Calendar

Carry Characterization

June 2013
- Week 3 (24-30 June)
  Amplification of new biobricks for cloning :
  BBa_K081008: rbs Lux I,
  BBa_I0462: rbs Lux R term,
  BBa_C0061: cI,
  BBa_R0065: pLux
  BBa_K081014: rbs RFP term,
  
  We tested a new method for midipreps using kits : alkaline lysis with spin columns. Results were really better in terms of samples purity. Cloning can start!!!
July 2013
- Week 4 (1-7 July)
  The first necessary construction for carry characterization is the following : Planning of cloning and study of the available methods for biobricks assembly.
  
  Assembly of first biobricks thanks to the iGEM 3A Assembly method :
  - strong prom/rbs + Lux I (698 bp) in PSB1K3
  - pLux + rbs RFP term (936 bp) in PSB1A2
  Restriction of parts, ligation and then transformation in DH5 α.
  
  2 mL liquid cultures of 4 clones for each cloning.
  Miniprep after 8 hours of incubation. The electrophoresis of the digested plasmids with EcoRI and PstI confirmed the presence of the wanted constructions for all clones (at 689 and 936 bp). A 1,5% gel was also run as reference to confirm the assembly. Small parts like promoters had been indeed added.
  
  Cloning for carry characterisation keeps going. New constructions have been considered useful to study AHL diffusion.
  
  New round of 3A cloning :
  - strong prom/rbs Lux I + rbs Lux R term (1634 bp) in PSB1C3
  - strong prom/rbs + rbs Lux R term (991 bp) in PSB1K3
- Week 5 (8-14 July)
  2 mL liquid cultures of 6 clones for each cloning and then minipreps.
  0,8% gel was run to confirm clonings success.
  - correct insert for strong promoter strong rbs + Lux R terminator
  - cloning failed for strong prom/rbs Lux I + rbs Lux R term (1634 bp) in PSB1C3
  
  New try for cloning of 08/08.
  Two new assembly with 3A method :
  - strong promoter/rbs Lux R + pLux RFP in PSB1C3
  - rbs cI term + pLux RFP in PSB1K3
  
  2 mL liquid cultures of 6 clones for cI pLux RFP cloning and then minipreps.
  Bacterial carpets were obtained for the two other clonings : transformation with the remaining 5 µL ligation of 09/07.
  0,8% gel : Cloning of cI failed. It seemed to have a problem with the biobrick’s size. May be the biobrick from the plate is wrong.
  
  Stand by because of a lack of plasmids backbones...
August 2013
- Week Z
- Week Y