Team:Cambridge/Diary/Week 6

Monday

We are suffering from some setback on the PCR front. Tom and Emmy's PCR re-runs for the large vectors did not work despite lengthening the elongation time to 5 minutes per cycle. After sequence alignments we discovered that the primers may anneal to various sites on the primer, producing a range of undesired products. We will carry out touch down PCR tomorrow to make the annealing conditions more stringent.

PCR of the mOrange gene last Friday also yielded no visible products. Looking at the primers' designs again, we think it might be due to problems with the annealing temperature. Therefore we are setting PCR reactions across a temperature gradient today to find the optimum annealing temperature.

For the first time, we actually did some Gibson assembly in a realistic setting! We (hopefully) fused together the Mg2+ fragment purified last week, along with the riboswitch DNA purified two weeks ago. If all goes according to plan, the transformation on bacillus that we later performed should provide us with some responsive bacteria by tomorrow morning. If not, we'll try again in e.coli, and see if we can make a new biobrick from the riboswitch in this chassis.

Tuesday

A day of abject failure. Bacillus from yesterday has failed to grow at all. We're leaving them in the incubator for another day, but don't hold out much hope of getting any useful results.

The problem may have been our transformation, in which one of the steps may not have been at the correct 37 °C temperature. Consequently, we're re-running the experiment, as well as trying to get the plasmid working in some TOP10 e.coli.

Gel electrophoresis of the mOrange PCR products give no visible bands. Tom discovered that a base was missing from the reverse primer- so we have ordered a revised version of it. Curiously, we also seem to have problems with our positive and negative controls.

Colony PCR of the luciferase vector followed by gel electrophoresis gave a very faint band that seems to be of the right size (the expected product was over 9kb), so the DNA is extracted. We will gibson it with the mOrange once the new primer arrives and the mOrange successfully PCR-ed up.

Touch-down PCR of pSJ150 for the fluorescent constructed yielded a single product- but not one that we expect. We will have to find a new solution.

The biobricks from the registry also appeared. Charlie streaked out the bacteria for later use.

Wednesday

Apparrently, writing off yesterday as a complete failure was somewhat pre-emptive, as it now appears that our bacillus was actually growing, albeit slowly. Given our bacteria seem to have been transformed therefore, we streaked out potentially functional colonies for testing later.

PJ suggested that pSJ150 is too large (~8.2kb) to be PCR-ed up as one-piece. He therefore gave Tom and Emmy split primers, which will amplify the vector in two chunks (3.5kb and 4.5kb). We will run the PCR products tomorrow to see if they work.

Thursday

Two deliveries today, one from our Yale contact and one from Starlabs. On the one hand, enough fluoride related bacillus to make us a couple of biobricks, on the other, enough tips to last us a lifetime. Thanks, you two!

Not all the day was so joy filled though. After growing up our streaked out bacillus, they have turned out to be e.coli. This makes our assessment that our original assessment was to hasty itself too hasty, which, while gratifyingly meta, is none the less frustrating. We're now going to try and debug our Gibson and PCR, and will probably try to rerun the vector fragments that only seemed to partially work.

Furthermore, the plates that we made for our (hopefully) transformed e.coli have nothing growing on them, meaning we probably will have to start from the beginning.

The PCR from yesterday has, however, worked! Having everything in place to perform a Gibson transformation to produce our fluorescent construct, we performed said reaction and transformed it into some TOP10. I will reserve judgement on this until next week.

Friday

It would appear that our isothermal reaction buffer was not correctly mixed, meaning all our Gibson assembly reactions had no chance of success. Having remixed the buffer, we now hope that the Gibson should work properly.

Unfortunately, we have now wasted all our TOP10 e.coli on non-functional Gibson assemblies, so now we have to create many more competent e.coli. Consequently, Jolyon got onto ordering all the materials we will need for electroporation, TOP10 being prohibitively expensive for a puny undergraduate research project.

We have also discovered that the primers for mOrange and luciferase vector (pSB1C3) have been mixed up due to incorrect labelling- that has probably caused all our problems with amplifying up the two over the past week. Nonetheless our new mOrange reversed primer has arrived, so with the corrected labelling and revised primer the mOrange has been amplified up successfully.

So the faint bands we had on Tuesday for the luciferase vector were probably not our desired product afterall, but learning our lesson from attempting to PCR pSJ150 backbone, Tom has designed split primers for the luciferase backbone and they should be delivered on Monday. With those and the correctly labelled primers hopefully we will obtain the right product that we can gibson to the mOrange.

Oli has decided to use the split primers for pSJ150 to PCR the pSJ150 backbone for the Mg riboswitch construct as well- the "product" from last Friday's PCR was rather ambiguous and it doesn't seem to have worked in the Gibson. Primers for Charlie's biosensor constructs have also arrived, so parts of the construct are PCR-ed. Unfortunately, none of these PCRs yielded any products- possibly due to a problem with the PCR master mix. We should try these again later.

Saturday

Unfortunately there are no visible colonies on our ampicillin plates, which means there might be some problems with the Gibson-ing of the fluorescent construct, or the transformation of E. coli. We will observe for another day.

Jolyon also started culturing some cells for electroporation so we could start the protocol on Monday.

Sunday

Still no colonies for the fluorescent construct plates. Meanwhile we redo Oli and Charlie's PCR: half of Oli's vector has came out, as well as Charlie's RFP. We suspect the reasoning for our failure to PCR the Mg promoter might be due to sequence inconsistency of the biobrick from the registry. Oli will attempt the failed PCRs again tomorrow.