Creation and characterisation of a high-copy-number version of the pAL5000 mycobacterial replicon.
The majority of mycobacterial plasmid vectors are derived from the pAL5000 replicon and maintained at approximately five copies per cell. We have devised a method that directly selects for high-copy-number plasmids. This involves enriching for high copy number plasmids by repeatedly isolating and retransforming plasmids from a mutant library. Using this method we have selected a copy-up version of the pAL5000 replicon. In Mycobacterium smegmatis the copy-number was shown to have increased 7-fold to between 32 and 64 copies/cell, and the plasmid remained relatively stable after 100 generations in the absence of antibiotic selection. The plasmid also has a high-copy-number phenotype in M. bovis BCG and can be used to increase expression of cloned genes, as we have demonstrated with the green fluorescent protein. The mutation was found to be the deletion of an alanine residue in the C-terminal end of the RepA replication protein. We argue that the mutation exerts its effect through altered RNA folding, thereby affecting the translationally coupled RepA-RepB expression.
Bourn
William R
WR
DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Health Sciences, Stellenbosch University, P.O. Box 19063, Tygerberg 7505, South Africa.
Jansen
Yvette
Y
Stutz
Helen
H
Warren
Robin M
RM
Williamson
Anna-Lise
AL
van Helden
Paul D
PD
D001483:Base Sequence; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009161:Mycobacterium / Q000235:genetics*; D009163:Mycobacterium bovis / Q000235:genetics / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D012093:Replicon / Q000235:genetics*; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D049452:Green Fluorescent Proteins