Clostridia perfringens, or C. perfringens is a pathogenic bacterium well known to scientists as having the shortest generation time of any organism and well known to the general population as the leading cause of food poisoning. Its ability to rapidly repopulate makes it a promising research tool and perhaps allows it to redeem its poor reputation.
Description
Inactivating a gene in the C. perfringens chromosome by traditional mutagenesis methods is very difficult. Instead, Group II intron-based Targetron technology is used to disrupt the alpha toxin-producing (plc) gene. PCR amplification followed by sequencing of the flanking region of the plc gene shows that the Targetron is successfully inserted into the alpha toxin gene, and western blot analysis showed no production of the pathogenic alpha toxin protein in the culture supernatant of the plc-mutant. Thus, intron insertion is highly stable. Furthermore, plc-mutant is electroporatable and expresses high levels of foreign proteins from the cpe promoter cloned onto a shuttle plasmid, making it highly useful as a vaccine vector for the development of a vaccine against HIV and SIV. Advantages of this technology, such as site specificity, relatively high frequency of insertion and introduction of no antibiotic resistant genes into the chromosome, could facilitate construction of C. perfringens with multiple knockout mutations.
Applications
· Inactivation of toxin-producing genes in C. perfringens and other gram positive bacteria without introducing antibiotic-resistant genes
· Construction of a C. perfringens with multiple knockout mutations
· Development of vaccine delivery vectors for use in chemotherapy and vaccine development
Advantages
· High site specificity
· High frequency of insertion
· No introduction of antibiotic-resistant genes into the chromosome
· Intron insertion is very stable
Invention Readiness
Proof of concept – alpha toxin gene deactivated
IP Status
Research Tool
