Wednesday 29^th June

Lab work

Visualization

Culture of clones with gBlocks

By Lea and Marion

6 clones of each construction were selected (transformed cells are supposed to grow white on xGal IPTG) and grown overnight in 3.5mL of LB (50µg/ml Amp) at 37°C, 180rpm.

BioBrick characterization

BL21 electro-competent cells preparation and transformation

By Charlene

200µL of BL21 pre-culture was added to 15mL of LB and incubated for 4h at 37°C. When OD_600nm=0.68, cells were centrifuged for 10 minutes at 4000rpm and washed twice with addition of 10mL glycerol (10%) followed by 10 minutes of centrifugation at 4000rpm. Cells were mixed with 200µL glycerol and kept -80°C.

We realized four transformations with the following plasmids:

• K1372001
• K1372001+pcl_TAA
• K1372001+pcl_TAG
• K1372001+pcl_Tq

50µL of cells were added in the cuvettes with 1µL DNA (diluted 1/10). Everything was done at 4°C.

Transformation did not work for K1372001 and K1372001+pcl_TAA because the cuvettes were broken (electroporation time constant was equal to 1.6ms. Cells died during transformation. Transformation worked for K1372001+pcl_TAG and K1372001+pcl_Tq (time constant was equal to 6ms. We added 1mL of LB to the cells and incubated them for 1h at 37°C.

The following cells were plated on Petri dishes (30µg/mL Cm + 50µg/mL streptomycin):

• BL21|K1372001+pcl_TAG : 50µL of transformed cells
• BL21|K1372001+pcl_TAG : 500µL of transformed cells concentrated 5x.
• BL21|K1372001+pcl_Tq : 50µL cells
• BL21|K1372001+pcl_Tq : 500µL of transformed cells concentrated 5x.

K1372001 plasmid digestion

By Naiane

K1372001 plasmid was digested with BglI using the following quantities: 3 µL DNA, 2µL buffer (O), 1 µL enzyme (BglI), 14µL water. The mix was incubated for 1h at 37°C and 10 min at 55°C and kept at -20°C until the gel electrophoresis was done. Fragments are expected to size 1.2kb and 2.4kb.

BL21 cell culture

We added 200µL of the BL21 culture made on 28/06 in 15ml of LB and incubated it overnight at 37°C, 180rpm. We need a new BL21 culture to redo the experiment because two of the transformations (K1372001 and pcl_TAA) did not work out (broken electroporation cuvettes).

Bringing DNA closer

Plasmids digestion

By Naiane

Plasmids received from Addgene were digested with AvrII.

The mix was incubated for 1h at 37°C and 10 min at 55°C and kept at -20°C until the gel electrophoresis was done. Fragments are expected to be as follow:

The migration's result was:

[[File:T--Paris_Saclay--160629_bringingDNAcloser_casdigestion_échelle.jpeg]|200px|thumb|left|TD,NM,SP,ST1 electrphoresis]

pUC19 digestion