The Xanthophyll Cycle in Photosystem II

Thursday, 29 May 2014

First day in labs!

Oh the excitement, I actually didn’t know what to expect! I’ve been in labs before obviously, but this was a project of my own now! Where I would be the only student doing this and basically discovering new results for my supervisor!

So starting at 9am and ending at 6pm, I can honestly say it was a long day! Obviously I had a lunch break and everything, but still very long!

Thing is with these types of experiments, there is a lot of centrifuging and treatment that needs to be done in order to get the final, correct medium that can be finally analysed via the chromatography machine- FPLC: fast protein liquid chromatography. In our experiment we are basically only wanted to separate the different supercomplexes in the photosynthetic membrane; to mainly get the fraction of photosystem II, so we can further analyse the pigments in the photosystem II via HPLC: high performance protein liquid chromatography that are involved in light stress. Obviously, understanding the underlying mechanisms of the photosystem II will really aid in my future plans of further being a researcher in artificial photosynthesis J

Anyways here is photos to explain the day:
This is a device which basically spins the spinach around in hand controlled speeds, hence drying washed spinach rapidly! We obtained the spinach fresh from Whitechapel market in the morning.

The nitrogen tank! Looks so scary :3 It actually looks like a nuclear bomb or something in real life.... stuff which i just generally think should have never been created in this world; we should all try to live in harmony, but sadly this is not the case in our world right now :( Anyways, the technician showing me how to switch on the supply/ off.
This is me chopping up the stems and midrib of the spinach preparing them for light treatment. I was initially quite slow as I didn't want to damage the spinach leaf and lose good material.

The spinach leaves ready for light treamtment.

The spinach leaves covers in cling film and starting light treatment.

Measuring the light intensity for the light treatment~ it was between 450-500 micro Eisenstein. This device is so cool it like an botanist stethoscope, haha!

Further measuring of light intensity and you can see the other plants grown by other researches at Queen Mary, in the plant room :)

Reading through the instructions and adding extra notes for next time.
While waiting for the centrifuge cycle, preparing of the buffers and mediums ready to add to the pellet of the centrifuge tubes.
The osmotic medium added after the break medium which breaks the photosynthesis membrane into smaller pieces, the break medium is added for 30 seconds, before the osmotic medium is added.
A centrifuge! You always learn about them throughout education in science. I always never really understood the true importance of them, till i actually used them in this studentship. They are such amazing devices and I've learnt to absolutely love this one in Ruban's lab! :D
Centrifuge walkthrough, left to right- Time: 1.41 seconds remaining of the spin, Rotations per minute: 4000, Temperature: 4 degrees and not sure about what the 9 means, think its pre-set!
Love my university lab coats, how they embroided the logo on every single one. Luckily for me X-smaller is in the colour of red and i love the colour red :) It's chinese lucky colour! However, if I'm honest pink is my favourite, it just makes me feel so happy and girlie, hahah...
Taking buffer from the FPLC machine~which we need for further steps in the instructions.

Other picture of the FPLC machine, looks pretty cool~ overwhelming at first.

So basically watching the different separation of fractions in the FPLC on the PC screen. The different wavelength of lights indicate what is observed. As we know wavelength of 670λ are of chorophyll A, 480λ is for both chorophyll A and B and pigments, 280λ gives proteins.
So by the end of the first full day in lab I have completed pretty much nearly all the steps in the protocol plan for the 8 weeks! I was initially worried if I would have enough time in the 8 weeks to do it all, as I didn't know anything about how long each step will take. However, after today I understand the whole project alot more! That 8 weeks is plenty time to get really nice results in this project. That mainly the 8 weeks will be analysing the fractions that I obtain from solubilisation, to find the efficiency of Violaxanthin de-epoxidation in PSII supercomplex. 

I am quite excited to move onto outline step B. I always think working with mutants is interesting as they can clearly indicate the purpose of certain complexes in each organisms.

Tomorrow I shall be analysing the results obtained from FPLC

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