Cloning of the Brucella gene suspected to express a DNA protection during starvation protein

Brucella is described briefly as an aerobic, gram negative, non-motile prokaryote. The disease caused by Brucella when infection occurs in the host is termed brucellosis. DNA protection against starvation proteins, Dps, have been discovered in several prokaryotes, such as Escherichia coli and Agrobacterium tumefaciens. The role of Dps protein in general is to protect a cell exposed to starvation conditions. The Dps protein enables the bacteria to maintain its stationary phase in a harsh host environment and thus maintain virulence. Recent research of Brucella suggests the existence of a Dps protein. The goal of the research was to clone the gene of the suspected Dps protein in Brucella melitensis. The Dps gene was cloned to allow functional studies of the proposed Dps protein, such as DNA binding. The Brucella genomic DNA was acquired and polymerase chain reaction, PCR, was the technique utilized for isolation, amplification, and purification of the target DNA. Specifically designed primers were used for accurate replication and to introduce mutations for restriction enzyme sites into the gene of interest. These sites were utilized in the digestion and ligation of the vector, pET-16b, with the insert, suspected Dps gene. A restriction enzyme, R.E., digest was then performed on the insert gene and the vector, pET-16b, separately under the same conditions using the restriction enzymes Bam HI and Nde I. A QIA PCR purification kit was used to deactivate the enzymes and purify the product from unwanted fragments in the insert digest. The amount of digested insert and plasmid present in the samples was analyzed by agarose gel electrophoresis, AGE., and comparison of band intensities to ladders of known concentration. The digested vector and target gene were then combined together via ligation. The competent host cell, BL21(DE3), was transformed with the recombinant DNA under appropriate laboratory conditions. The transformants were then selected by plating on media with ampicillin that only allowed the successfully transformed cells, which would express the ampicillin resistance gene, to grow. Further, the transformants were induced to allow expression of the gene of interest and the proteins present after induction were analyzed by SDS PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, to separate the proteins based on size and visualize the protein of interest