Note Book - iGEM Bordeaux 2012

Day 50 : 17-09-2012

Today we did the ligations IAIB , IA'IB' , ICIE , IVAIIC5B , 5AIIICIIIEIIID , 5AIVDIVEIVC , IA IIC5B , IVA5AIVB , IVEIVC with the digestions from last friday.
We inactivated the ligase after 1 hour 20°C incubation and pute the tubes overnight at 4°C

Day 51 : 18-09-2012

Today we transformed by electroporation the ligations from yesterday.

Day 52 : 19-09-2012

Today we put the transformants into preculture.
Nothing on IA'IB' , ICIE , IVAIIC5B , 5AIVDIVEIVC plates so we redid ligations for those assemblies.

Day 53 : 20-09-2012

Today we did a miniprep of the precultures

Day 54 : 21-09-2012

Today we tested the plasmids on agarose gel.

Day 55 : 22-09-2012

We started a new strategy using 3 operons instead of 4. As the first operon does not seem to assemble, we will simply put its promoter before the operon II. Thus, our system should work sooner.
We also rethink operon IV and removed LsrR and LsrK. We removed as well the CIbox from operon III.
We digest, ligate and transform : IVAIVEIVC, IIIA5AIIIC, IIIEIIID.

Day 56 : 23-09-2012

Culture of single colonies from plates displaying single colonies.