[Study on gene knock-out in Mycobacterium BCG].
OBJECTIVE
To establish the methodology of plasmid for gene knock-out in Mycobacterium BCG.


METHODS
We designed two pairs of primers for amplification of MDP1 gene and inserted two fragments into pKO plasmid, and then the recombinant plasmid for MDP1 gene knock-out was obtained, and named pKO-MDP1. Gene exchange took place within the genome of BCG after pKO-MDP1 plasmid was transformed into Mycobacterium BCG. The strain of Mycobacterium BCG with MDP1 gene knock-out was selected, and the curve of growth rate was studied.


RESULTS
The target strain was that of the positive strain by two step PCR and one step sucrose counter selection, without growth in culture media with hygomycin. The value of A(600) per 12 hours was detected for sixteen days. A "S" shaped growth curve was detected. However, there was no significant difference in the growth rates between the wild type Mycobacterium BCG strain and the gene knock-out Mycobacterium BCG strain.


CONCLUSIONS
The plasmid of pKO was a useful tool for gene knock-out in Mycobacteria. MDP1 maybe one of the factors influencing the growth rate, but it was not the only one.
Chen
Xiao-you
XY
Department of Bacteriological Immunology, Beijing Tuberculosis & Thoracic Tumour Research Institute, Beijing 101149, China.
Li
Chuan-you
CY
Ma
Yu
Y
Liu
Chong
C
Wang
Jing-hui
JH
Zhang
Xue-feng
XF
Chang
Zeng-yi
ZY
D001500:BCG Vaccine; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D009163:Mycobacterium bovis / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010957:Plasmids; D011995:Recombination, Genetic; D014169:Transformation, Bacterial
D001500:BCG Vaccine; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C403010:MDP1 protein, Mycobacterium