Molecular cloning with bifunctional plasmid vectors in Bacillus

Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses

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435 68, 435253, 435317, 435839, 935 27, 935 56, 935 74, C12R 1125, C12N 1500, C12N 120, C12N 100, C12P 2100

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045956600

ABSTRACT:
A mutant of Bacillus subtilis has been isolated which greatly facilitates gene cloning in this nonpathogenic microorganism. B. subtilis is a known protein secretor and can be used efficiently in commercial operations. Unlike the more commonly used clone-propagating organism E. coli., B. subtilis has the advantage of lacking pyrogenic substances in its cell envelope. However, chimeric plasmids for infection of B. subtilis have been difficult to prepare, and if E. Coli is used as an intermediate host to provide plasmid forms suitable for Bacillus transformation, the B. subtilis treats any E. coli-propagated DNA as foreign and preferentially attacks the insert portion of the plasmid. This attach results in loss of cloned genes and limits the use of B. subtilis as a cloning system. The B. subtilis recipient strain of this invention is, on the other hand, stably and efficiently transformed by E. coli-propagated plasmid DNA at high frequency. The discovery of this mutant and the substantially higher frequency stable transformants obtained from it greatly facilitates the use of B. subtilis as a host for the expression of cloned genes.

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