Factors affecting transformation efficiency of BCG with a Mycobacterium-Escherichia coli shuttle vector pYUB18 by electroporation.
BCG has been one of the vehicles for multi-recombinant vaccine. However, low transformation efficiency of BCG with plasmid DNA hampered studies involving expression of foreign antigens in BCG. In an effort to determine the optimal conditions, this study was initiated to investigate factors involved in the transformation of BCG with a Mycobacterium-Escherichia coli shuttle vector, pYUB18, by electroporation. Mycobacterium bovis BCG (strain 1173P2) was grown in Middlebrook (M) 7H9 broth containing albumin-dextrose-catalase and 0.05% tween 80, and transformed BCG was grown in M7H10 agar containing kanamycin for counting viable cells. Pretreatment of BCG with 10 mM CaCl2 improved the transformation efficiency, but overnight incubation of BCG with 1% glycine did not. The transformation efficiency in BCG also varied depending on voltage, resistance, and DNA concentration. The maximum transformation efficiency was obtained when the infinity resistance, 12.5 Kv/cm, and 100 ng of DNA were used, and reached 1.4 x 10(5) CFU/microgram of plasmid DNA, which is about 3-100 times greater than those from previous reports. The transformation conditions described in this study, therefore, will give us a better position for employing BCG as a vehicle for developing multi-recombinant vaccines.
Cho
S N
SN
Department of Microbiology, Yonsei University College of Medicine, Seoul, Korea.
Hwang
J H
JH
Park
S
S
Chong
Y
Y
Kim
S K
SK
Song
C Y
CY
Kim
J D
JD
D002122:Calcium Chloride / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004594:Electrophysiology; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D005998:Glycine / Q000494:pharmacology; D009161:Mycobacterium / Q000235:genetics*; D009163:Mycobacterium bovis / Q000235:genetics*; D009994:Osmolar Concentration; D014169:Transformation, Bacterial / Q000187:drug effects / Q000502:physiology*
D004247:DNA; D002122:Calcium Chloride; D005998:Glycine