From 2007.igem.org

Work Progress

---

← 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...

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

.

---

October →