[A new resolution vector with cry1Ac10 gene based on Bacillus thuringiensis transposon Tn4430]

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Bacillus thuringiensis (Bt) is a key biological pest control agent.
• Development of Bt-based biopesticides faces regulatory hurdles, often related to foreign DNA like antibiotic resistance genes.
• Efficient methods for genetic manipulation of Bt are crucial for improving biopesticides.

Purpose of the Study:

• To develop a novel resolution vector for creating genetically clean Bt strains.
• To eliminate antibiotic resistance genes and non-Bt DNA from Bt strains.
• To enhance the potential for regulatory approval of Bt biopesticides.

Main Methods:

• A resolution vector, pBMB801, was constructed using the TnpI-mediated site-specific recombination system of Bacillus thuringiensis transposon Tn4430.
• The cry1Ac10 gene (insecticidal protoxin) and ori1030 gene were inserted into RES sites on the vector.
• The vector was introduced into a crystal-negative Bt host (BMB171) for selective DNA elimination.

Main Results:

• The developed system successfully eliminated antibiotic resistance genes and other non-Bt DNA from the host strain.
• The resulting recombinant plasmid (pBMB801) demonstrated high stability, even without antibiotic selection pressure.
• The final Bt strain contained only Bt DNA, rendering it free of antibiotic resistance markers.

Conclusions:

• The TnpI-mediated site-specific recombination system provides an effective strategy for generating genetically defined Bt strains.
• This method facilitates the removal of undesirable DNA, simplifying the genetic makeup of the biopesticide.
• The resulting clean Bt strains are more amenable to regulatory approval for commercial biopesticide applications.