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 Monday 10th October 1.1 Visualization 1.1.1 Plasmids extraction 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 BbaB0015 1.1.2 Digestion of extracted plasmids by EcoRI 1.2 BioBrick K2039000 and K2039001 characterization 1.2.1 Protein extraction 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 1.2.2 Protein electrophoresis 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 1.2.3 Transfert of protein extracted from 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 Monday 10th October Visualization Plasmids extraction 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 BbaB0015 By Sylvie The OD were measured for each overnight cultures: Sample OD (1 ml) FRB - GFP 11 in pSB1C3 (pPS16_019) 3 FKBP - GFP 10 in pSB1C3 (pPS16_018) 3.9 GFP 1.9 in pUC19 (pPS16_009) 4.2 BbaB0015 5 Then the plasmids were extracted using a "standard Plasmid Miniprep". Digestion of extracted plasmids by EcoRI By Maxence & Caroline Extracted pPS16_019, pPS16_018, pPS16_009 and BbaB0015 from the 10th October were digested by restriction enzymes EcoRI in order to verify the concentration as following: 2 µL of plasmid 2 µL of buffer FD 2 µL of restriction enzyme EcoRI 14 µL of water The mix were incubated for 1 hour at 37°C. Then, 20 µL of digestion products and 15 µL of DNA ladder were placed in wells and migrated at 100V during 30 min. Result of the migration The digestions were good for pPS16_019, pPS16_018 and BbaB0015, but we did not obtained the expecting band size for pPS16_009. BioBrick K2039000 and K2039001 characterization Protein extraction 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 In order to perform a Western Blot, the proteins were extracted from clones (pPS16_018 clone 6, pPS16_019 clone 4, pPS16_009 clone 1 and PhB1040 strain 594) put on liquide culture the 7th October. For that purpose, the protein extraction protocol was used: Sample OD (1 ml) Volume of lysis buffer FRB - GFP 11 in pSB1C3 (pPS16_019) 5.1 127.5 μl FKBP - GFP 10 in pSB1C3 (pPS16_018) 5.2 130 μl GFP 1.9 in pUC19 (pPS16_009) 5.3 132.5 μl PhB1040 strain 594 2.8 70 μl Protein electrophoresis 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 A protein electrophoresis of the extracted protein was done using the protein electrophoresis protocol. For that purpose, the gel cassette Bolt 4-12% Bis-Tris Plus 10W was used and the power was set for 1 hour at 65V. Transfert of protein extracted from 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 The proteins on the electrophoresis gel were transfered on blotting membrane using the protein transfert protocol.
By Sylvie
The OD were measured for each overnight cultures:
Then the plasmids were extracted using a "standard Plasmid Miniprep".
By Maxence & Caroline
Extracted pPS16_019, pPS16_018, pPS16_009 and BbaB0015 from the 10th October were digested by restriction enzymes EcoRI in order to verify the concentration as following:
The mix were incubated for 1 hour at 37°C. Then, 20 µL of digestion products and 15 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
The digestions were good for pPS16_019, pPS16_018 and BbaB0015, but we did not obtained the expecting band size for pPS16_009.
By Maxence
In order to perform a Western Blot, the proteins were extracted from clones (pPS16_018 clone 6, pPS16_019 clone 4, pPS16_009 clone 1 and PhB1040 strain 594) put on liquide culture the 7th October. For that purpose, the protein extraction protocol was used:
A protein electrophoresis of the extracted protein was done using the protein electrophoresis protocol. For that purpose, the gel cassette Bolt 4-12% Bis-Tris Plus 10W was used and the power was set for 1 hour at 65V.
The proteins on the electrophoresis gel were transfered on blotting membrane using the protein transfert protocol.
