Construction of an Escherichia coli to Bacillus thuringiensis shuttle vector for large DNA fragments.
Shuttle vectors for Bacillus thuringiensis or Bacillus cereus usually cannot hold fragments larger than 20 kb. With the development of genome research, shuttle vectors with higher loading capacity are necessary. We constructed an Escherichia coli to B. thuringiensis shuttle vector, pEMB0557, with a large loading capacity. This vector incorporated the ori60 replicon from B. thuringiensis subsp. kurstaki YBT-1520, erythromycin resistance (B. thuringiensis), and chloromycetin resistance (E. coli) genes. A bacterial artificial chromosome library of B. thuringiensis strain CT-43 was constructed and pEMB0557 was able to accommodate at least a 70-kb DNA fragment. Simultaneously, the cry1B gene on a 40-kb fragment could express a 140-kDa protein in plasmid-cured B. thuringiensis BMB171. Due to its high capacity and utility in expressing exogenous genes, pEMB0557 will be useful in cloning (especially silencing genes) and expressing large DNA fragments (e.g., gene clusters) in B. thuringiensis. Plasmid pEMB0557 provides a new tool for B. thuringiensis genome or B. cereus group research.
Liu
Xiaoyan
X
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, People' s Republic of China.
Peng
Donghai
D
Luo
Yi
Y
Ruan
Lifang
L
Yu
Ziniu
Z
Sun
Ming
M
D001413:Bacillus thuringiensis / Q000235:genetics*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014169:Transformation, Bacterial
D004269:DNA, Bacterial