshift mutation in the gene encoding the repressors Pip. To solve these problems, we (i) constructed a new integrative vector in which the gene encoding the integrase was deleted to increase its stability, and (ii) developed a new integrative vector carrying the gene encoding Pip to introduce a second copy of this gene in the chromosome. The use of these new tools was shown to reduce drastically the selection of escape mutants.

SUBMITTER: Boldrin F

PROVIDER: S-EPMC6453970 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Improving the stability of the TetR/Pip-OFF mycobacterial repressible promoter system.

Boldrin Francesca F Anoosheh Saber S Serafini Agnese A Cioetto Mazzabò Laura L Palù Giorgio G Provvedi Roberta R Manganelli Riccardo R

Scientific reports 20190408 1

Tightly regulated gene expression systems are powerful tools to study essential genes and characterize potential drug targets. In a past work we reported the construction of a very stringent and versatile repressible promoter system for Mycobacterium tuberculosis based on two different repressors (TetR/Pip-OFF system). This system, causing the repression of the target gene in response to anhydrotetracycline (ATc), has been successfully used in several laboratories to characterize essential genes ...[more]

PMID: 30962489

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Project description:The genetic elements regulating the natural quorum sensing (QS) networks of several microorganisms are widely used in synthetic biology to control the behaviour of single cells and engineered bacterial populations via ad-hoc constructed synthetic circuits. A number of novel engineering-inspired biological functions have been implemented and model systems have also been constructed to improve the knowledge on natural QS systems. Synthetic QS-based parts, such as promoters, have been reported in literature, to provide biological components with functions that are not present in nature, like modified induction logic or activation/repression by additional molecules. In this work, a library of promoters that can be repressed by the LuxR protein in presence of the QS autoinducer N-3-oxohexanoyl-L-homoserine lactone (AHL) was reported for Escherichia coli, to expand the toolkit of genetic parts that can be used to engineer novel synthetic QS-based systems. The library was constructed via polymerase chain reaction with highly constrained degenerate oligonucleotides, designed according to the consensus -35 and -10 sequences of a previously reported constitutive promoter library of graded strength, to maximize the probability of obtaining functional clones. All the promoters have a lux box between the -35 and -10 regions, to implement a LuxR-repressible behaviour. Twelve unique library members of graded strength (about 100-fold activity range) were selected to form the final library and they were characterized in several genetic contexts, such as in different plasmids, via different reporter genes, in presence of a LuxR expression cassette in different positions and in response to different AHL concentrations. The new obtained regulatory parts and corresponding data can be exploited by synthetic biologists to implement an artificial AHL-dependent repression of transcription in genetic circuits. The target transcriptional activity can be selected among the available library members to meet the design specifications of the biological system.

Project description:Bacteria frequently rely on transcription repressors and activators to alter gene expression patterns in response to changes in the surrounding environment. Tet repressor (TetR) is a paradigm transcription factor that senses the environmental state by binding small molecule effectors, the tetracyclines. However, recently isolated peptides that act as inducers of TetR after having been fused to the C-terminus of a carrier protein, suggest that TetR can also regulate gene expression in a signal-transduction pathway. For this shift in regulatory mechanism to be successful, induction of TetR must be sensitive enough to respond to an inducing protein expressed at its endogenous level. To determine this regulatory parameter, a synthetic Tet-regulated system was introduced into the human pathogen Salmonella enterica serovar Typhimurium and tested for inducibility by a peptide. Reporter gene expression was detected if the peptide-containing carrier protein Thioredoxin 1 was strongly overproduced, but not if it was expressed at a level similar to the physiological level of Thioredoxin 1. This was attributed to high steady-state amounts of TetR which was expressed by the promoter of the chloramphenicol acetyl transferase gene (P(cat)). Reducing P(cat) strength either by directed or by random mutagenesis of its -10 element concomitantly reduced the intracellular amounts of TetR. Sensitive and quantitative induction of TetR by an inducing peptide, when it was fused to Thioredoxin 1 at its native locus in the genome, was only obtained with weak P(cat) promoter variants containing GC-rich -10 elements. A second important observation was that reducing the TetR steady-state level did not impair repression. This permits flexible adjustment of an inducible system's sensitivity simply by altering the expression level of the transcription factor. These two new layers of expression control will improve the quality and, thus, the applicability of the Tet and other regulatory systems.

Dataset Information

Improving the stability of the TetR/Pip-OFF mycobacterial repressible promoter system.

Publications

Improving the stability of the TetR/Pip-OFF mycobacterial repressible promoter system.

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