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

End of Week 2

It's hard to believe that my second week in the lab is almost over- safe to say it's flown by! It's been a fortnight of varying successes - but I guess an important lesson that one must learn as a research scientist is that things don't always work. Actually, quite often they don't work. But sometimes they do and it's ace.
Here's what I've been up to:

Week 1

Week 1 started fairly slowly, which gave me a chance to read up on several papers about research already carried on PcaK and the AAHS family. I began by culturing E. Coli which had been transformed with a plasmid containing the PcaK gene of the extremophile Haemophilus ducreyi, and then induced these via addition of arabinose (to turn on T7 RNA polymerase expression) and IPTG (to turn on protein expression). Unfortunately, the cells started to die on induction of protein expression - a possible explanation for this being that the protein is toxic to E. coli growth. This may lead to a low final concentration once purified, as it's possible that the cells that survived were those that were not expressing the protein!  I proceeded to harvest the cells via centrifugation and then resuspend the pellets in PBS buffer and TCEP (reduces disulphide bonds). Next I got to use a cell disrupter to lyse the cells and, after an inital centrifugation to remove debris and unlysed cells, I pelleted the membranes via ultracentrifugation. These were then resuspended in detergent.

HisTrap column for protein purifcation

From here the real fun began as I purified PcaK via a HisTrap column - this contains nickel Sepharose beads which bind the ten histidine tag that PcaK was expressed with. Having learnt about this process in undergrad lectures, it was interesting to put the theory into practice! After measuring the absorbance of the purified protein at 280 nm, I calculated that a final protein concentration of 0.27 mg/ml had been achieved - alas, too low for crystal trials.

As a final 'polishing step' we performed size exclusion chromatography

Size exclusion chromatography

which separates the column contents based on size. Ideally, one would see a single, well resolved peak (following the column void) corresponding to elution of the purified protein.
Unfortunately it seemed most of my protein eluted in the column void (this usually indicates aggregation) and due to the low concentration no visible peak was observed. Nevertheless, it was a good opportunity to use equipment I hadn't seen before. Finally, I ran an SDS PAGE gel of fractions from the purification process, which verified the low protein concentration I'd achieved - only a very faint band (see below) of purified protein can be seen on the gel.



All in all, a really interesting and enjoyable week. It feels like I've learnt a LOT already and I'm looking forward to what the next few weeks will involve.

 

 

Week 2

Dave the autoclave - my nemesis

The initial aim of week 2 was to express and purify PcaK from the extremophile Sulfolobus tokodaii by following a similar procedure used last week. However, due to contaminated media (uh oh) and a pesky autoclave with a handy habit of boiling dry I was set back a couple of days.
Luckily, one can never do enough reading so this gave me an opportunity to further research aromatic transporters and also look in more detail at the mechanisms of the equipment I'm using. I'm not in the lab tomorrow as am off to do the National Three Peaks Challenge (eek) so will update next week on how the purification process comes along.

For now, I'll leave you with this review (Cao, B., K. Nagarajan, and K.-C. Loh, Appl. Microbiol. Biotechnol.) on the biodegradation of aromatic compounds if you fancy a read - I think it offers a great insight into the mechanisms and possible applications of bioremediation.

A Brief Intro to PcaK

This summer I'll be spending eight weeks investigating the aromatic acid transporter PcaK under the supervision of Dr. Paul Curnow at the University of Bristol. I'll be using this space to document my progress/experiences/ramblings, but before I get started I figured some brief background info on what I'm actually doing may make it all a bit easier to comprehend.

What is PcaK?
PcaK is a bacterial membrane transporter that utitlises the proton motive force to import aromatic acids, primarily 4-hydroxybenzoate and protocatechuate. It belongs to the vast major facilitator superfamily as a member of the aromatic acid:H+ symporters, and the use of bioinformatics has identified PcaK homologues across multiple bacterial phyla. Interestingly, PcaK has also been shown to play a role not just in transport but also chemotaxis - though the mechanism by which it does so remains unknown. AAHS are thought to comprise of 12 transmembrane helices, with conserved sequences at the 2-3 and 8-9 cytoplasmic loops. The predicted membrane topology of PcaK from Pseudomonas putida can be viewed here (Ditty, J.L. and C.S. Harwood, J. Bacteriol., 2002.) which displays proposed hydrophobic regions, functionally important residues and conserved regions between the AAHS family.

Little is known for certain about the molecular structure and mechanism of PcaK so it's pretty exciting that I may contribute towards this!

What's the relevance of the research?
By understanding how aromatic acids are acquired from the environment we can gain further insight into the the degradation of aromatic compounds - this may lead to development of novel bioremediation strategies of aromatic pollutants. In addition, there are potential consequences for advances in biofuel production. The plant heteropolymer lignin, which is the most abundant aromatic compound on earth, may provide a favorable source for biofuels such as bioethanol if an efficient degradation and production method can be designed to do so.

The chemical structure of lignin - a significant constituent of Earth's biomass

I will initially be spending my summer project expressing and purifying two PcaK homologues from different extremophile organisms, which if successful will be used for crystal trials. Exciting stuff! Time to get started.

Ready to rock and roll

References and further reading
http://www.sciencedirect.com/science/article/pii/S1046592814001120
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC179362
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC93997/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3560178/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC134867/