Contents 1 Notebook : August 8 1.1 Lab work 1.1.1 A - Aerobic/Anaerobic regulation system 1.1.1.1 Obtaining biobricks in pSB3K3 1.1.1.2 1 - Digestion of BBa_J04450 by EcoRI/PstI 1.1.1.3 2 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion 1.1.1.4 3 - Electrophoresis of BBa_J004450 digested by EcoRI/PstI 1.1.1.5 4- Electroelution of PSB3K3 digested by EcoRI/PstI 1.1.1.6 Objective : obtaining BBa_K1155007 1.1.1.7 1 - Colony PCR of BBa_K115007 in DH5α 1.1.1.8 2 - Electrophoresis to check the colony PCR products : BBa_K1155007 1.1.1.9 Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 1.1.1.10 1 - Tranduction of Km in MG1655Z1 1.1.2 A - Aerobic/Anaerobic regulation system / B - PCB sensor system 1.1.2.1 Objective : obtaining FNR and BphR2 proteins 1.1.2.2 1 - Extraction of plasmid of BphR2, FNR, RBS-FNR 1.1.2.3 2 - Gel purification of RBS-BphR2 Part I

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 let our digestion 1h30 at 37°C.

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 obtain 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/Pst1 Gel : 0.8%

PSB3K3 : 2750bp

We obtain 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

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

We mixed our colonies in 10µL of H2O.

Used quantities :

• DNA : 2µL
• Mix  : (it was divided in 25 tubes for each promotor with 23µL of mix in each on)
  • Oligo ... : 3.5µL
  • Oligo ... : 3.5µL
  • Buffer Dream Taq : 70µL
  • dNTP : 28µL
  • Dream Taq : 5µL
  • H2O : 590µL

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 obtain fragment 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 bacteria was called BW : Δfnr::Km. Our wild type bacteria was called MG1655Z1.

We did the first step of the protocol : bacteriophage stock which packed Km gene.

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

Objective : obtaining FNR and BphR2 proteins

1 - Extraction of plasmid of BphR2, FNR, RBS-FNR

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

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

We lost fragment. We will do the PCR again.

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