Team:Cambridge/Diary/Week 8

Monday

Got the results from the transformations today. Guess what - they didn't work. Given the cells are definitely competent (the transformation of Jim's YFP DNA into the e.coli seems to have worked), the master mix may be suspect. However, the positive control plates we made yesterday of sfGFP were plated on the wrong type of antibiotic, so we'll change that before we assume anything too rash.

Tom miniprepped the lux plasmid out of the e. coli and tried to PCR the split backbone again- only half the vector came out. Interestingly, he tried to induce light in some of his overnight cultures of E. coli with the lux plasmid, but found that they don't glow.

In other news, Oli and Paul began thinking about how to tune our system using an outside parameter, for use with our standardized output system. It may be valuable to spend some time modelling such a system in visual GEC. See what we did here need to link.

Tuesday

Looks like our master mix for Gibson isn't working, given that only three colonies grew on one of the three control plates. We'll beg some more off Paul G. which should work, and retry the Gibson reaction with his master mix. If that doesn't work either, we'll have to look for something else to explain our failures...

The fluoride riboswitch plasmid sent to us was tested by Jolyon again, this time with a higher concentration of X-Gal to try and get a greater colour contrast for our pictures.

Paul also managed to get some of the tuning ideas up and running in GEC. Surprisingly enough, they do seem to show stability, though more so in the buffered system. If we ever manage to get Gibson to ever work, it will be excellent if we can make such a system.

On the lux construct front, we started to suspect that some of the colonies (which have been on agar plates in the fridge for almost a month now) have lost part of their plasmid. After consulting Jim H, we decided to do a patch test on agar plates containing arabinose. We should be able to see light on plates tomorrow.

Wednesday

No colonies today from the positive controls, either with Paul G.'s master mix or our master mix. So it looks like it might be a problem with the DNA itself. Emmy is re-running the PCR of the positive control fragments, but we will concentrate it more than usual using a modified gel extraction protocol, as suggested by Paul. We also try using a smaller amount of T5 exonuclease, as we read from a paper that the amount we are currently using is for 150bp overlaps while we only have 40. Fingers crossed, we should get some (admittedly useless) colonies tomorrow.

Light from cells on the arabinose plate! So they still have the lux plasmid afterall- looks like the lack of light from Tom's liquid culture might have been due to arabinose concentration problems. In the 4th attempt to PCR out the second half of the lux vector, Tom tried to use GC buffer- still no luck! What is going on...

On the instrumentation front, further improvements were made on the hardware to improve stability. Andreas and Paul would like to thank the personnel of the Instrument shop of the Engineering department for their help.

London approaches quickly, and we had to get on with the presentation. As learning experiences go, it was most informative. For example, we learnt that trying to do a presentation between eight people takes an extremely long time.

Oli also try re-running the β-galactosidase assay on the fluoride riboswitch, as the initial run gave rather variable results.

Thursday

Successes! The positive control has produced many colonies, now that we used more concentrated DNA. We'll change the protocol to reflect this.

The X-Gal assay also produced a beautiful Dulux™ colourwheel of blues.

Final preparation for London tomorrow! We had another long session discussing and fine-tuning our presentation, with many of our advisors present we were starting to feel the pressure! But their advice had been invaluable. Our poster is also printed and ready.

Friday