Team:Cambridge/Diary/Week 9

Monday

Tested the Ratiometrica cultures for YFP fluorescence and none visible yet... However, we remain optimistic and our current theory is that the YFP we are using produces a very poor signal. Also, there may have been some CFP fluorescence (Although this is hard to tell as there is some autofluorescence of the cells under this excitation) meaning we might have put our proteins int he wrong way around (which doesn't matter too much thankfully). In order to know for sure whats going on we restreaked one of our colonies to grow up over night in preparation for a restriction digest and IPTG induction test.

Meanwhile Andreas and Stuart started deciphering Jolyon and Oli's information for turning the riboswitches into BioBricks. Andreas also continued work on the Arduino software. Paul and Andreas also started construction of the cuvette holders for the hardware part of the instrumentation.

Tuesday

Mylar reflective film has arrived and the first cuvette holder has been completed!

Ratiometrica still not showing any YFP fluorescence (CFP hard to tell) but some of the restreaked cultures have now been place in amp resistant liquid medium for growth in preparation for IPTG induction. Some cultures were also prepared for a restriction digest tomorrow!

Emmy ran a positive control on the PCR machine with an increased loading dye concentration and despite producing the weirdest gel I've ever seen, did locate the band we wanted! PCR back on track! In light of this, Stuart and Andreas started a PCR on some B. subtilis, the fluoride riboswitch template and the Magnesium riboswitch template. Unfortunately they only discovered today that they require gibson primers for the linearised standard vector for the registry so the entire construct will be a little delayed.

PCR results: Due to lack of primers only the colony PCR and the two halves of the Mg RS vector could be run. Only the smaller fragment of the vector showed on the gel! After diagnosis we concluded that we'd used completely the wrong protocol for colony PCR and that it was a 5.5kb fragment that didn't run on the gel for the Mg RS vector was difficult PCR conditions right.

We also discovered we'd been using 10 times as much primer as we needed in all our PCR reactions which resulted in a severe lack of primers today! more delay as we order more!

Wednesday

Emmy ran gels for ratiometrica and the lux-mOrange fusion. Of these we managed to extract the smaller fragments for the fluorescent construct and got a quarter of the vector for the lux construct... slow progress is being made! just one quarter of a vector and two more fluorescent fragments to get and we'll all be set for gibson (which always works...). As the band wasn't too clear for the lux construct Emmy has rerun the pcr for this and also tried to get the other quarter of the vector we're still missing. Funny enough only the other quarter came out this time- PCR is indeed very temperamental. But we have kind of got all the fragments we want now- a verification PCR will be done tomorrow to make sure these are actually what we want.

Andreas designed some primers for the amplification of linearised plasmid backbone of Gibson, sent them to Oli and they are ready to be ordered. In addition, Andreas made new diluted to 10mM primer stocks, so that the protocol used can be followed exactly (i.e. 2.5 μl primers in each tube). He also progressed with the PCR reactions of the Fluoride riboswitch aimed to be made into a biobrick by standard assembly, however this did not work. What we might need to do now is to do the PCR again with shorter elongation times, as Emmy thinks this is the problem. Furthermore, he did the colony PCR with the right protocol (i.e. Taq polymerase was used as an enzyme in the master mix instead of Phusion), yet this did not work either. Only the positive control worked in those PCRs unfortunately.

Stuart meticulously finalised his list for sporulation medium and aims to have the ingredients by friday. He also did a major (really?) rehall on our freezer stocks as things were getting a little unorganised.

Paul had lots of fun designing his own protocol for IPTG induction of ratiometrica in E.coli but has to wait a night to see his results - These are unlikely to be positive and not just because of Paul's ineptitude but mainly because we're pretty sure this isn't actually ratiometrica. This is because today Emmy also mini-prepped our ratiometrica cells and did a restriction digest - results were inconclusive but its starting to look like our two colonies were the result of a transformation of the template vector that might have got into the gel well we extracted! A more elaborate restriction digest will tell us more. Meanwhile there's no harm in attempting gibson again!

A PLAN Of ATTACK FOR RATIOMETRICA HAS BEEN DEVISED:

1) Perform 3 2-piece gibsons and then piece these together in a 3-piece gibson (done, will know tomorrow)

2) PCR the two smaller fragents from the 3 pieces and gibson with the two vector fragments in a 4-piece reaction

3) PCR up the 3 fragments as well to be used in future gibsons (in case the above doesn't work)

4) Gibson together each of the 2 small fragments with halves of the vector and then do a final 2 piece gibson

Even if all this fails we will have learnt a lot!

Thursday

Verification PCR was carried out for all the lux-mOragne construct fragments- we ran samples in triplicates and most of them came out- so we take it that we have finally got all our fragments and are ready for Gibson! Learning from Ratiometrica that Gibson is not as simple as we thought it would be, this time we are extremely prudent about our plan of attack. Anyhow- we thought it doesn't hurt to first try a 4-piece Gibson anyway, we will know the results tomorrow.

Meanwhile, we get on with our Gibson plan of attack for the fluorescent Ratiometrica. We are running low on the vector backbone fragments, so we thought we would PCR more out from the existing pJS130 template. PCR is as temperamental as usual for long fragments- it was only on our third attempt that we manage to get both vector backbone fragments out. Only then (and it was already very late at night!) we were able to carry on with our plan of attack. Special thanks to Jolyon who stayed behind till 6am to finish all the Gibson and transformation! Unfortunately it also means that we won't know the result until quite late on Friday/early Saturday...

THE PLAN OF ATTACK:

1) Perform 3 2-piece gibsons and then piece these together in a 3-piece gibson (failed- no growth of transformed cells)

2) PCR the two smaller fragents from the 3 pieces and gibson with the two vector fragments in a 4-piece reaction (PCR of two smaller fragments succeeded! but we decided not to go for the 4-piece and head straight into (4)...)

3) PCR up the 3 fragments as well to be used in future gibsons (in case the above doesn't work) (we decided it's probably not feasible to PCR out the vector backbone fragment as it is ~8kb, so we proceeded straight onto (4))

4) Gibson together each of the 2 small fragments with halves of the vector and then do a final 2 piece gibson (done, will know later tomorrow)

Jolyon is also carrying out the Miller Assay to characterise the fluoride riboswitch- that needs to go on for 16 hours, so he should get data tomorrow.

Friday

Unfortunately, there doesn't seem to be any growth on the lux-mOrange plates nor the Ratiometrica plates... except for positive control. Nonetheless we have kind of got used to it now- new plans of attacks devised for both lux-mOrange and Ratiometrica.

LUX-MORANGE PLAN OF ATTACK:

1) 2 x Two-piece Gibson to join the quarters of vector backbone together, and the mOrange and the other half of the vector (Attempted)

2) PCR out each half (PCR completed... Should run gel on Monday to confirm)

3) Two-piece Gibson the two halves

RATIOMETRICA PLAN OF ATTACK:

1) 2 x Two-piece Gibson to join each of the smaller fragments to the adjacent half of the vector backbone. (attempted)

2) PCR out each half (done- only one half came out... will reattempt the other half on Monday)

3) Two-piece Gibson the two halves

Saturday

Stuart came in and tidied up the lab!