Molecular
Biology Experiments Utilizing the lux Genes of Vibrio fischeri and
gfp Gene of |
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SDM of Dark Mutation of LuxR Gene
Introduction Site-directed mutagenesis (SDM) is a powerful tool that is used to discover the relationship of the protein structure to function. SDM can be used to introduce a defined mutation into a known DNA sequence. In the experiments described here, a single nucleotide of a gene (luxR or gfp) is mutated. The SDM technique used here is referred to as the overlap-extension PCR method. It requires three PCR amplifications and four primers. SDM of Dark Mutant of luxR Gene The plasmid pDV743 is a dark mutant of pHK724’s luxR gene. Again, the luxR gene is composed of 750 bp and therefore codes for 250 amino acids. The luxR gene of pDV743 has a single mutation at bp #589 (G-598 to A). This mutation converts amino acid #197 from glycine to arginine thus yielding a non-functional LuxR protein. SDM is used to convert the dark mutation Arg-197 back to wild type (Gly-197). The three PCR amplifications are outlined as follows: Note that Rx #3 uses the products of Rx #1 and #2. The only exponential amplification product of Rx #3 is (d.). This product is the wild type luxR gene. The luxR is then cloned into pUC 18 (Eco RI and Kpn I restriction sites).^top
Polymerase Chain Reaction Amplification Reaction #1 Amplification Reaction #2
Amplification Reaction #3 Amplification Program
Results This experiment accomplished the following: 1. PCR primers were selected. Summary This teaching exercise demonstrates the power, elegance, and simplicity of PCR. In addition to PCR, this system teaches students primer design, mutagenesis, cloning, transformation, plasmid and agarose gel electrophoresis.^top
©2009 James Slock, Ph.D |
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