Molecular Biology Plasmids and Cloning

Plasmids

"Construction of the mobilizable plasmid pMV158GFP"

"Construction of the mobilizable plasmid pMV158GFP" is an article that describes the construction of a new, mobilizable plasmid, based on the pMV158 plasmid; but containing the gene which codes for green fluorescent protein (gfp) and is controlled by a maltose inducible promoter (Pm). The new pMV158GFP plasmid allows for bacteria which contain it to glow green under UV light, indicating whether or not the plasmid has been incorporated into, or mobilized, the Gram-positive bacteria. This new plasmid will allow for a better understanding of the processes and development of infectious bacteria in their natural environment.

Plasmids are circular, self-replicating pieces of DNA which are used to insert other pieces of DNA into cells and can be extremely useful in biological research. Nieto and Espinosa use one particular plasmid, pMV158, to create a new one which is designed for their specific needs. They intend to study the progress and development of infectious bacteria, but first needed to develop a mobilizable plasmid which could be used to mark specific ones. They did this by combining a well-known plasmid with new pieces of DNA which added a controllable marker to the plasmid. This marker was the gfp gene; a gene which could be induced into producing fluorescence in the presence of maltose. In other words, bacteria that contained the new plasmid, when grown in the presence of maltose, would fluoresce.

The authors began with two variants of the naturally occurring streptococcal plasmid pMV158, called pLS1GFP and plS1RGFP, which have the gfp gene under control of the malM gene. The malM gene negatively controls the production of the gfp gene's ability to produce fluorescence in the bacteria when grown in the presence of maltose. Unfortunately, neither of these two plasmids had the ability to mobilize into other bacteria, so the first thing the authors needed to do was to add this ability to the plasmids. They did this by taking the gene cassette containing the pMV158-mob M. gene, amplifying it through PCR, and cloning it into the pCR2.1-TOPO plasmid (Invitrogene). The mob M. gene should give the plasmid the ability to transfer, or mobilize, between bacteria. After isolating a purified DNA fragment containing the mob M. gene and its promoter, flanked by two restriction sites: XhoI and SmaI, it was fused to the pLS1PMGFP plasmid which had been digested with the AvaI restriction enzyme. In order to then be able to link the pLS1MGFP plasmid with the mob M. gene, the plasmid's overhanging ends were filled in with PolIK and then digested with the enzyme SalI This allowed for the mobM gene to be inserted into the pLS1PMgfp plasmid that had its erythromycin resistance removed, thus creating the pMV158gfp plasmid. "This plasmid has all the genes of its parental pMV158 and harbors the gfp gene under the control of the Pm inducible promoter." (Nieto, 2003, p. 283) In other words, this plasmid is the pMV158 plasmid but with a gfp gene under to the control of maltose inserted into it.

With the pMV158gfp plasmid created, the authors then had to test whether or not this plasmid had the ability to transfer between bacteria, or mobilize, and at what frequency, if any, this occurred. The plasmid pMV158 had previously been mobilized between pneumococcal and lactococcal strains of bacteria by means of incorporation into a separate auxiliary plasmid. The authors needed to use such an auxiliary plasmid to test whether their pMV158gfp plasmid could be transferred between three different species of Gram-positive bacteria: S. pneumoniae, L. lactis, and E. faecalis. They used pAM?1 as their auxiliary plasmid with the pMV158 and their pMV158gfp used as the replicons to be transferred. Nieto and Espinosa then used a newly developed mobilzation assay "based on the nutritional differences between pneumococcal and lactococcal" to select for transconjugants, or bacteria that have incorporated the new DNA into their cells. (Nieto 2003, p. 283) And by growing the bacteria in maltose, fluorescence could be used to test for the incorporation of the plasmids. According to the authors, all three strains of bacteria showed a measurable fluorescence "although cells of E. faecalis/pMV158GFP exhibited such a high level of fluorescence that the color was appreciable when colonies were illuminated with an UV lamp." (Nieto, 2003, p. 284)