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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 1.1.1.3 PCR 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 1.1.3.2 Digestion 1.1.3.3 Electrophoresis

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 15ml 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 15ml 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 cuvettes:

• A control cuvette(without DNA): 50µl of E. coli MG1655Z1 or E. coli MG1655.
• A second cuvette: 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 cuvette and transfer in 2 tubes.

Spread on 8 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.

PCR

by Sean

Cultures used: MG1655Z1 10I and MG1655 100I.

Protocol

Note: although we have 2 cultures to prepare, it is recommended to prepare 3 tubes because of probable uncertainties when measuring volumes.

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 Cl.1
• BBa_J61051 Cl.2
• BBa_K228001 Cl.1
• BBa_K228001 Cl.2

from Bacterial Culture made the 25th July.

At the end of the process, we collected 4ml of each clone 1 plus 1ml of glycerol (80%) and stored at -80°C.

Protocol

Digestion

by Fabio

Once we have a good amount of our BioBricks' plasmid, it's time to do the digestion.

TODO: Digestion's protocol

Electrophoresis

by Fabio

The Electrophoresis was used to verify the success of the Digestion and the success of the plasmid DNA purification at the same time.

1. BBa_J61051 Cl.1
2. BBa_J61051 Cl.2
3. BBa_K228001 Cl.1
4. BBa_K228001 Cl.2

Results:

(TODO: Photos from LU000101 to LU000105)

Members present:

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

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