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Team:UGA-Georgia/Plan
From 2013.igem.org
General Purpose and Goal
The purpose for this study is to determine if a gene for geraniol synthase and acetyltransferase can be properly expressed in the archaea Methanococcus maripaludis via a plasmid shuttle vector. Successful transformation will result in Methanococcus maripaludis producing geraniol from geranyl diphosphate, an intermediate in a pathway of lipid biosynthesis, creating a rose-like odor.
This research will create Methanococcus maripaludis that will produce a rose-like fragrance. Salt marshes tend to have a very distinct and bad smell from the production of methane from methanogens. If Methanococcus maripaludis can successfully produce geraniol then these marshes can be made to have a nice floral fragrance. The production of geraniol is made possible by the successful expression of a gene coding for geraniol synthase. Geraniol synthase produces geraniol from geraniol diphosphate which is an intermediate in a lipid biosynthesis pathway. To express the geranyl synthase gene a plasmid shuttle vector was created. This was a continuation of a project done last summer and geranyl synthase colonies produced in during prior were screened. As expected the results came back negative. New colonies of Methanococcus maripaludis with the acetyltrasferase gene and geranyl synthase plus acetyltransferase gene were created. The second part of this experiment involved transforming the geranyl synthase gene into Escherichia coli (E. Coli) which was successful. Currently we are working on the transformation of mcherry which codes for a fluorescent protein into E.Coli. We will use mcherry to check geranyl synthase in later research over the summer.
The purpose of this research is to successfully transform the geraniol synthase gene into the archaea Methanoccocus maripaludis with high levels of expression. M. maripaludis is an anaerobic methanogen, and was the predominant methanogen isolated from a salt-marsh in South Carolina. The ideal living condition for M. maripaludis is in moderate temperatures, usually around 20-45°C. It has an irregular cocci shape and utilizes flagella for movement. M. maripaludis is an ideal research candidate because of its rapid, reliable growth rate, effective genetic tools and complete genome sequence. (Whitman et al., 1997)
Genetic transformation will be accomplished through the use of a plasmid shuttle vector, pAW50. The DNA sequences encoding for geraniol synthase and mcherry will be added to the pAW50 vector and transformed into E. Coli. The sequences were slightly altered for better expression in M. maripaludis by way of codon optimization. This technique involves using the most frequent base pair triplets which appear most often for a certain codon in the M. maripaludis genome. After the genes are inserted into the vector they are transformed into cells of the E. coli strain XL-1 Blue to replicate the plasmid. The E. coli cells will then be lysed to extract the plasmids for transformation into M. maripaludis. The plasmid will also contain a gene encoding for a resistance to the antibiotic puromycin. This will serve as a selectable marker in order to confirm successful transformation of the plasmid. Cells which have not taken up a plasmid will die when grown on media that contains puromycin.
Over the summer the team failed to get expression of geraniol. This semester we retested those results, inserted the geraniol synthase with the pAW50 plasmid into E. Coli and inserted the mcherry gene into E. Coli with the pAW50 plasmid.
