Tuesday 26^th July

Lab work

Visualization

High fidelity PCR on bacteria transformed with pPS16_001, pPS16_003, pPS16_005 and pPS16_009

By Alice

PCR peformed on July 25 with bacteria containing plasmids pPS16_001, pPS16_005 and pPS16_009 did not give us expected results. We performed again this PCR, adding bacteria transformed with pPS16_003 (clone 3) that got good sequencing results. The PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following this protocol. Specific primers were used for each plasmids (table below). Annealing temperature calculated was 72°C. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put into a well of the gel. A last well contained 10µL of DNA ladder. PCR products were migrated 25min at 100V.

PCR products expected were :

Low Fidelity Dreamtaq PCR of DH5α|pPS16_002

By Laetitia

We made a DreamTaq PCR on DH5α|pPS16_002 transformed cultures containing the Gblock 1.2.

The PCR was done on 6 clones of transformed cells: 4 clones of the Petri dish from 19/07 and 2 clones of the Petri dish from 22/07. The usual protocol was used with TM at 57°C.

These 6 clones were re-plated on a Petri dish containing LB, Ampicilin, IPTG and X-Gal.

Biobrick Characterization

BL21 electrocompetent cells preparation and transformation

By Léa, Charlène and Sylvie

We did an electro-transformation of BL21 with two solutions of glycerol different. iGEM team's glycerol :

• pcl_TAA + K1372001 (time constant equal to 5.8ms)
• pcl_TAG + K1372001 (time constant equal to 6,2 ms)
• pcl_Tq + K1372001 (time constant equal to 6 ms)
• K1372001 (time constant equal to 6 ms)

Sylvie team's glycerol:

• pcl_TAA + K1372001 (time constant equal to 6 ms)
• pcl_TAG + K1372001 (time constant equal to 6 ms)

For the third conditions with iGEM team's glycerol, cells were displayed on LB + Streptomycin (50µg/mL) + Chloramphenicol (30µg/mL) medium. For each condition, we made a dish with 50µL of cells and another with 500µL of cells. 150µL of BL21|K1372001 were displayed on LB + Chloramphenicol medium.

For BL21 transformed with Sylvie team's glycerol, 150µL were displayed on LB + Streptomycin + Chloramphenicol medium.

Two controls were made with 100µL of BL21 which were displayed on LB + Chloramphénicol or LB + Streptomycin medium.

Cells grew overnight at 37°C.

Culture of BL21 electrocompetent cells

By Léa

A BL21 colony was put into 4mL of liquid LB medium, and grown at 37°C, 180 rpm overnight.