Valencia/September
From 2007.igem.org
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Latest revision as of 08:01, 11 October 2007
Work Progress
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← August
03/09/07
we have done a first electrophoresis, and we have seen no DNA... but we know that the cultures from friday had very few cells, so we will grow them overnight and hopefully tomorrow we will have some growth and we'll be able to detect the false positives from the good ones....
04/09/07
we're doing the minipreps... and to our anxiety we've seen that we only have false positives, vectors that have religated themselves.
looking ahead, we have a question to be solve regarding how do we want the total system to be implemented into the cell. we could try to put all the constructs in one single plasmid (total 7 kb of inserts) or we could use two plasmids, changing one plasmid 'backbone' to another resistance cassette and do a double selection. after some talk, we have decided to go for the double selection, because we think it will be easier to have transformants if we keep the inserts low (3,5 kb) and because we want the plasmid's copy number to be high...
we have an answer to the ligation failure. and, as always, the answer came up from the most common place: we have read again our lab notebooks. lab notebooks are important told me once a teacher, and you never now when you will need them. there's a saying in Valencia that states that 'a short pencil is better than a long memory'. today we have seen that truth with our bare eyes... in one of the vector's digestion from last week we forgot to digest it with SpeI. this has an explanation: we have temporarly ran out of the regular SpeI enzyme and we're using one from a friend's lab. our enzymes are from Roche, his from Fermentas. buffers are not compatible, so we have to first digest it with one enzyme, inactivate it and then with the other. we just forgot the second part... life gives you experience (sometimes as a slap on the face, I should add) and experience makes you better...
05/09/07
on the morning we have a group meeting in order to decide where we head our comparator. there are many ideas, but, at the end we decide to characterise well our device and use it as a promoter calibrator. this way we might have a useful and well characterised device so that everyone can use it on their projects and Synthetic Biology systems.
we are digesting again (now taking care of using all the enzymes) and purifying. the plan is the same as last week: having enough digested and gel purified DNA in order to ligate it with some guarantees, keeping the ligation mix volume as low as possible.
06/09/07
we continue digesting and purifying
if the purification efficiency is high, we might be able to ligate this afternoon.
we have enough DNA so to reach a good ligation mix. let's hope that tomorrow we have some transformants...
07/09/07
we have colonies!!
we do the minipreps, but the DNA is quite low... so we'll inoculate them on sunday and do more minipreps on monday...
10/09/07
we have miniprepped the inoculation we had from yesterday and we have ran the gel... only to know that they are false positives, ligated vector...
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we have digested correctly the vectors and the inserts, but the problem could be on using enzymes from two different companies...
11/09/07
we are doing two works on parallel: on one hand, we keep digesting and purifying the vector and inserts to build our comparator and, on the other hand, we are changing the vector's backbone to one that bears a kanamicine resistant cassette
we have transformed another kanamicine-resistant plasmid into DH5-alpha cells, as the previous plasmids had resistance to kanamicine and ampicillin !!
12/09/07
not a single colony on the plates... we are eager for the commercial cells, that are said to be here next wednesday. in the meanwhile, we will make chemically competent DH5-alpha cells...
more digestions & purifications...
13 & 14/09/07
we are transforming other BioBricks that have ONLY kanamicine (or are said to have ONLY that resistance)...
and we keep digesting and purifying...
17/09/07
we have received a mail from our friends who will ship us the XL1-blue competent cells... they tell us that the package will arrive on friday, not on wednesday as it was supposed...
today we have done chemically competent DH5-α cells... it's a quite long protocol... we have decided to make them competent with DMSO...
18/09/07
we have ligated our comparator with the different fluorescence...
today we have tried to transform some plasmids with our recent competent cells... the plasmids were a kanR plasmid, pUC as a control, today's ligations... we transform a total of 15 plates...
19/09/07
there are no colonies... not a single one... snif, snif...
the primers we ordered have arrived... now we will be able to sequence our new BioBricks!!
we're preparing everything so, when the cells arrive (on friday or, if there is any problem, on monday) we will work 24/7...
20/09/07
we keep preparing the ligations in order to transform on the run once the cells arrive...
we have done the sequencing experiments using a BigDye protocol... we leave it overnight...
21/09/07
today is a said day for our team... for some bureaucratic problems, the cells have not arrived yet and are not expected to arrive today, it's 20.00 and there are no deliveries at that time on a friday...
everything has begun at 9 in the morning when Raúl has started the ligation thinking ahead on the time when the cells would probably arrive... our ligations protocol states 3 hours for it with the Rapid Buffer, so at noon we expected to transform...
at noon the delivery was not done, neither at 12.30 nor at 13.00. at that time Arnau has called the company which sells the cells, back in Barcelona, so to ask for the cells. they have said that the cells were at Valencia, but were not delivered because of a local holiday at the University of Valencia... indeed, today was a holiday for undergraduates as it is the first day of the course, they call it the 'opening day'... but every single lab is working that day and the bench are still crowded... so, the delivery company did not even call us...
anyway, Arnau has explained that, even though it was a holiday for bureaucracy, labs were still opened... after an hour or so of phone call, the company in Barcelona has sworn to insist to the ones in charge of their deliveries in Valencia, in order to have the cells this evening... well, the delivery company must have thought that a late-afternoon delivery was not a good idea...
24/09/07
after a fight that have lasted all morning, we keep with the delivery problem... each company says that the cells are in the other company's warehouse...
the phone call has been shorter than friday's, but Arnau has finished as angry as yesterday... he's mumbling some things about going himself to Barcelona to get the cells and kill people... anyway, the rest of the team could calm him down, without using any taser... ;-P
we're starting to grow DH5a in order to make our own chemically competent cells...
25/09/07
the delivery issue is still present...
now the delivery company has told the company in Barcelona that they have already send the cells... and has send them a dellivery note from last 13 of September!! seems like each of them is arguing that it's the other's fault, and we are right in the middle...
Arnau has started looking at each delivery guy that enters the corridor with hungry eyes...
we have send the construction to the sequencing facilities.
we have finished the chemically competent cells and have transformed one in order to try its efficiency.
26/09/07
looks like our home made competent cells have transformed pUC!! we will start our transformations with our construction right away...
at the end of the morning, as a silent cat, a dry-ice package with two boxes of competent cells have appeared at our lab... a lab technician has told us that a delivery person had just dropped by... Arnau has not ran after him, but, instead, has smiled (for the first time since last week)... :-D
at the end of the evening, we have two sets of ligations to transform in home made and commercial cells, we hope that some of them will have colonies...
27/09/07
yesterday we transformed our constructions with DH5-α and XL1-blue, as well as a pUC control with the commercial XL1-blue. this morning we have some colonies of each construction, but we have to be cautious, as they could be false positives: contaminating bacteria that grow on our ampicillin plates. we inoculate them and leave them growing until late afternoon, when we do the minipreps... we will do the gel tomorrow...
at the same time, we are preparing the next BioBrick we want to ligate: pOmpR and pOmpRm. thus, we will be able to make the characterisation of our promoter calibrator.
28/09/07
this morning we have made the gel in order to see that our clones have plasmids that are heavier than the controls... looks like they are not false positives, but, still, they have a weird gel running behaviour... next week we will do some PCRs in order to investigate this...
