Team:Cambridge/Diary/Week 12

Monday

PCR of the biobrick backbone is still proving troublesome, as no bands are coming out. Oli's trying the PCRs at ever higher temperatures on the backbone, but we've realised that there's a CG palindrome in both the prefix and suffix sequence, which may be causing our primers to self-anneal and form primer dimers. If these continue to fail, we'll probably have to switch to traditional ligation.

But at least both a restriction digest and a colony PCR confirmed the identity of our Magnesium Riboswitch construct. One of the colonies seems to be fluorescing far more green than the other, which may be down to a mutation that has upregulated our sfGFP. We'll need to do some characterization before we can confirm that, though.

We've also started producting spores, with some different bacillus strains cultured up in sporulation medium. Who would have thought that inducing these bacteria to form what they should do naturally would be so complicated. Apart from everyone who's worked with bacillus in the past.

Tuesday

Our freeze dried shipment of allivibrio fischeri from the States came in today, and Paul started waking them up with some rather curious marine broth. Looks like it's mostly just sea salt with some amino acids thrown in, but better to avoid messing up this rather expensive consignment we got.

Andreas gets back on Thursday, so hopefully he should be able to use these cells in conjunction with the normal luciferase containing e.coli to demonstrate the correct function of his prototype set up. If it is able to detect the tiny quantities of light that are coming off these cultures, there's a good chance they'll be able to work with the construct we should be getting from DNA 2.0.

Wednesday

Let me tell you about stains. They get everywhere. Get some in dust form on one side of the lab, expect to see mysterious blue or red spots appearing in your experiments on the other side.

At least they do seem to have stained our bacillus properly, along with lots of other things that happened to be lying around. Alas, the confocal microscope was in full use today, so we will have to wait until tomorrow to see if our bacteria have formed spores.

And, at around noon, a rather special package arrived. Our construct from DNA 2.0 has finally arrived! We're quickly replating, and hope to get some rather lovely ratiometric data from it as soon as possible.

Thursday

Jolyon and Andreas came back today, allowing us to share the workload between plenty of people again. On top of that, the construct from DNA 2.0 turned up, along with a load of high quality plate reader plates. The construct containing cells don't look hugely happy, but looking at them in the dark room shows that they do seem to be expressing the lux operon. Now we just need a strategy to get it into bacillus...

Oli also decided to use the new plates in anger, setting up an experiment to test the efficacy of the magnesium riboswitch construct. PJ claims that the resultant storm of pipetting is a 'rite of passage'.

Friday

Analysis of the sequence sent to us of the biobricks we had hoped to make indicate that the riboswitches have inserted - backwards. The current sequence is Backbone-Front of Suffix-End of Prefix-Riboswitch-End of Suffix-Start of Prefix. This almost success is somewhat mocking. However, Jolyon is starting to try to assemble our biobricks with standard assembly instead - hopefully less can go wrong with this. It's a shame to give up on Gibson, but it has been a cruel master this Summer.

Andreas and Paul started getting some images of the spores we made over the last few days. We should be able to use this microscopy as an assay when we are testing the efficacy of the new promotor we're hoping to submit. Speed of germination should be fairly simple to measure with this capacity.

But there was some more bad news, as ever. After getting the data back from the 20 hour run of the magnesium riboswitch construct in the plate reader, it turns out that our medium autofluoresces. The first sign was when Oli saw that the fluorescence actually decreased with time, instead of increasing. Easily explainable if the e.coli was degrading the amino acids that were causing the fluorescence in the first place. Some M9 minimal medium has been ordered in, but time is running scarily short for waiting for reagents to appear.

Saturday

At least the tried and tested technique of ligation appears to work. We got lots of colonies on the plates that should contain our riboswitch biobricks, which is a good sign. Emboldened by this success, Jolyon started trying to insert our DNA 2.0 construct into the backbone by ligation, while also growing up some liquid cultures of some of the successful colonies. We should have some plasmid DNA for sequencing by Monday morning, and a confirmation of biobrick construction by Tuesday morning. Which will be a considerable weight off our minds.

Also, the vibrio we woke up a few days ago seem to have started fluorescing, if very dimly. We'll give them a bit longer in the incubator, to allow their quorum sensing to kick in.