The purpose of this experiment was to create a new plasmid (map GAFF), which lacks the mall protein and is mobile and has the GAP 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 mall regulatory protein which imposes a problem because when active, the mall gene is not induced, so maltase is not utilized and the gap gene is not expressed when maltose isn’t present similar to our lab experiment when GAP is not expressed when rabbinate is not present). Other plasmids previously available were not capable of transforming Gram-positive bacteria that are low G, but were primarily used to track invasion of tissue cultures. Additionally, neither of the current plasmids are embroiled.
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 inflammation would be beneficial in order to track the transfer of genes between bacterial cells (in the case of this experiment, between Streptococcus pneumonia ells and other bacteria). To construct a embroiled plasmid, the procedure starts with plasmid plus MPEG (which carries the gap under control of the PM promoter, which then controls Mall). Then, this plasmid replicates.
The GAP source is detected from the plasmid by being grown on a 2% maltose medium (meaning if maltose is present, then GAP is expressed). Next, a gene cassette with the umpped-mob gene was amplified by PC and cloned into APPC. 1-TOP. The resulting plasmid (PC TOP mob) contains the mob gene (which has two restriction sites: Cool and Small). The PC TOP mob lassie was digested with Cool and Small enzymes in order to isolate the mob fragment. This digestion causes this mob piece to become purified of NP, blab and PUC while retaining its own promoter.