Morning: Did a mass spec run with thylakoid membranes and HC03 with the new thin membrane. Worked well but data was a bit fuzzy at end indicating that there might've been a bubble in there.

Then workshop from 10:30-12:30. Annie from Academic learning centre took the class and was really great. Very funny, knowledgeable and insightful. The session could've been boring and useless in the hands of a less capable person, but she really did a great job. Two big things I took out of it were:

1. Explaining my project to the person next to me. One of our tasks was to explain our projects using a template of "background, issue, solution, implications". So the idea was that the research project would be founded on a background of knowledge (background) that either had some kind of gap in it, or a controversy, or a problem. The research project would then attempt to address that issue, and the results of the project would have implications for future research/applications.

I found myself unable to explain my carbonic anhydrase assay (I really did not know what the purpose of doing these mass spec measurements was), but impressed my partner with my story of what the general research direct of the photobioenergetics group was (I interpreted it as being the quest for an artificial photosynthesis H2 production method).

2. Write an academic journal documenting your progress. I think this is a great idea (and am obviously doing one now). I find it really helps me understand things when I have to synthesise the ideas in my head and put them down on (virtual) paper. It'll also mean I can look back on my work months/years from now and recall what exactly I did. I plan on documenting pretty much everything: treating it like an electronic lab book. When I do reading, I'll do a bit of a summary in here too.

Afternoon:

Infused with vigour from the workshop (and the coffee), I 'upwardly managed' Warwick by sitting down with him and asking him to help me explain what the point of it all was. It was really good: I actually understand the purpose of doing these mass spec measurements now and feel a lot more motivated to do the work. He also indicated that since it's not going to take that much longer to do all of this, that I'd be able to do another mini project afterwards. I'm hoping it'll be something mol bio based: possibly expression of the recombinant BFR protein (or dare I hope a directed evolution experiment?).

Mini report draft:

- debate about whether bicarbonate has a part to play in photosynthesis

- initially thought by some to be the oxidative substrate that provided O2 and electrons. Now conclusion is that it is actually water

- possibly has a role in CO2 concentrating mechanisms in some algae or with photoprotection/PSII donor side efficiency

- reactions with HCO3 alone are very slow: need carbonic anhydrase to speed things up

- CA deficient mutant of algae used as model organism to examine role of CA/HC03

- Shutova et al have shown that there is a link between CA activity and O2 evolution as well as photoprotection by comparing the ca3- PSII mutant and the wild type. O2 evolution can't proceed until H+ ions removed, HCO3 buffering reaction allows this to happen and CA catalyses the reaction. O2 evolution rates shown to increase by 40% (back up to normal levels) in the mutant when exogenous CA + HCO3 are added.

- Other paper showed photosynthetic efficiency goes way down with far more rubisco molecules needed to assimilate the same amount of CO2

- However, actual CA activity has not been measured in this organism. This data is needed to provide more evidence that it is actually the CA activity that is having the effect, and not a protein-protein interaction etc.

- Experiment will use membrane inlet mass spectrometry (MIMS) to measure isotopic mass ratios of CO2 species released by the association of O18 labelled HCO3- and H+. Changes in O18 mass ratios in CO2 detected by the MS can be entered into a model to determine the rate of the equilibrium reaction.

- Background rate measured first and then subtracted from the rate constants for the CA3- PSII mutant and the wt PSII complexes to determine the level of CA activity

Lit reviewy stuff:

"HCO3- accelerates proton removal from PSII" 2008 T Shutova et al

Carbonic anhydrase (at donor side): CO2 + H20 <-> HCO3- + H+

Inverse relationship btw H+ removal and O2 evolution

Adding CA or HCO3- accelerates O2 evolution in CA deficient algal species

Evidence of CA binding to PSII complex

Probable role of HCO3- is as proton receptor: NR pH buffer also accelerates O2 evolution to same level as HCO3- and CA

CA/HCO3- deficiency -> photodamage. Assumed to be due to proton accumulation at PSII

Also evidence that CA actively stabilises proton concentration and thus optimises ATP production from H+ gradient

Hanson et al 2003 Plant Phys:

CAH3 located in thylakoid lumen

CA possible rule as Carbon concentrating mechanism

CA deficient plants require more rubisco to process same amount of CO2