Wednesday, 9 July 2014

Week 4 Update

My next two weeks in the lab have been a blur - there's lots to update on yet it does not feel as though I'm already half way through! After successfully scaling the three highest peaks in England, Scotland and Wales in 36 hrs (feel free to sponsor us here) it was straight back into the lab on Monday to continue the expression and purification of PcaK from Sulfolobus tokodaii in E.coli.
Celebrations in the clouds at the summit of Ben Nevis
Following some trial and error, expression was successful and the protein was purified via the same method described in week 1. A final concentration greater than that of PcaK from Haemophilus ducreyi was achieved, but unfortunately not high enough to proceed with crystal trials with this homologue. However,  I have now been given the responsibility of writing a protocol for the expression and purification process for future use by my supervisor.

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:
Mutagenesis products: M=DNA marker, C=control (no polymerase). Smears in lanes 3-5 suggest incomplete elongation products, potentially due to insufficient elongation time or an annealing temperature that was too low - this may have facilitated non-specific primer binding.

It's important to remember that no visible band on a gel after a mutagenesis reaction does not automatically indicate no product has accumulated. Quite often there is just not enough of it to visualise.
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.

Agarose gel showing successful excision of BenK gene from shipping plasmid via double restriction enzyme digest. This generates two non-complementary cohesive ends to prevent vector religation and insertion of the gene the wrong way round during ligation into pET28.
Next week we'll continue to investigate the AAHS family member BenK. We'll also be heading off to Oxford to visit my supervisor's collaborator to deliver purified Acinetobacter PcaK and initiate crystal trials. Hopefully there will be some exciting news to report! Until then here is one last photo from the Three Peaks Challenge - our view from the summit of Snowdon certainly made me question why I don't spend more time 3500ft above sea level...
Ain't no mountain high enough