Proposed use
Conditional gene expression systems are powerful tools for studying essential genes in bacteria. Tetracycline and its analogues are ideal inducer molecules as they regulate gene expression at very low concentrations (nM range), show good bio-availability (ie. they can penetrate both mycobacterial and animal cells), are non-toxic at the necessary levels and are stable over the required length of study.
Problem addressed
There is a lack of genetic tools available to researchers to study the function of essential mycobacterial genes, we have therefore constructed a range of improved tetracycline inducible vectors. We have shown that changing the terminators upstream of the inducible promoter does not affect background transcription, and that repressor binding to the operator is a more critical step in controlling regulation. We have eliminated further read-through problems by changing the backbone vector used. The TetRO promoter can be induced in a dose-dependent manner by tetracycline, anhydrotetracycline and doxycycline.
Technology overview
| Plasmid | Description |
| pMEND-Lx | pMIND-Lx with Gly40 to Thr40 change in TetR protein; episomal |
| pMEND-Lx-Int | Integration version of pMEND-Lx |
| pKW08-Lx | TetRO promoter from pMEND-Lx inserted into the backbone of pSE100; episomal |
| pKW08-Lx-Int | Integration version of pKW08-Lx |
Additional information
These can be purchased for academic use directly through Addgene.
Publication
Williams KJ, Joyce G, Robertson BD. Improved mycobacterial tetracycline inducible vectors. Plasmid. 2010 Apr 29.
Inventor information
Dr Brian Robertson, Reader in Systems Microbiology, Department of Infectious Disease, Faculty of Medicine
