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Notebook : August 8

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining biobricks in pSB3K3

1 - Digestion of BBa_J04450 by EcoRI/PstI

Nadia

Used quantities :

DNA : 5µL
Buffer FD : 2µL
EcoRI FD : 1µL
PstI FD : 1µL
H2O : 11µL

We incubate our digestion at 37°C for 1h30.

2 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion

Damir, Nadia

Well 1 : 6µL DNA Ladder
Well 2 : 5µL of BBa_J004450 digested by EcoRI/Pst1
Gel : 0.8%

Expected sizes :

pSB3K3 : 2750kb
GFP : 1069kb

We obtained fragments at the right size. We will purify the highest band which contains the pSB3K3 plasmid.

3 - Electrophoresis of BBa_J004450 digested by EcoRI/PstI

Anaïs

Well 1 : 6µL DNA Ladder
Well 2 : 45µL of BBa_J004450 digested by EcoRI/Pst I
Gel : 0.8%

pSB3K3 : 2750bp

We obtained fragments at the right size. We will purify the highest band which contains the pSB3K3 plasmid.

4- Electroelution of pSB3K3 digested by EcoRI/PstI

Nadia

Protocol : Electroelution

We lost our plasmid. We will do the digestion again.

Objective : obtaining BBa_K1155007

1 - Colony PCR of BBa_K115007 in DH5α

Anaïs

Transformation of 08/07/13 works. we will make a PCR Colony.

We took a single colony and resuspend in 10µL H2O. For each Biobrick, we did 25 PCR Colonies.

Used quantities :

PCR preparation mix for 25 different colonies including:

- Oligo 44 : 3.5µL
- Oligo 45 : 3.5µL
- Buffer Dream Taq : 70µL
- dNTP : 28µL
- Dream Taq : 5µL
- H2O : 590µL

PCR reaction:

DNA : 2µL
Mix :23µL

Total volume: 25µL

PCR Program :

2 - Electrophoresis to check the colony PCR products : BBa_K1155007

Anaïs, Damir

Well 1 : 6µL DNA Ladder
Well 2 to 24 : 10µL of BBa_K1155007 + 2µL of 6X loading dye
Well 25 : 6µL DNA Ladder
Well 26 : 6µL DNA Ladder
Well 27 : 6µL DNA Ladder
Well 28 : 10µL of BBa_K1155007 + 2µL of 6X loading dye
Gel : 0.8%

Expected size :

BBa_K1155007 : 3583 bp

We obtained fragments at the right size for colonies 10, 14 and 15. We will extract BBa_K1155007.

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Tranduction of Km in MG1655Z1

Anaïs, Nadia

We didn't obtain colonies from the transduction of 08/07/13. We will do it again.

Protocol : Transduction

Our Mutant strain is BW1328 (Δfnr::Km) and the wild type strain is MG1655Z1.

We did the first step of the protocol : bacteriophage stock on BW1328 (Δfnr::Km).

A - Aerobic/Anaerobic regulation system / B - PCB sensor system

Objective : obtaining FNR and BphR2 proteins

1 - Extraction of plasmid pSB1C3 containing BphR2 ,FNR and RBS-FNR in competent cell DH5α

Damir

Transformation of 08/02/13 works. We will extract plasmids.

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

We lost our plasmids. We will do the Gibson assembly again.

2 - Gel purification of RBS-BphR2 Part I

Nadia, XiaoJing