Genetic transformation of Chainia and heat attenuation of its restriction system.
A PEG-mediated transformation system for Chainia (NCL 82-5-1) was developed using a broad host range Streptomyces vector, pIJ702. Protoplasts prepared from Chainia (NCL 82-5-1) were regenerated with 5% efficiency. Transformation of the protoplasts with pIJ702 gave 10-20 transformants/micrograms DNA. The low efficiency of transformation is attributed to a restriction system in Chainia; this could be inhibited by treating the protoplasts at 42 degrees C for 10 min just before transformation. The yield of transformants increased 100-fold when pIJ702 was modified by passage in Chainia. Because the plasmid replicon was functional in Chainia and the modified plasmid was stably maintained, the transformation system should be useful for self-cloning in Chainia NCL 82-5-1 of the many commercially important enzymes this strain is known to produce.
Chauthaiwale
V M
VM
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Vyas
P R
PR
Deshpande
V V
VV
D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006358:Hot Temperature; D010957:Plasmids / Q000235:genetics; D011092:Polyethylene Glycols; D011523:Protoplasts / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics*; D013696:Temperature; D014169:Transformation, Bacterial / Q000235:genetics*
D011092:Polyethylene Glycols; D004262:DNA Restriction Enzymes