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From 2014.igem.org

Contents 1 Monday 28th July 1.1 Lab work 1.1.1 A - The frame 1.1.1.1 Preparation of electrocompetent cells 1.1.1.2 Transformation of electrocompetent cells 1.1.2 C - Lemon scent 1.1.2.1 Liquid Culture 1.1.3 E - Salicylate Inducible Suppressing System 1.1.3.1 Plasmid DNA Purification

Monday 28th July

Lab work

A - The frame

Preparation of electrocompetent cells

by Arnaud & Romain

Strain used: E. coli MG1655Z1 and E. coli MG1655.

Protocol:

Two dilution of 200µl of bacterial culture MG1655Z1 and E. coli MG1655 in 30ml of LB at 30°C for each strain.

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 Arnaud & Romain

Strain used: E. coli MG1655Z1 and E. coli MG1655. Plasmid used: BT640 (code for a flipase for E. coli)

Make 2 électroporations in cold electroporation tanks:

• A control tank (without DNA): 50µl of E. coli MG1655Z1 or E. coli MG1655.
• A second tank: 50µl of E. coli MG1655Z1 or E. coli MG1655 culture + 1µl of BT640 plasmid.

Electroporation : 2500V, 132W, 40µF.

After that, add 1ml of cold LB in each tank and transfer in 2 tubes.

Spread on 4 dishes LB + Cm:

• 20µl of control E. coli MG1655Z1 (without plasmid)
• 50µl of transformed E. coli MG1655Z1 with BT340.
• 100µl of transformed E. coli MG1655Z1 with BT340.
• Transformed and concentrated E. coli MG1655Z1 with BT340.

• 20µl of control E. coli MG1655 (without plasmid)
• 50µl of transformed E. coli MG1655 with BT340.
• 100µl of transformed E. coli MG1655 with BT340.
• Transformed and concentrated E. coli MG1655Z1 with BT340.

Incubate for a night at 30°C.

C - Lemon scent

Liquid Culture

by Fabio

Due to an widespread infection of the cells made the 25th July, we collected the original strain DY330 to verify it's legitimacy.

2ml LB + 20μl of DY330. We incubate cultures at 30°C.

E - Salicylate Inducible Suppressing System

Plasmid DNA Purification

by Fabio

• BBa_J61051
• BBa_K228001

from Bacterial Culture made the 25th July

Protocol

Members present:

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

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