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From 2014.igem.org

Contents 1 Tuesday 29th July 1.1 Lab Work 1.1.1 A - The frame 1.1.1.1 PCR 1.1.1.2 Electrophoresis 1.1.2 C - Lemon scent 1.1.2.1 PCR of BBa_K762100 1.1.3 E - Salicylate Inducible Suppressing System 1.1.3.1 Digestion 1.1.3.2 Electrophoresis

Tuesday 29th July

Lab Work

A - The frame

PCR

by Romain

StrainS used: E. coli MG1655Z1 and E. coli MG1655, and oligonucleotides used: FTR-Apra-F and FTR-Apra-R.

Protocol:

• 218µl of H20 MilliQ
• 80µl GoTaq buffer 5X
• 16µl oligonucleotide FTR-Apra-F (10mM)
• 16µl oligonucleotide FTR-Apra-R (10mM)
• 12µl DMSO 10mM
• 32µl MgCl2 (25mM)
• 8µl dNTP (10mM)
• 2µl of GoTaq enzyme
• 2µl of different bacterial cultures in each tubes:

1. Tube 1: MG1655Z1 10I
2. Tube 2: MG1655 100II
3. Tube 3: MG1655 50I
4. Tube 4: MG1655 100I
5. Tube 5: MG1655Z1 10II
6. Tube 6: MG1655 50II
7. Tube 7: MG1655Z1 10I positive control with plasmid

Electrophoresis

by Romain

C - Lemon scent

PCR of BBa_K762100

by Sean

Oligonucleotides used: iPS66, iPS67

Oligonucleotides were diluted twice from 100µM to 50µM.

Protocol

Add into a 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	variable	55°C	25 - 30
Extension	72°C	45 s	25-30
Final extension	72°C	10 min	1
Final extension	8°C	hold	1

E - Salicylate Inducible Suppressing System

Digestion

by Fabio

BioBrick BBa_J61051 (Salicylate promoter + NahR)

Electrophoresis

by Fabio

Results:

First process to verify the digestion

Second one to separate both parts (only with J61051).

Members there:

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

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