Site-Directed Mutagenesis

Two types of mutagenesis were performed. One type was a silent mutation to eliminate internal biobrick cut sites (EcoRI, NotI, XbaI,SpeI, PstI) in the genes and the other type was to introduce a mutation in the gene that changes an amino acid in the final protein product.

In the first type, primers were designed in order to change a base pair in a codon without affecting the amino acid coded for by the codon (a silent mutation). In the second type, one base pair is changed to change a codon so that it codes for a desired amino acid while also introducing a non-biobrick cut site in the gene so that successfully mutated genes can be screened for by cutting with that enzyme. For this type it might be necessary to mutate more than one base pair to create the cut site.

Designing primers (based on stratagene quick change mutagenesis):

Two primers are designed in a way so that they are complementary to each of the DNA strands except for the one base pair that is going 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:

The next step is the PCR reaction. During the PCR the DNA strands dissociate, the primers bind to them and the DNA polymerase synthesizes the whole plasmid. In order to find out the right proportions, primer concentrations must be kept in excess and different concentrations of DNA template must be tried to find the optimum concentrations. We used the Kappa Hifi kit for the PCR reactions. The following illustrates how the PCR reactions were set up:

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.