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From 2014.igem.org

Contents 1 Wednesday 30th July 1.1 Lab Work 1.1.1 A - The frame coli Odor free 1.1.1.1 Preparation of electrocompetent cells 1.1.1.2 Transformation of electrocompetent cells 1.1.2 C - Salicylate Inducible Suppressing System 1.1.2.1 DNA purification gel agarose 1.1.2.2 Ligation 1.1.3 D - Lemon scent 1.1.3.1 PCR of BBa_K762100

Wednesday 30th July

Lab Work

A - The frame coli Odor free

Preparation of electrocompetent cells

by Romain

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

Protocol:

Two dilution of 500µl 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 Romain

Strain used: E. coli MG1655Z1 and E. coli MG1655. Plasmid used: BT340 (code for a flipase for E. coli)

Make 2 electroporations 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.

Incubate during 1h at 30°C.

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.

C - Salicylate Inducible Suppressing System

DNA purification gel agarose

by Fabio

Once the segregate process made yesterday by electrophoresis, the DNA correspondent to the core of BBa_J61051 was purified from the gel agarose.

Segregate Process Protocol

Ligation

by Fabio

The final step to have a BioBrick Assembly is the Ligation reaction.

TODO: illustration of the process

• BioBrick BBa_J61051 (Salicylate promoter + NahR) as Part A
• BioBrick BBa_K228001 (RNA suppressor) as Part B

BioBrick Assembly - Ligation Protocol

D - Lemon scent

PCR of BBa_K762100

by Sean

This was a second attempt at yesterday's PCR, with several changes in parameters. Two tubes were prepared: one with yesterday's enzyme and another with a newer version of the same enzyme in order to determine if yesterday's enzyme was one of the causes of the rather unsatisfactory results.

Oligonucleotides used: iPS66, iPS67

Oligonucleotides were diluted twice from 100µM to 50µM.

Protocol

Add into each PCR tube the following:

Component	For a total volume of 50μl
H2O	35.5μl
Phusion buffer 5X	10μl
dNTPs	1μl
iPS66	1μl
iPS67	1μl
BBa_K762100	1μl
Phusion DNA polymerase	0.25μl

Tube was placed in PCR machine with the following parameters.

Cycle step	Temperature	Time	Cycle
Initial denaturation	98°C	1 min	1
Denaturation	98°C	15 s	25 - 30
Annealing	52°C	25 s	25 - 30
Extension	72°C	45 s	25-30
Final extension	72°C	10 min	1
Final extension	8°C	hold	1

Members there:

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

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