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TetR-controlled recombination-based in vivo expression technology (TRIVET) vector

Invented by Stefan Schild
Invented at University of Graz
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A resolvase-based screen to identify in vivo repressed genes. (A) Schematic illustration of the genetic components of the TetR-controlled in vivo expression technology (TRIVET). Chromosomal sequences are highlighted in light gray, tetR-phoA-cat (tpc) cassette in black, integrated pTRIVET in dark gray, and the res cassette parts in open shapes. The tpc cassette is integrated into a V. cholerae hypothetical geneX via Tn10 mutagenesis, resulting in a transcriptional fusion of tetR and phoA to the chromosomal promoter of geneX. Subsequently, pTRIVET is integrated downstream of lacZ. Thus, the expression level of TetR via the geneX promoter dictates the expression of TnpR, which catalyzes the excision of the res cassette. The IS10 elements and constitutive cat promoter of the tpc cassette, the TetR-controlled tnpR, mobilization (mob), origin of replication (oriR6K), and ApR (bla) regions of pTRIVET, as well as the gene for KmR (neo), SucS (sacB), and the target sites of resolvase (res) of the res cassette are indicated. (B) Shown are the effects of iron availability on resolution frequency (red) and alkaline phosphatase (PhoA) activity (black) of strain Vc_res1_TRIVET irgA::tpc, as well as the relative expression of irgA (green) in WT determined by qRT-PCR. LB was supplemented with varying amounts of 2,2′-bipyridyl or FeSO4 as indicated to mimic low and high iron concentrations, respectively. Resolution frequencies and PhoA activities were determined after 8 h while samples for gene expression analyses were taken during the exponential phase. Data are presented as the median with interquartile range (n ≥ 6). Adapted from Cakar F, Zingl FG, Moisi M, Reidl J, Schild S. In vivo repressed genes of Vibrio cholerae reveal inverse requirements of an H+/Cl- transporter along the gastrointestinal passage. Proc Natl Acad Sci U S A. 2018 Mar 6;115(10):E2376-E2385. doi: 10.1073/pnas.1716973115. Epub 2018 Feb 20. PMID: 29463743; PMCID: PMC5877934.
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Catalogue Number 160861
Bacterial Resistance pTRIVET - ampicillin, tpc reporter cassette - chloramphenicol
Selectable Markers PhoA activity
Vector Type Reporter
Synonyms TRIVET
Antigen/Gene or Protein Targets Cholera
Relevance Vibrio cholerae gene expression
Research Area Bacteriology
Notes This system is based upon recombination-based in vivo expression technology (RIVET) and allows the detection of transient gene silencing events even in a subset of a complex, heterogenous bacterial population. It requires all three components; the tpc reporter cassette, pTRIVET and pTRIVET1. It can be used as a random approach to identify in vivo repressed genes during intestinal colonization and as a specific approach to study conditional transcriptional silencing of a gene of interest. The protocol for which can be found at PMID: 33659431.

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References: 2 entries

33659431

Cakar et al. 2018. Proc Natl Acad Sci U S A. 115(10):E2376-E2385. PMID: 29463743.

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References: 2 entries

33659431

Cakar et al. 2018. Proc Natl Acad Sci U S A. 115(10):E2376-E2385. PMID: 29463743.

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