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 reduced amount of T5 didn't really help (there were fewer colonies on those plates), so we will stick with our original recipe for Gibson mastermix.

Learning from positive control's success, Paul, Emmy and Andreas went ahead to concentrate the DNA fragments for the fluorescent construct. Paul did minivac, which evaporated some of the liquid from the existing tubes, while Emmy and Andreas worked on re-PCR the fragments from the existing fragments, and using the new gel extraction protocol. Running both solutions at the same time just show how desperate we are... So the minivac-ed fragments are gibsoned together, cells are transformed and plated, and we should know tomorrow if it has worked. The PCR attempt, however, yielded some interesting results: only the fluorescent proteins came out. However we have gotten rather used to the temper of PCR, so we will just try again on Saturday.

Tom performed a restriction digest on the Lux vector miniprep product- and yes, the restriction map was exactly what we expected. This is followed by a 5th attempt on second half of lux vector- and an unsurprising no. Tom designed sub-split primers and they should come in on Monday...

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

UK team meet-up! We had a great day in London meeting people from other UK teams and also hearing about their ideas. Everyone seemed to be working very hard on their projects, so it's good motivation for us to work hard too! Nonetheless it was a nice day out of the lab...

Emmy dropped by the lab on her way home from London to check on the ratiometrica cells- nothing. Not even the controls... but then, she's realized she has made the oldest mistake in the book- plating the positive controls on amp plates. So she replated some of the leftover culture on Kanamycin plates. We will know tomorrow... (perhaps we should engineer cells that grow extra fast for next year's iGEM project)

Saturday

The replated positive control on kanamycin plates worked- so the Gibson was working, which means it is possible a design problem. More research into the Gibson protocol tells us we should really not be using Gibson for any fragments shorter than 250bp in the danger of the T5 exo chewing the entire fragments away... which we have two. We will try to tackle the problem by putting in a smaller amount of T5 exo, or/and putting them in later (when the reaction mixture is very close to 50 degrees). Also, we found out that we should use 20x insert to vector. With all these changes, Emmy tried Gibson again with the minivac concentrated DNA. We will know if that has worked tomorrow.

Emmy re-attempted the PCR from Thursday, with increased DMSO concentration, slightly lower annealing temperature, and some of our new polymerase (velocity from bioline)... no bands at all, not even the positive control. It is probably a problem with the mastermix (see Lab book for details). And on a second look at the gel photos from Thursday- there might actually be some other products, but were mistaken for primer dimers. But nevermind- we will try again tomorrow...

Sunday

RATIOMETRICA!!!! We *might* finally have it. There are two tiny colonies on one of the plates from yesterday- though we still cannot tell if they are colonies or bubbles they are DEFINITELY COLONIES. Hopefully more will grow tomorrow.

Third attempt at PCR (even though we might have it already we are running out of the original DNA fragments... so it will be good if we have some more anyway)- not much luck, positive control was non-existent/very faint. Looks like we have to troubleshoot PCR again...