Team:Cambridge/Diary/Week 11

Monday

We continue trying to debug the Gibson reaction, with incremental success. The Gibson control plates from yesterday showed that there is no significant difference between the competency of our cells, or our master mixes. However PJ obtained a 10x greater efficiency.

We decided to rule out a problem with the plates. Prewarming does not appear to be a factor in the success of the plating step.

Paul's been working on developing his germination protocols. Germination medium has been determined and the protocols for imaging the sporulation and germination of B.subtilis have been ironed out.

Oli also began checking if the growth of our bacteria would be seriously impacted by removing all the magnesium from the growth medium. It's only used for everything to do with ATP, probably not too important.

Tuesday

Gibson control plates from yesterday showed no difference between PJs plates and ours.

Paul Grant, another member of the Haseloff lab, suggested that he observe Tom assemble and transform, but failed to detect any gross incompetency. We thought that the problem might be the PCR machine - it takes a little while to load ours, whereas the Haseloff lab's is a hotblock that can be preheated, minimizing the time the T5 exo has to digest our fragments.

New control fragments were obtained, some with different "minelute" gel extraction kits. One assembly was set up with mineluted control, the other with normally extracted control. Additionally, a reaction was set up to assemble our 4-part ratiometric construct. We used the Haseloff hotblock PCR machine to assemble. Transformation was done with our competent cells, and they were plated.

Also, Paul began designing his first Gibson primers! He's aiming to make a biobrick out of a plasmid containing the spoVAA gene under control of the PssB "fast" promoter. Peter Setlow's team from the University of Connecticut have kindly offered to send us the B. Subtilis strain containing the modified chromosone and two E.coli cultures containing plasmids used to facilitate the single crossover recombination event that placed spoVAA under the control of PssB. Designing Gibson primers may prove a little tricky though as we don't have access to the full sequence.

Lastly, the results from the platereader came through. B.Subtilis needs more magnesium to grow than e.coli, as expected, but there is still this weird peak at around 50mM MgCl2 in the growth rate. Oli will... Investigate. In particular, he'll check if there are any simelar peaks when he repeats the experiment in bacillus.

Wednesday

Only one of the control plates from yesterday (the minelute control) has colonies on it, and still a low number. The other has none on it at all. The ratiometric construct plate has a couple of colonies on it, but their lack of fluorescence implies that they are not what we want.

A gel was run to check the control fragments. (5ul of each 10ul elution). No significant differences observed between fragments, all were visible and correctly sized.

We quite urgently need to get some biobricks submitted, so Oli began attempting to make the fragments we'll need to Gibson together to make biobricks of the riboswitches we've been trying to test. Hopefully we can make some successfully transformed colonies quite quickly...

And it turns out that the ridiculous peaks at around 50 - 300 mM magnesium are still around, even in bacillus. We'll try rerunning the experiment with a bit more concentration resolution - if these results are real, we may have managed to make these bacteria double in two minutes! Unlikely, to say the least.

Thursday

Paul and Tom had a large handover session before Tom left on holiday. The fate of the project may now lie in the hands of an engineer!

A Gibson from yesterday produced lots of colones, possibly indicating that we have had our first successful construct produced! We'll need to check it somehow (see the options that were open to us on the protocols page), but with a little bit of luck, we should be able to start characterizing our magnesium biobrick.

Higher magnesium concentration resolution growth run in both e.coli and bacillus indicated that there is a smooth transition from the slow 'growth' to the fast 'growth'. However, the error bars that have been plotted with this data indicates that the results are far too reproducible for biological data. Since this implies some physical cause, the experiment will be rerun, minus the addition of cells. We may well get the same results, and assuming we don't have ghost bacteria in our culture, this would imply that some sort of magnesium based percipitate is forming in our growth medium. Still doesn't explain why the OD drops off later, or at higher concentrations, but at least we will know our weird results aren't coming from our bacteria.

Friday

The strains for sporulation arrived and have been plated. the long range pcr of the flu and lux 9kb fragments was run again but no gel has been run yet.

Unfortuneately, not all the fragments that were needed for the production of the riboswitch fragments came out on Wednesday, so we did another PCR with lower temperature settings to try and get them out. And lo and behold, it worked! Some of those bands were bright enough to see with the naked eye, with no special illumination at all! Gel extraction is proving to be... unpleasant however. Initial estimates indicate we're still loosing almost 99% of our DNA in the extraction step. There must be some better way to resolve this.

Oh, and the results of the control run of the bacterial growth curves came through. Turns out some sort of percipitate is forming in the medium at high magnesium concentrations, producing the spuriously steep growth curves. Our best guess is that it is magnesium phosphate. Considering that for a few seconds some of us thought we'd managed to get the doubling time of our bacteria down to a couple of minutes, somewhat disappointing.

Saturday

Well, weekends have pretty much disappeared for Oli and Paul, the only two people left in the lab. While everyone else is sunning themselves in such far away lands as Canada and Cyprus, they worked away at making some of the biobricks and constructs we need.

Sunday

The magnesium riboswitch PCRs were redone at the same conditions as before, and surprisingly enought we got the same great yields. However, we decided to try simply purifying the PCR mix instead of dealing with the whole gel extraction protocol. Yields increased around 50 times. We're now going to try and do this for all our products, if they are known to be fairly pure, as this result is so much better than the normal DNA extractions we do.