Monday 22 August
Great news, we must have left some food in the dustbin and the lab has been invaded by an army of ants! So it takes us some time to clean them all before starting to purify some digestions. We've introduced several changes in the protocol, following the advice of people from the department of Genetics. Let's hope it goes better, because we're starting to lose patiente...
In the mean time, we try to transform some of our ligations...again.
Tuesday 23 August
We're giving up ethanol purification, it deffinitely doesn't work as it should. This means we have to re-define everything in order to start with the standard assembling, so that we only have to purify directly from the gel. Designing new digestions means having into account the size of every insert, where to cut them, in which order, which will be the vector, etc. so we spend the whole evening examining all the details carefully in order not to spoil it all tomorrow.
Wednesday 24 August
So, standard assembling, there we go. Lots of digestions with the corresponding inoculums to regenerate the mini-preps we've spent. Heat shock, electrophoresis,transiluminator...the same story...and yet we're supposed to have a good command of this protocols, the results are not always as expected.
And things are not going better with some easy stuff like transforming with a BioBrick...we're not able to get T9002, but where's the problem? Our competent cells? Our transformation protocol? The BioBrick from the kit? What the...?
Science is not an exact science, no matter what people tell you...
Thursday 25 August
We finally got to transform T9002, using different competent cells and a slightly different protocol. It will be useful for the project but firstly we need to test it. It's supposed to detect C6-3-oxo-HSL and generate GFP, but which concentration, how long it needs to be exposed to the signal...we have no clue about that. We'll have to design some basic experiments, but before that, we need to know whether our AHLs are OK, since they came a month late and probably not in the best conditions, given the 45ºC of Seville these days...
So we go upstairs, but not to disturb the people from de department of Genetics, but the microbiologists in the third floor this time. We've heard of a bacterium, Chromobacterium violaceum, that can detect a wide range of AHLs and turn violet. This will be the perfect indicator. Charo Espuny kindly seeds for us a plate with C. violaceum 026 and gives us some useful instructions to start testing it. But for now, let's leave it growing o/n...
Friday 26 August
As half of the group is studying for their exams of September, the girls take control of the lab and put some order - this was becoming quite a jungle with pictures of electrophoresis, empty racks, notebooks and gloves all over the place.
While we keep trying to get our digestions and ligations right - the endless, tedious cycle of mini-prep, digestion, electrophoresis, purification, ligation, transformation... - we also dedicate our time to C. violaceum and the AHLs. We've seeded the strain in plates and added drops of our AHLs with different concentrations. First we try C6-3-oxo-HSL and C4-HSL. C12-3-oxo-HSL needs to wait until we know whether C6 is alright.
C. violaceum 026 test:
1.Prepare an inoculum with several colonies of C. violaceum and leave it growing at 29ºC for a couple of hours.
2.Then take about 200 ul of that culture and extend them all over the plate until it's dry.
3.Add three drops of 5, 15 and 25 ul of a solution of C6-3-oxo-HSL 5 ug/ml in absolute methanol.
4.Leave growing o/n at 29ºC.