CcaS (continues) and luciferase assay
Thank you very much for your reply and your kindness!
For the luciferase, we have three mutations and they will probably
work but need to be checked today. We will tell you about the test
after the results come out.
For the LovTAP, Marta is working ont that and she keeps on touch with
your guy who is working on that.
If there is anything more you are interested in, please let us kknow
and we can share between us. After we modify and make more details of
the green sensor design, I will send you the whole design plan.
We have finished testing the luciferases and our S284T and 365R
mutants glow. They seem to be more red than the wild type we used as a
control, but it is hard to tell as they are not very bright. We are
going to to test their spectral output next week, and sequence them to
confirm the mutation is present. In mutant 356K we didn't see as much
Will and Lu
Dear Will and Lu,
Thank you very much for your information, which is really useful. What I
commented you the other day during the skype reunion is that I modified the
RBS on both luciferases (the wild type and the S284T) as described in the
parts registry http://partsregistry.org/Part:BBa_K216015:Design. In theory,
this modification may improve the output.
As soon as I test the mutation, we can also compare the results to check if
this modification is worthy.
I am planning to use the pBSKII (+/-) vector to check the luciferases
before putting them into pSB1C3 with a J23101 promoter, in which plasmid
did you checked them and with which promoter?
One of our advisors has worked with luciferase before and he is willing to
help us during the assay, if you could please tell us more of your
experiment we would very much appreciate it (for example, the
concentrations of luciferin), as he may start with a better background and both
teams will gain knowledge while trying to
optimize the assay :D.
Wish you too the best,
Mariana Ruiz Velasco Leyva
Licenciatura en Ciencias Genómicas
LuxAB - Vibrio Fischeri - LuxCDE - Problems
I'm Augusto and I'm working with LuxAB; I amplifyed LuxAB from Vibrio
fischeri's genomic DNA without many problems but I've spent a lot of time
trying to ligate LuxAB to some vector from iGEM kit plate 2009 (because
iGEM kit plate 2010 arrived later than we expected).
My lab partners had the same problems with the vectors.
Rigth now we are using commercial vectors that our advisor uses in his
daily resarch in order to make some bioluminescence tests (pSB11k5(+)73
from Stratagene) and we are gonna use pSB1C3 Parts Registry vector from
iGEM kit plate 2010 to build our bio-bricks.
I don't have any result yet but I can send you an e-mail during the
weekend to inform you about our results.
Sorry for the delay answering you, have you got new results during these
days? Do you have experience about bioluminescence assay protocols?
I'm Maria, I'm doing most of the work on the lux DNA for our group, so
I reckon I'm best off chatting to you.
So far only Chris French, our supervisor, has managed to succesfully
amplify luxCDE. He's not being incredibly open about what he's done
differently to us, so I'm not entirely sure what we did wrong, but if
you like we can continue working on that here and send it to you in
biobrick form later on so that you don't have to keep wasting time on
it if it's not working for you either.
LuxAB will amplify out and is clearly the right size on the gel. The
problem is, it's not transforming properly. We're inserting it into
our old vector, Edinbrick I (we have to use that because of the
restriction sites that the luxAB primer has, and to be honest I'm not
really sure why we can just replace it's restriction sites with the
proper restriction sites, but like I said, Chris doesn't really tell
us anything), and we're using blue-white selection on Xgal. I got
white colonies for the first time yesterday, but my subcultures from
those didn't grow, so I'm doing those again today. Chris, however,
says he can improve transformation efficiency by digesting the vector
then running on a gel and taking the DNA from the band rather than
just using the supercoiled DNA directly.
It's up to you if you want to continue trying with lux. I'm honestly
so close to giving up on it, but we can't really afford to - it's sort
of central to our project. I think whoever gets it to work first
should let the other know how they did it, then we needn't send the
DNA, which will take so long that half of us will have gone by the
time it gets here (we're on a much stricter time scale than you, by
the sound of it).
So that's where we are really. I got the impression that luxCDE was
giving you a headache and you were planning to start luxAB next week.
Let me know how it's going and whether you have or need any advice.
Nice to see/meet all of you :)
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