Site-Directed Mutagenesis

We used two types of mutagenesis protocols. The first method introduces a silent mutation to eliminate a biobrick cut site (EcoRI, NotI, XbaI,SpeI, or PstI) in the gene of interest; the second method introduces a mutation in the gene to change an amino acid in the final protein product.

To introduce a silent mutation (first method), primers were designed in order to change a base pair in a codon without affecting the amino acid sequence (a silent mutation).

In the second method, one base pair in a codon so the codon now codes for a (new) desired amino acid while also introducing a non-biobrick restriction site in the gene. Successfully mutated genes can be screened for by cutting with the particular restriction enzyme whose site has been created. In some cases, it mmay be necessary to mutate more than one base pair to create the restriction site.

Primer Design

(Modified from Stratagene's QuikChange Site-Directed Mutagenesis Kit)

Two primers (forward and reverse) are designed to be complementary to the target gene sequences except for the base pair to be mutated. The primers must adhere to the following criteria:

1. The mutation must be in the middle of the primer with 10-15bp on each side.
2. The primers must be between 25-45bp.
3. The primers must introduce the same mutation.
4. The primers must have a GC content of more than 40% and they must end on each side with at least a G or C.
5. Primers must have a Tm of at least 78°C based on the following formula:

PCR reaction

During the PCR reaction, the mutagenesis primers bind to the dissociated DNA strands of the plasmid, and the DNA polymerase synthesizes the whole plasmid. To find out the right proportions of primers to plasmid DNA, primer concentrations is kept in excess and different concentrations of the DNA template are used to find the optimum concentration. We used the KAPA HiFi PCR Kits. The PCR reaction is set up as follows:

Transformation

10 µL of PCR products are run on a gel. If the amplification is observed, 1 µL of DpnI enzyme is added to the PCR tube directly and it is incubated at 37°C for one hour. DpnI enzyme degrades the methylated parental DNA. The newly synthesized DNA from the PCR is not methylated so it does not get digested by DpnI.

Afterwards, the PCR product is transformed into E. coli. 1 µL of the PCR product is added to 50 µL of competent cells, after which the regular transformation procedure is followed.

Screening

Make overnight cultures of colonies of the transformed cells. Afterwards, plasmid isolation is carried out, followed by digesting the plasmid with the appropriate enzyme for screening. Unmutated plasmid should be digested as a control for comparison. Digested products are run on gel electrophoresis in order to confirm mutations are present. Based on the bands observed on the gel the success of the mutagenesis is determined. It is also necessary to send the plasmid for sequencing since in some instances mutations or insertions happen. Insertions near the primer binding sites are probable.