M Lertcanawanichakul | PubMed Health

PubMed2000

Improved shuttle vector for expression of chitinase gene in Bacillus thuringiensis.

A 6.96-kbp plasmid vector pBCX was constructed from the plasmid pBC16 (4.4 kbp) and a 2.56-kbp fragment of pBluescript II KS. The bifunctional plasmid pBCX conferred ampicillin and tetracycline resistance in Escherichia coli but only tetracycline resistance in Bacillus thuringiensis. It has unique sites for BamHI, SmaI, PstI, HindIII, SalI, XhoI, DraII, ApaI and KpnI derived from pBluescript II KS and was lost at a low rate in B. thuringiensis subsp. israelensis when cultured in Luria-Bertani broth without antibiotic. The chitinase gene from B. circulans number 4.1 (pCHIB1) was subcloned into the HindIII sites of this vector and designated as pBX43 (9.56 kbp). This plasmid produced three times as much chitinase in B. thuringiensis subsp. israelensis strain c4Q272 as pHYB43, which comprises the commercial shuttle vector pHY300PLK plus the chitinase gene.

Bacillus thuringiensis↗

PubMed1996

Expression of chitinase-encoding genes from Aeromonas hydrophila and Pseudomonas maltophilia in Bacillus thuringiensis subsp. israelensis.

Fifty isolates of chitinase (Cts)-producing bacteria were collected from soil samples and tested for their ability to degrade chitin using colloidal chitin agar as the primary plating medium. The results indicated that three isolates could degrade chitin at high pH. Further studies also demonstrated that crude Cts preparations from Bacillus circulans (Bc) No. 4.1 could enhance the toxicity of Bacillus thuringiensis subsp. kurstaki (Bt-k) toward diamondback moth larvae. Thus, it might be useful to increase the toxicity of B. thuringiensis (Bt) toward target insects by introducing a Cts-encoding gene (cts) into Bt. To investigate the expression of cts in Bt, cloned cts from Aeromonas hydrophila (pHYA1) and Pseudomonas maltophilia (pHYB1, pHYB2 and pHYB3) were cloned into the shuttle vector pHY300PLK and transformed into Escherichia coli DH5 alpha using 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside (4-MUF GlcNAc) as the detecting substrate. The four plasmids were then introduced into B. thuringiensis subsp. israelensis (Bt-i) strain c4Q272 by electroporation. Various transformants harboring cloned cts were selected, and expression and stability of the plasmids in Bt were studied.

Aeromonas hydrophila↗