|Celebrations in the clouds at the summit of Ben Nevis|
Across these two weeks I've also had a chance to explore some molecular biology techniques - something I've definitely enjoyed. Being able to put lecture content (and things i'd never heard of!) into practice in a research based environment has been ace.
The first of these was site directed mutagenesis via the Quikchange method. I was given the task of designing forward and reverse oligonucleotide primers to alter residues on Acinetobacter PcaK that are hypothesised to be important for transport. When designing these, one must consider a variety of factors including optimum primer length, melting temperature and %GC content. Once desgined and ordered, we were ready to proceed with mutant strand synthesis via thermocycling to denature the DNA, anneal mutagenic primers and then extend them using a thermo-stable polymerase. The methylated template plasmid was then digested by dpn I endonuclease. The products of the mutagenesis were visualised on an agarose gel:
The DNA was then used to transform competent cells, which were cultured on LB agar plates, and individual colonies used to innoculate overnight cultures. Only the R131A mutant grew - suggesting this was the only successful quikchange mutant. Short products of the plasmid (generated in the other reactions) will not contain the gene for kanamyn resistance so will be unable to grow on kanamycin media. This mutant has been sent for sequencing, and my next steps will be to alter and optimise the thermocycling conditions (elongation time and annealing temperature) of the other mutations, and if this fails then we will re-design the primers. I am learning the importance of trial and error!
I have also initiated investigations of another member of the AAHS family, the benzoate transporter BenK. After successfully excising it from the plasmid it arrived in via a double restriction enzyme digest (see gel below) it has been ligated into the vector pET28 and this has been used to transform competent cells, ready for expression and further analysis next week.
|Ain't no mountain high enough|