EPF-Lausanne/18 October 2009

Wet Lab

Broth culture

We did a culture of every plate from yesterday (only the 4x diluted, because otherwise it was too concentrated). RO1 transf., RO2 transf., D transf. RO2.4 + ILE 427 PHE #10, D transf. RO1.1 +ILE 427 PHE #10, RO2.4 + BB1, RO1.1 + BB1. Finally this culture has not been used.

The following clones have been put in culture for the afternoon characterization : 2ml LB + antibiotic + 2 mM IPTG, tips from plates :

RO1.1 + BB1 #3 (from the plate), RO2.4 + BB1 #3 (the original, from culture 1ul inoculation), RO2.4 + BB1 #2 (new plate), RO1.1 + BB1' #1,2,3 and RO2.4 + BB1' #1,3,6 with mutated LovTAP gene : ILE 427 PHE.

We also did a glycerol stock of ILE 427 PHE #1 and #10.

We then did a colony PCR, using the protocol with Taq platinium. We did the following clones : RO1.1 + ILE 427 PHE #10 dil. 4x n° 1,2,3,4,5,6; D transf RO2.4 + ILE 427 PHE #10 dil. 4x n°1,2,3,4,5,6; RO1.1 + BB1 dil. 4x 1,2,3; RO2.4 + BB1 dil. 4x 1,2,3,4. We then did a gel of the colony PCR. The gel is curved, so not too good. Morever it has a strange colour... we will do a new gel tomorrow ! From what we can see, RO2.4 is missing, RO1.1 seems okay (lengths are right) but not for all the clones (RO1.1 + BB1' #3,4,5,6 okay, RO1.1 + BB1 #13,14,15 okay).

We then put into culture some other clones : the tips induced culture are very low... it is quite normal as we were not able to take a lot out of the plates. After about 3h30, all tubes were clear (except RO2.4 + BB1 "original"). We switched the light on for 1h30 (for +light clones). We did the plate experiment even though we couldn't see any turbidity in the tubes.

Characterisation experiment :

We did two experiments :

1. Static with about 1h30 light induction and negative control in the dark.

2. Experiment to assess the stability of mutants. They were grown in the dark and then induced for 45 s by light. Try the difference in fluorescence evolution between BB1 and BB1'.

Note : both plates were done at the same time. Loaded in the dark under red LEDs.

For the stability assess : 1 minutes of light was shined on the plate just before taking them to the plate reader.

Results

Double transformants

Double transformants : static experiment

RO2.4 + BB1

181009

Conclusion (TO TRANSLATE):

Columns :

1=RO1.1+BB1/3-->ok selon gel

2=RO1.1+BB1(muté)/1-->pas ok

3=RO1.1+BB1(muté)/2-->pas ok

4=RO1.1+BB1(muté)/3-->ok

5=RO2.4+BB1/2-->pas ok

6=RO2.4+BB1(muté)/1-->pas ok

7=RO2.4+BB1(muté)/3-->pas ok

8=RO2.4+BB1(muté)/6-->pt ok?

En gros, si vous regardez c'est un peu la fête du slipe... je sais pas trop quoi penser. Il faut quand même noter, que les culture n'étaient même pas turbide quand on a fait les mesures (on avait pris des plaques mais trop chaud de piquer...). Donc sauf les RO2.4+BB1 "original" qui vient de la culture overnight re-inocculée, c'est pas la joie...^^ Mais c'est toujours ça! Une expérience reproductible! Et au moins ça montre que quand je suis là, il n'y a pas TOUT qui foire n'est-ce pas Gab;-) Ou alors c'est toi Heidi qui a une influence positive^^

Et en tout, il y a quand même quelques résultats qui se tiennent... enfin j'espère!

People in the lab

Heidi, Basile, Caroline