Thursday, 19 June 2014

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.

Wednesday, 18 June 2014

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