Ralstonia eutropha H16 Soluble Hydrogenase complex:

- NiFe active site deeply embedded within protein meaning that ligands must pass through protein channels

- Oxygen insensitivity perhaps due to the active site having cyanides bound to the Ni/Fe

- contained on PHG1 mega plasmid

- HoxHY dimer (HoxH and HoxY genes) = hydrogenase

- HoxFU (HoxF and HoxU genes) = NADH-dehydrogenase

- HoxE subunit?

- two additional subunits (HoxI gene)

- FMN (Flavin mononucleotide) cofactor also essential

- hypX necessary for assembly of active site

- HoxI perhaps has a role in the activation of the enzyme by binding NADPH

- (S/T)RRxFxK leader causes peptide to be translocated to periplasm through the Tat mechanism

Maturation of SH:

- HypABCDEF(and F1) req'd for maturation of all NiFe h2ases

- SH also requires HypX (involved in binding of CN- to active site)

Project:

- is it possible to express functional SH in E coli by transfecting with the individual genes? Would be good for future purposes plus for screening (high expression!)

- If not can PHG1 be expressed in E coli?

- If not, use Ralstonia eutropha as host

Just had a stroke of inspiration:
could actually finish my degree halfway through next year! Do honours second semester.

Would only have to do 3 subjects:)

Climate symposium @ 9:30: topic = adaptation:
- contention: we already have ESD/disaster management/equity policies that achieve same aims as adaptation (up to a point: 4.5 centigrade warming). No point reinventing the wheel.
- need to be multidisciplinary and take examples from other disciplines

Presenting your research project workshop @ 10:30
- "Three reasons" structure
- Strong conclusion
- Stand up straight
- Open hands
- Positioning yourself and your audience
- Time management + communicate time "in the next 10 minutes.."
- Handling questions
- Appropriate level of complexity/language depending on auidence

Introducing a topic: 4 moves:
1. What discipline?
2. Previous research
3. Problem/controversy
4. Your research + hypothesis and how you will test it

Concluding: three moves
1. Results - was hypothesis supported or disproved?
2. What does this mean?
3. What will these results lead to: implications of research.

Accessibility:
- speak slowly and clip ends of words for ease of clarity (and especially if there are hearing impaired people in the audience)
- visual aids also important for this reason but focus should still be on the presenter not the powerpoint presentation

Talked with Robbie:
- Gene synthesis for $0.35/BP! I didn't know you could do that! Opens up a whole new range of opportunities.
Alternative: culture collection: eg. http://www.ncimb.com/results.php?parent=culture

Talked with Warwick: important point: h2ase requires oxidant to provide source of electrons

Researched different hydrogenases: e.g.
- Soluble Hydrogenase (SH) of R. eutropha - bidirectional. reduces NAD. oxygen insensitive (due to peculiar active site?)
- Hyperthermostable and oxygen resistant hydrogenases from Aquifex aeolicus: only H2 uptake not evolution, but is oxygen insenstive (not to do with active site but rather with the whole enzyme being very slowly inactivated by oxygen?)

One degree matters:
http://www.eea.europa.eu/cop15/bend-the-trend/one-degree-matters-movie

Phloem loading:
- http://www.sciencedaily.com/releases/2007/12/071221163216.htm
Sugar transport driven by polymerisation of sucrose molecules in leaves. If sucrose backs up, photosynthesis is paused/slowed down. Therefore, accelerating sucrose polymerisation could lead to faster ps rates

Cool technologies currently developed:
- ebook readers
- bidets
- power meter
- bheestie bag (dries out wet devices)
-

Ideas for the future

Below are what I see as game changing technologies that are currently very immature/entirely theoretical

CO2 sequestration:
- GM Rubisco with enhanced CO2 uptake efficiency -> super efficient trees

Farming:
- vertical farming (more efficient use of space, resources) -> aeroponics, GM crops
- growing native crops in Australia: e.g. Alpine rice (better drought tolerance, higher yields, higher protein content, gluten free)
- greening the deserts: permaculture, desal plants running off solar power

Energy:
- H2 production using artificial photosynthesis (oxygen evolving complex + hydrogenase)/photolysis
- Nuclear fusion

TILLING (Targetting Induced Local Lesions In Genomes) Technology for
Plant Functional Genomics
R. Soner SİLME, M. İlhan ÇAĞIRGAN http://www.nobelonline.net/UserFiles/File/PDF.pdf

Journal of Applied Biological Sciences 1 (1): 77-80, 2007

TILLING used to detect gene lesions obtained through chemical mutagenesis

Isolate DNA, PCR amplify using wild type primers.

Mutant and WT DNA will hybridise, but areas of difference will cause the DNA to loop out.

Add S1 nuclease, which will cleave mismatched strands.

Fluorescent end labelling will make it easy to identify mutant strands when run on a gel (look for RFLPs)




H2 directed evolution experiment:
Mutate cells
Transfer to microplate with cell lysis chemicals, universal pH indicator and an acid
H2ase activity will use up protons and result in colour change?

Another idea:
Transform E coli with combinatorially mutagenised H2ase. Grow in LB and then plate out on acid agar (pH 4.3). H2ase will convert protons to H2 and have protective effect?

Next gen sequencing:
Next-Generation DNA
Sequencing Methods
Elaine R. Mardis 2008
- blunt end ligate adaptor sequences to DNA fragments
- these adaptors can be used to PCR amplify the DNA, eliminating the requirement for a time consuming plasmid amplification step
- long read times: 8hrs to 10 days (shitloads of microreads of 25-30bp): each run could yield 10s of millions of sequence reads eg Illumina system or Applied Biosystems SOLID
- due to long read times without operator intervention and reduced preparation reqs, labour intensity = very low

Pacific biosystems:
- 'human genome in minutes for under $100'
- multiplex of ZMW fluoroscopy microcells: phosphonucleotides (4 different fluorophores attached to 3' phosphate group) are added to ZMW chamber with DNA polymerase.
- when a NT is added by DNA Pol, flurophore is excited and the emission spectrum measured by ZMW to determine which NT was added

Chromatin Immunoprecipitation Sequencing (ChIP-Seq)

Chromatin Immunoprecipitation: Look at epigenetic properties of genes (eg whether a particular gene is bound to histones and thus unlikely to be expressed): Crosslink DNA and DNA binding proteins using Formaldehyde; break the DNA apart by sonication; Add an antibody specific to the DNA binding protein (eg histone); run it all through an antibody affinity column; and tada: you have all the DNA that is bound to a particular DNA BP. Using next gen sequencing, this DNA can then be sequenced to find out which genes are affected by epigenetical modifications.

SAGE: (Serial analysis of Gene expression):
- SAGE can monitor levels of gene transcription by looking at mRNA levels
- each mRNA transcript can be adequately codified by a 'tag' that is composed of 26 nucleotides (SUPER SAGE using type III-endonuclease EcoP15I of phage P1) long enough for the sequence to be pretty much unique
- so you tear a bit off each mRNA (or more correctly cDNA) molecule and then, (and here's the beauty of the technique), you ligate all the tags together to form one long DNA strand and then sequence it! Heaps easier than PCRing each individual cDNA!

Ancient DNA sequencing:
- because NGS only requires single DNA molecules, degraded DNA from bones can be sequenced
- neanderthal genome is being sequenced through this method

Metagenomics:
- high throughput, low cost is making metagenomic studies possible.
- Can look at RNA present in a particular site (eg. acid mine) and look for genes involved in metabolisation of toxic/waste products

http://www.nature.com/nrm/journal/v3/n12/pdf/nrm975.pdf
"Protein engineering 20 years on" Brannigan J, Wilkinson A

Combinatorial mutagenesis:
- mix DNA fragments derived from multiple sources (eg. mutants/wild types, or closely related genes) and PCR it up. 'sexual PCR'
- SHIPREC (Sequence Homology Independent Protein Recombination) and ITCHY (Independent Truncation for the Creation of HYbrid enzymes) -> allows distantly related genes to be put together in chimeras. SCRATCHY (shuffling) then uses sexual PCR to further shuffle things up
- Random Insertion Deletion (RID) mutation? Remove arbitrary number of bases and replace with arbitrary number of bases?
http://www.nature.com/nbt/journal/v20/n1/full/nbt0102-76.html
- read "The blind watchmaker" and "The selfish gene" by Richard Dawkins
- phage display?
- application: artificial blood substitutes for blood transfusions (based on re-engineered Haemoglobin)
- long lasting insulin that doesn't clump together and lose its activity
- COMPLETELY NOVEL PROTEINS BASED ON UNNATURAL AMINO ACIDS: DESIGN NEW aminoacyle-tRNA synthetase enzymes to 'incorrectly' activate tRNAs!

http://en.wikipedia.org/wiki/Weasel_program
R Dawkins' Weasel program
Try to arrive at the phrase "Methinks it is like a weasel"
Purely by chance it would take a very long time (26^28 combinations). However, by coupling random changes with a 'fitness advantage' (ie. phrases that more closely resemble MIILAW are selected for), the change is possible

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