JGG 1984, 11(6): 416-422 DOI:   ISSN: 1673-8527 CN: 11-5450/R           

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Clonig of Lys3+ Gene of Bacillus amyloliquefaciens in Bacillus subtilis

Guo Sandui, Men Dapeng, Jia Shifang

Institute of Microbiology,Academia Sinica,Beijing

Abstract

Chromosomal DNA of Bacillus amyloliquefaciens H AS.1.1099 and plasmid pUB-110 DN A were digested with restriction endonuclease EcoRI and ligated with T4 DNA liga se.Bacillus subtilis BR151 (trpC2 metB5 lys3 Neo s) competent cells were trans-f ormed by ligated mixture.Cells were plated onto complete medium containing 5μg/ ml neomycin.A total of 3,180 Neo r colonies were obtained.These colonies were pi cked picked onto minimal medium containing 5μg/ml meomycin and lacking the tryp tophan,meth-ionine,or lysine separately.74 resistant-neomycin and only complemen t BR151 lys3 mutation colonies were obtained.Agarose gel electrophoretic analysi s of plasmid DNAs from these clonies showed that 13 of them contained inserts of identical size.One of these recombinant plasmid,pB-L29,was studied further.Upon retransformation into BR151 competent cells and protoplasts,pB-L29 was found to confer upon the host resistance to neomycin and complement lys3 mutation indica ting that Lys3+ phenotype is associated with the inserted fragment.Electrophoret ic mobilities of plasmid DNAs isolated from the secondary transformants were ide ntical to pB-L129.It is a reliable proof that recombinant plasmid pB-L29 consist s of plasmid pUB110 with an insert of B.amyloliquefaciens H Lya3+ fragment at it s EcoRI site.According to this DNA fragment migration,it was calculated that the MW of pB-L29 is about 5.0 kb,and the MW of Lys3+ fragment is about 0.5 kb.

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Received 1900-01-01 Revised 1900-01-01 Online 1984-11-10 
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