Micrococcus luteus crtE Plasmid
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Dormancy is a mechanism by which bacteria are viable but do not grow, called a Viable But Not Culturable (VBNC) state. Our lab has identified 18 distinct proteins that are up-regulated during dormancy; in order to further study these, we want to generate knock-out mutations in Micrococcus luteus and reintroduce mutated genes into the Micrococcus luteus genome. To this end, my project is to use the pigment operon (genes: crtE, B, I, E2, Yg and X) and use the first gene (crtE) as a neutral site to insert mutated gene sequences for analysis. I will describe here the amplification and cloning methods that I am utilizing to use the crtE gene as a neutral site for the insertion of mutated DNA into the Micrococcus luteus genome. For example, we want to insert green fluorescent protein into the neutral site to use as a biomarker for dormancy and resuscitation. This provides a rapid method for measuring the degree of resuscitation based off the return of the green fluorescence.