GGS LIVE - Site Directed Mutagenesis

Hello BioFreakers!!

Today in the GGS - LIVE section the Site Directed Mutagenesis technique. Wanna see how it is done? Lets roll then.

Method: Site Directed Mutagenesis (SDM).

About: Allows for introduction of point mutations in DNA and thus sometimes in protein sequence.

What: Introduction of STOP codon in order to generate C-terminal deletion in protein X.

         Site directed mutagenesis is performed on circular DNA substrate. In our study case, the cDNA of protein X will be inserted into GST vector. Cloning of the protein X was performed analogously to study case described in a different post (click here GGS LIVE - Makinga fusion protein).

         To mutate protein X cDNA sequence we will need a set of primers that will be used in PCR reaction (GGS LIVE - Polymerase chain reaction (PCR)). SDM primers overlap with the target sequence, whereas regular PCR primers flank the target sequence (see cartoon below).

          The idea behind SDM is that each primer will mutate a single DNA strand of the pPLAS + X cDNA plasmid giving a product of the mutated pPLAS + X cDNA. Naturally, the template will be also present in the final mixture. Tamplate is removed by DpnI, a restriction endonuclease that digest modified DNA (DNA methylation). Such modification of the DNA occurs within bacterial cells, therefore SDM product will not wear it, as it was formed in vitro. After PCR reaction and overnight digestion with DpnI, SDM  reaction is analysed by agarose gel electrophoresis (see figure below).

         As you can see from the gel electroporesis analysis, desired product is present in both DpnI treated and untreated samples (R and R+D). Template +/- DpnI was used as a negative control. Additionally, amplification of 0.3kb fragment served as template control.
         You probably, wonder why SDM product and template are not observed as single band. This is due to different conformations of these plasmids. Template is supercoiled, therefore more packed and travels through gel faster and thus appears smaller on the gel. SDM product is not and therefore migrates slower.

         The R+D sample is then used to transform bacterial cells. In this process specific E.coli cells (for example Top10) uptake R+D plasmid and replicate it. This allows obtaining workable amounts of DNA, which is then send for sequencing. Several different E.coli clones are tested and when sequencing confirms mutation of pPLAS + X cDNA, DNA from correct clone is used to transform yet another specifc E.coli strain (for example BL21 pLysS).
          We then express X and X-truncation proteins using these BL21 cells (see figure below). For the protein expression in E.coli tutorial, please go here GGS LIVE - Protein expression in E.coli.

As you can see, this way we can generate a mutant protein KA-CHING. 

I hope you enjoyed it.

Maciek

GGSTEAM