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TransFLP — A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination
Published on: October 8, 2012
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A modular chromosomally integrated toolkit for ectopic gene expression in Vibrio cholerae
Triana N Dalia1, Jennifer L Chlebek1, Ankur B Dalia2
1Department of Biology, Indiana University, Bloomington, IN, 47405, USA.
Scientific Reports
|September 22, 2020
Summary
We developed a new gene expression system for Vibrio cholerae, enhancing control over gene activity. The PBAD-riboswitch system offers tight regulation with no detectable promoter leakiness, ideal for bacterial genetic studies.
Area of Science:
- Microbiology
- Molecular Biology
- Synthetic Biology
Background:
- Ectopic gene expression in bacteria is crucial for research and industry.
- Regulated promoter systems require tunable induction and minimal basal expression (leakiness).
- Vibrio cholerae research benefits from advanced genetic tools.
Purpose of the Study:
- To create and assess a modular, chromosomally integrated platform for ectopic gene expression in Vibrio cholerae.
- To compare the performance of established promoters (Ptac, PBAD) with their riboswitch-modified versions.
- To evaluate both the inducibility and leakiness of these novel promoter constructs.
Main Methods:
- Constructed and integrated promoter variants (Ptac, PBAD, Ptac-riboswitch, PBAD-riboswitch) into the V. cholerae chromosome.
- Utilized a sensitive reporter system to quantify gene expression levels and promoter leakiness.
- Assessed promoter performance based on induction levels and absence of basal expression.
Main Results:
- All tested constructs showed unimodal, titratable gene expression.
- Maximum induction varied: Ptac > PBAD > PBAD-riboswitch > Ptac-riboswitch.
- Promoter leakiness followed: Ptac > Ptac-riboswitch > PBAD; PBAD-riboswitch exhibited no detectable leakiness.
- The PBAD-riboswitch system demonstrated utility in studying dynamic gene activity in V. cholerae.
Conclusions:
- The PBAD-riboswitch system provides highly regulated, inducible ectopic gene expression in V. cholerae with zero leakiness.
- This modular, chromosomally integrated toolkit enhances genetic manipulation capabilities for V. cholerae.
- The platform is adaptable for use in other bacterial species, broadening its applicability.
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