Wednesday 29th 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
By Charlene
200µL of BL21 pre-culture was added to 15mL of LB and incubated for 4h at 37°C. When OD600nm=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.
| Plasmids used
|
Description
|
Use
|
| pcl_TAA
|
Promoteur_RBS_LacZ_TAA_Luciferase_Terminator
|
Negative control = base level
|
| pcl_TAG
|
Promoteur_RBS_LacZ_TAG_Luciferase_Terminator
|
Our interest plasmids allowing us to measured the biobrick activity
|
| pcl_Tq
|
Promoteur_RBS_LacZ_CodonSens_Luciferase_Terminator
|
Positive control = luciferase 100% activity
|
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.
| Component
|
Volume (µL)
|
| Plasmid
|
5
|
| Tango buffer
|
2
|
| Water
|
12
|
| AvrII enzyme
|
1
|
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:
| Plasmid name
|
Plasmid size (kb)
|
Expected digestion product size (kb)
|
| DS-NMcas
|
6
|
2.2 and 3.7
|
| DS-SPcasN-
|
6.8
|
2.2 and 4.5
|
| DS-ST1casN-
|
6
|
2.2 and 3.8
|
| DS-TDcasN-
|
6.9
|
2.2 and 4.6
|
