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From 2014.igem.org

Contents 1 Friday 25th July 1.1 Lab work 1.1.1 C - Lemon scent 1.1.1.1 Results PCR targeting 1.1.1.2 Preparation of electrocompetent cells 1.1.1.3 Transformation of electrocompetent cells 1.1.2 E - Salicylate Inducible Suppressing System 1.1.2.1 Bacterial culture: 1.2 Reunion

Friday 25th July

Lab work

C - Lemon scent

Results PCR targeting

by Romain

After the night, the electrophoresis revealed the oligonucleotides iPS70 and iPS71 prepared with the GoTaq enzyme.

We made a PCR Clean-up. Protocol

After that, we made another electrophoresis which revealed successfully the purification.

Preparation of electrocompetent cells

by Romain

Strain used: DY330.

Protocol:

Dilution of 300µl of bacterial culture DY330 in 30ml of LB at 30°C.

When the culture OD₆₅₀ = 0,6:

• put in ice during 10min.
• centriguge at 4°C, 5min, 4000rpm.
• Discard the supernatant, and resuspend the pellet in 30ml glycerol 10% COLD.
• centriguge at 4°C, 5min, 4000rpm.
• Discard the supernatant, and resuspend the pellet in 15ml glycerol 10% COLD.
• centriguge at 4°C, 5min, 4000rpm.
• Discard the supernatant, and resuspend the pellet in 200µl glycerol 10% COLD.

Transformation of electrocompetent cells

by Romain

Strain used: DY330. Plasmid used: pJBEI-6409 (code for enzymes to produce limonene from glucose in E. coli)

Make 2 électroporations in cold electroporation tanks:

• A control tank (without DNA): 50µl of DY330.
• A second tank: 50µl of DY330 culture + 1µl of pJBEI-6409 plasmid.

Electroporation : 2500V, 132W, 40µF.

After that, add 1ml of cold LB in each tank and transfer in 2 tubes to incubate during 1 to 2h at 30°C.

Spread on 4 dishes LB + Cm:

• 100µl of control (without plasmid)
• 50µl of transformed DY330 with pJBEI-6409
• 100µl of transformed DY330 with pJBEI-6409
• The rest of transformed DY330 with pJBEI-6409 (concentrate in approximately 150µl)

Incubate for the weekend at 30°C.

E - Salicylate Inducible Suppressing System

Bacterial culture:

by Fabio

8 liquid cultures with 5ml of LB and 5µl of Amp, 4 with BBa_J61051 and 4 with BBa_K228001 (at 11am - 37°C - 150 rpm), all from the concentrated colony made the 24th July

Reunion

With Arnaud, Fabio, Sean, Terry, Alice, Solenne, Sylvie & Philippe

Members present:

• Instructors and advisors: Solenne.
• Students: Arnaud, Fabio, Juliette, Pierre, Romain, Sean and Terry.

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