Identification of Knock-Out Genes to Create a Temperature Sensitive Mutation in pMR10 Plasmids

Abstract

Brucella spp. is a gram-negative, cocobacillus, non-motile bacteria that causes a zoonotic disease called brucellosis. Cases of brucellosis remain prevalent in Middle Eastern countries, and it is the most common laboratory-acquired infection in the United States. It typically causes a mild infection but if left untreated it can lead to chronic problems in both humans and animals. There is currently no vaccine against brucellosis approved for human use; because of its highly communicable nature and global prevalence, one is needed. Finding the knock-out genes for temperature sensitivity in a broad-host-range plasmid would be an asset in developing a Brucella mutant for use in a vaccine. In this study random mutation with hydroxylamine was preformed in vitro on the broad host range plasmid pMR10. Mutated plasmids were then transformed into DH5-α competent cells and individual colonies were isolated and grown in nutrient broth. The broth was stamped onto agar plates and grown at selective (42°C) and nonselective (37°C and 30°C) temperatures. The plates were then screened for growth at the lower or non-selective temperatures and no growth at higher or selective temperatures. Of the 23,000 colonies screened in the project none were found to be temperature sensitive.