Toggle navigation Home Team Team Media Collaborations Sponsors Acknowledgements Project Background Design CRISPR/Cas9 Strategy Experiments Notebook Results Perspective Interlab Study Parts Parts Basic Parts Composite Parts Human Pratices Overview Societal Issues of CRISPR/Cas9 Responsible Research and Innovation GMO regulation Integrated Practices Engagement Model Attributions Safety Contents 1 Thursday 6th October 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 1.1.1.2 Transformation of DH5a cells with and FKBP - GFP 10 (pPS16_018) and FRB - GFP 11 (pPS16_019) 1.1.2 BioBrick X characterization 1.1.2.1 Culture of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and PhB1040 strain 594 Thursday 6th October Lab work Visualization Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 By Maxence & Victor The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit: pPS16_018 (FKBP - GFP 10) clone 6 pPS16_019 (FRB - GFP 11) clone 4 After extraction, 5 µL of each plasmids and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min in order to assess their concentration. GEL Transformation of DH5a cells with and FKBP - GFP 10 (pPS16_018) and FRB - GFP 11 (pPS16_019) By Maxence & Victor As we were not able to obtain good plasmid concentration after culture of glycerol stocks and plasmid extraction, transformations were done. Dh5a cells were transformed with pSB1C3 containing FKBP - GFP 10 (pPS16_018) and FRB - GFP (pPS16_019) using the usual protocol. BioBrick X characterization Culture of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and PhB1040 strain 594 By Maxence & Victor As we have met several issues in order to obtain plasmis pPS16_023 (gBlock ATG linker FRB + GFP 11 + gBlock ATG linker FKBP + GFP 10 + gBlock GFP 1-9), we decided to characterize our BioBricks with a Western-Blot with antibodies against GFP. For that purpose, the following clones were put on solid and liquid cultures (LB + 30 µg/mL Cm ou Amp) grown at 37°C overnight: pPS16_018 (FKBP - GFP 10) clone 6 pPS16_019 (FRB - GFP 11) clone 4 pPS16_009 (GFP 1.9) clone 1 Furthermore, another strain containing the full GFP was cultures in the same conditions: PhB1040 strain 594 (lac-3350 galk2 galT22 2psL179 pGFP) (Amp resistance).
By Maxence & Victor
The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
After extraction, 5 µL of each plasmids and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min in order to assess their concentration.
GEL
As we were not able to obtain good plasmid concentration after culture of glycerol stocks and plasmid extraction, transformations were done. Dh5a cells were transformed with pSB1C3 containing FKBP - GFP 10 (pPS16_018) and FRB - GFP (pPS16_019) using the usual protocol.
As we have met several issues in order to obtain plasmis pPS16_023 (gBlock ATG linker FRB + GFP 11 + gBlock ATG linker FKBP + GFP 10 + gBlock GFP 1-9), we decided to characterize our BioBricks with a Western-Blot with antibodies against GFP. For that purpose, the following clones were put on solid and liquid cultures (LB + 30 µg/mL Cm ou Amp) grown at 37°C overnight:
Furthermore, another strain containing the full GFP was cultures in the same conditions: PhB1040 strain 594 (lac-3350 galk2 galT22 2psL179 pGFP) (Amp resistance).
