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 will redo the experiment for because two of the transformation did not work out (broken electroporation cuves).
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
|
