Biology Article Summary

The purpose of this experiment was to create a new plasmid (pMV158GFP), which lacks the malR protein and is mobile and has the GFP gene to be used to track the flow of genes between bacterial cells through conjugation. A plasmid with these qualities is necessary to create a plasmid that can be transferred to Gram-positive bacteria low in C&G (which are hard to transform with traditional means) by conjugation with other bacteria. Current vectors have the malR regulatory protein which imposes a problem because when active, the malM gene is not induced, so maltase is not utilized and the gfp gene is not expressed when maltose isn’t present (similar to our lab experiment when GFP is not expressed when arabinose is not present). other plasmids previously available were not capable of transforming Gram-positive bacteria that are low G&C, but were primarily used to track invasion of tissue cultures. Additionally, neither of the current plasmids are mobilized. Current vectors can only invade host cells and track host cell infections. Instead, this experiment uses vectors that can be conjugated between bacterial cells. This mobilization would be beneficial in order to track the transfer of genes between bacterial cells (in the case of this experiment, between Streptococcus pneumoniae cells and other bacteria). To construct a mobilized plasmid, the procedure starts with plasmid pLS1PMGFP (which carries the gfp under control of the pM promoter, which then controls MalM). Then, this plasmid replicates. The GFP source is detected from the plasmid by being grown on a 2% maltose medium (meaning if maltose is present, then GFP is expressed).
Next, a gene cassette with the pMV158-mobM gene was amplified by PCR and cloned into pCR2.1-TOPO. The resulting plasmid (PCR TOPO mob) contains the mobM gene (which has two restriction sites: Xhol and Smal). The pCR TOPO mob plasmid was digested with Xhol and Smal enzymes in order to isolate the mobM fragment. This digestion causes