Development of a novel Bacillus subtilis cloning system employing its neutral protease as screen marker.
Part of the pUC19 polylinker sequence (33 bp) was inserted into the pro-peptide-coding region of the Bacillus subtilis neutral protease-encoding gene to replace a 93-bp FspI-HindIII fragment. This in-frame sequence replacement had little effect on the expression and secretion of the neutral protease. This plasmid can therefore be used as a cloning vector, and recombinant clones can be directly identified on skim milk indicator plates by the loss of a clear ring (or halo) around the colonies. This novel cloning system offers several advantages over existing B. subtilis cloning vectors: (i) convenient direct screening of recombinants; (ii) the use of inexpensive indicator; (iii) no restriction on the use of host strains; and (iv) the availability of seven frequently used unique cloning sites: BamHI, XbaI, SalI, PstI, SphI, HindIII, and EcoRI. This system also has the potential to be used as an expression/secretion vector.
Wu
Z R
ZR
Department of Biology, East China Normal University, Shanghai.
Qi
B J
BJ
Jiao
R Q
RQ
Chen
F D
FD
Wang
L F
LF
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011995:Recombination, Genetic
D015500:Chloramphenicol O-Acetyltransferase; D008666:Metalloendopeptidases; C044194:microbial metalloproteinases