"

From 2014.igem.org

Contents 1 Friday 25th July 1.1 Lab work 1.1.1 A - The frame coli Odor free 1.1.1.1 PCR on odor-free E. coli 1.1.2 C - Salicylate Inducible Suppressing System 1.1.2.1 Bacterial culture: 1.1.3 D - Lemon scent 1.1.3.1 Results PCR targeting 1.1.3.2 Preparation of electrocompetent cells 1.1.3.3 Transformation of electrocompetent cells 1.2 Reunion

Friday 25th July

Lab work

A - The frame coli Odor free

PCR on odor-free E. coli

by Sean

We want to be sure that the odor-free E. coli really is E. coli. We will use a postitive control.

Protocol

C - Salicylate Inducible Suppressing System

Bacterial culture:

by Fabio

To insure a resonate quantity of DNA, we made two liquid cultures from each box of the last process, resulting in a total of 8 liquid cultures composed by 5ml of LB, 5µl of Amp, and respective colonies (4 with BBa_J61051 and 4 with BBa_K228001) (at 9am - 37°C - 150 rpm).

The cultures number 1 and 3 were collect from concentrated colonies' boxes and number 2 and 4, from undiluted colonies' boxes. At the end of the process, tubes 1 and 3 resulted in BioBricks' clones 1 and tubes 2 and 4, BioBricks' clones 2.

D - 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 cuvettes:

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

Electroporation : 2500V, 132W, 40µF.

After that, add 1ml of cold LB in each cuvette 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.

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.

Back to the calendar