Improved mycobacterial tetracycline inducible vectors.
We have previously reported on the development and assessment of the tetracycline inducible vector pMIND (Blokpoel et al., 2005). Here we report the development of improved pMIND vectors that exhibit both reduced basal transcription in the absence of inducer and increased fold induction in the presence of inducer. An amino acid change in the repressor protein, TetR(Z), produced a 6-fold reduction in basal transcription compared to the original pMIND-Lx and a 100-fold induction of LuxAB in the presence of tetracycline. An integration version of the improved vector (pMEND-Lx) was constructed which resulted in a 9-fold reduction in basal transcription compared to pMIND-Lx and a 17-fold induction of LuxAB in the presence of tetracycline. Further improvements were obtained by cloning the pMEND TetRO promoter into an alternative vector backbone. The resulting vector, pKW08-Lx, exhibited a 70-fold reduction in background compared to pMIND-Lx and a 230-fold induction of LuxAB in the presence of tetracycline. An integration version of pKW08-Lx was constructed and the basal transcription for this vector was zero; an 11-fold induction of LuxAB was observed in the presence of tetracycline. The construction of these improved mycobacterial vectors will prove extremely useful for genetic studies.
Williams
Kerstin J
KJ
Centre for Molecular Medicine and Infection, Room 3.40, Flowers Building, Division of Infectious Diseases, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom. kerstin.williams@imperial.ac.uk
Joyce
Graham
G
Robertson
Brian D
BD
D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D020102:Mycobacterium smegmatis / Q000235:genetics* / Q000378:metabolism; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology*
D001426:Bacterial Proteins; D012097:Repressor Proteins; D008156:Luciferases; D013752:Tetracycline; D004318:Doxycycline