Team:Cambridge/Diary/Week 7

Monday

Like many days, today was a good day for shopping. No shoes and salads for us though - we had chemicals to order. NAD, Iron (III) chloride, LB base and potassium acetate, all for the greater glory of our project.

Oli re-ran his vector fragment B PCR and, despite some slightly weak bands, extracted the DNA needed. A similar retry of the magnesium sensitive promoter failed, and we are now positive that the part (both in sequence and content) was unreliable. Irritating, but it will look good when we make our own really good magnesium sensor. Which we will. Don't worry.

Tom's split primers for the lux vector (pSB1C3 backbone) has arrived, so he retried the colony PCR. Unfortunately still nothing has came out. We will do another run tomorrow.

We've run out of competent e.coli cells, and we need some more. Unfortunately, the TOP10 cells that were grown up over the weekend were too old to use, so Paul is using our final vial of TOP10 to make some more that should be in the log phase by tomorrow morning. Then we can get on with the protocol to make them competent.

Tuesday

A very practical day today, as various people (Paul and Andreas in particular) undertook to perform the rather long electro-competent cell production protocol. We also recieved our delivery of reagents we ordered yesterday. Using some quite stinky NAD+, the isothermal reaction buffer for the Gibson assembly reaction was finished off and lots of aliquots of Gibson master mix stored.

Having made all this, we did some Gibson for the riboswitch and fluorescent constructs, which *should* work this time. Certainly, we're pretty confident we have all the ingredients in the mix this time. We'll transform some cells with the resultant DNA cocktail as soon as we have some competent cells. Hopefully tomorrow then, though such efficient hopes have had a way of failing in the last couple of weeks.

Tom struggled through his PCR again, retrying the split luciferase primers. No successes, alas...

Wednesday

We are all at that stage where our various pieces of DNA need putting into cells. To that end, we began transformations, and lots of them. We started using the electroporation device for the first time today, possibly making lots of cells with plasmids shoved through their walls. Or else we've just made a load of dead bacteria. Only a night in the incubator will tell.

Desperate to find out the reasons behind the failure to Gibson our fluorescent contruct, Emmy did a restriction digest on the Gibson product of the fluorescent components- and there were no bands. She suspects that there wasn't enough DNA in the sample to be shown in the gel. Looks like this diagnostic test can only be performed after we successfully transform E.coli with the construct, grow them up and miniprep the plasmid. With our lack of functioning competent cells looks like it's not going to happen until earliest next week.

We also began thinking about what we're going to show at the meet up in a week. We've been invited down to the Google campus in London by the UEA team to discuss (and more importantly, present) our projects. Paul and Emmy therefore began creating a poster for it, and Oli and Tom began talking about how to order our presentation.

Thursday

Well, some of the plates seem to have colonies. They are rather small though, so we will probably wait another day or so before we start doing anything with them.

We got started on making more chemically competent cells, for use in conjunction with the electrocompetent cells. Hopefully if one fails, the other will work. Naturally, this involved the transfer of various clear liquids into other clear liquids, as well as the production of clear liquids. Such is the life of a bacterial consommé chef.

Tom attempted the colony PCR for the third time with some changes in the PCR programme. Still no luck... After some research, we decided that it is probably a better idea to miniprep the plasmid out of the cells, since colony PCR seems somewhat unreliable. Tom has began preparing some overnight cultures of the E. coli.

More biobricks, as well, as Charlie both plated out and ordered some more. With luck these will be easier to use than the magnesium sensitive promoter.

Friday

There is even more growth on the plates today. Unfortunately it looks like most of what has grown is some sort of fungus in the agar. Nice halos surrounding dense black cores are a testament to our failure at aseptic technique.

It also looks like our ampicillin has been degraded due to incorrect storage, so we made up a fresh batch and stored it. This may explain the observation that we have many small colonies of e.coli growing where they should not. Hope still remains, however, that the large colonies are still representative of successful transformants that can be used by us. We're culturing them up, though we don't imagine they will be usable.

Paul made up some chemically competent cells as well, which can be used in conjunction with our electrically transformable cells. We made two batches (due to miscalculations in the number of autoclaved eppendorf tubes we would need), one of them ended up going through an extra freeze-thaw cycle. He has transformed the first batch with pUC19, so if all works out we should be able to see growth on agar plates tomorrow. Then we can do further tests over the weekend.

Tom amplified up the sfGFP-ampR and pSB4K5 provided by the Haseloff Lab for Gibson control. PCR worked out very nicely and the required DNA are extracted. He will Gibson some of them together so they could be used to test our chemically competent cells.

Additionally, we managed to get some of the actual charactarisation started, courtesy of the plasmid sent to us by Yale. Jolyon tested the sensitivity of the riboswitch by testing the rate of lac Z transcription with a β-galactosidase assay.

Saturday

The first batch of our chemically competent cells are competent! The cells transformed yesterday with pUC19 control has grown very nicely on agar plates.

Oli transformed some chemically competent cells with the Gibson product of the Riboswitch construct from a few days ago.

Sunday

More transformations of the Gibson products (Gibson control, fluorescent construct) by Emmy and Paul, along with the YFP donated by Jim H. No growth yet for the transformation done yesterday, so we'll leave it again overnight, along with the others.