Identification of Knock-Out Genes to Create a Temperature Sensitive Mutation in pMR10 Plasmids
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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.
Murphy, Sarah (2011). Identification of Knock-Out Genes to Create a Temperature Sensitive Mutation in pMR10 Plasmids. Texas A&M University. Available electronically from