Contents 1 Notebook : August 14 1.1 Lab work 1.1.1 A - Aerobic/Anaerobic regulation system 1.1.1.1 Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 1.1.1.2 1 - Gel purification of the digestion of BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI 1.1.1.3 2 - Extraction of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 from DH5α 1.1.1.4 3 - Digestion of BBa_K1155004,BBa_K1155005, BBa_K1155006 by EcoRI/SpeI 1.1.1.5 4 - Electrophoresis to check the digestion of BBa_K1155004, BBa_K1155005, BBa_K1155006 by EcoRI/SpeI 1.1.1.6 5 - Digestion of BBa_K1155000, BBa_K1155004,BBa_K1155005, BBa_K1155006 by SpeI and EcoRI/SpeI 1.1.1.7 6 - Electrophoresis to check the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI and EcoRI/SpeI 1.1.1.8 7 - Gel purification of the digestion of BBa_K1155000 and BBa_K1155006 by EcoRI/SpeI and BBa_K1155004, BBa_K1155005 and BBa_K1155006 by SpeI 1.1.1.9 8 - Electrophoresis of the digestion of BBa_K1155003, BBa_K1155007 by XBaI/PstI 1.1.1.10 9 - Gel purification of the digestion of BBa_K1155003 and BBa_K1155007 by XBaI/PstI 1.1.1.11 10 - Glycerol stock of MG1655Z1 Δfnr::Km 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 - Gibson assembly 1.1.2.3 2 - Electrophoresis of PCR products : BphR2 Part I 1.1.2.4 3 - PCR of BphR2 Part I

Notebook : August 14

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Gel purification of the digestion of BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI

XiaoJing

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

Nanodrop :

• NarG : 10.5ng/µL
• NarK : 16.8ng/µL
• NirB : 24.3ng/µl

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

2 - Extraction of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 from DH5α

XiaoJing

Protocol : High-copy plamid extraction

Nanodrop :

• NarK: 89.8ng/µL
• NarG : 80.7ng/µL
• Nir B :65.8ng/µL
• Pndh* : 227ng/µL

The extraction was good. We will digest BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006.

3 - Digestion of BBa_K1155004,BBa_K1155005, BBa_K1155006 by EcoRI/SpeI

Nadia

• Buffer FD : 2µL
• H2O : 6µL
• DNA : 10µL
• SpeI FD : 1µL
• EcoRI FD : 1µL

We let the digestion at 37°C during 15 minutes.

4 - Electrophoresis to check the digestion of BBa_K1155004, BBa_K1155005, BBa_K1155006 by EcoRI/SpeI

Anaïs, Nadia

Well 1 : 6µL DNA Ladder Well 2 : 5µL of BBa_K1155006 digested by EcoRI/SpeI +1µl of 6X loading dye Well 3 : 5µL of BBa_K1155005 digested by EcoRI/SpeI +1µl of 6X loading dye Well 4 : 5µL of BBa_K1155004 digested by EcoRI/SpeI +1µl of 6X loading dye Gel : 1%

Expected sizes :

• pSB1C3 : 2070kb
• NarK, NarG, NirB : 200kb

We obtained NarK, NarG, NirB fragments at the right size but in very few quantity. We do it again but this time we will use more quantity of enzymes.

5 - Digestion of BBa_K1155000, BBa_K1155004,BBa_K1155005, BBa_K1155006 by SpeI and EcoRI/SpeI

Anaïs, Nadia

• Digestion by SpeI :
  • Buffer : 2µL
  • SpeI : 2µL
  • ADN : 15µL
  • H20 : 1µL

• Digestion by EcoRI and SpeI :
  • Buffer : 3µL
  • SpeI : 2µL
  • EcoRI : 2µL
  • ADN : 20µL
  • H20 : 3µL

We let digestions at 37°C during 10 minutes.

6 - Electrophoresis to check the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI and EcoRI/SpeI

Anaïs, Nadia

Well 1 : 5µL of BBa_K1155000 digested by SpeI + 1µl of 6X loading dye Well 2 : 5µL of BBa_K1155006 digested by SpeI + 1µl of 6X loading dye Well 3 : 5µL of BBa_K1155005 digested by SpeI + 1µl of 6X loading dye Well 4 : 5µL of BBa_K1155004 digested by SpeI + 1µl of 6X loading dye Well 5 : 6µL DNA Ladder Well 6 : 5µL of BBa_K1155000 digested by EcoRI/Spe I + 1µl of 6X loading dye Well 7 : 5µL of BBa_K1155006 digested by EcoRI/Spe I + 1µl of 6X loading dye Well 8 : 5µL of BBa_K1155005 digested by EcoRI/Spe I + 1µl of 6X loading dye Well 9 : 5µL of BBa_K1155004 digested by EcoRI/Spe I + 1µl of 6X loading dye Gel : 1%

Expected sizes :

• Pndh* in pSB1C3 : 2200bp
• Nar K in pSB1C3, NarG in pSB1C3, NirB in pSB1C3 : 2270bp
• pSB1C3 : 2070kb
• NarK, NarG, NirB : 200kb
• Pndh* : 111bp

Well 1 : we have done one digestion so we have to obtain one fragment, we have two stripes, the digestion of BBa_K1155000 by SpeI wasn't good. Well 2, 3, 4, 6, 7 : we obtain NarG, NarK and NirB in pSB1C3 and Pndh*, NarK fragments at the right size, we can purify it. Well 8, 9 : we have done two digestions so we have to obtain two fragments, we have only one stripe, the digestion of BBa_K1155005 and BBa_K1155004 by EcoRI/SpeI wasn't good.

7 - Gel purification of the digestion of BBa_K1155000 and BBa_K1155006 by EcoRI/SpeI and BBa_K1155004, BBa_K1155005 and BBa_K1155006 by SpeI

Anaïs, Nadia

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

The cutting for the gel purification was good.

Nanodrop :

• Pndh* : 13.7ng/µL
• NarK : 27.9ng/µL
• NarK in PSB1C3 : 87.1ng/µL
• NarG in PSB1C3 : 39.2ng/µL
• NirB in PSB1C3 : 37.7ng/µL

The purification was good. We will ligate them.

8 - Electrophoresis of the digestion of BBa_K1155003, BBa_K1155007 by XBaI/PstI

Nadia

Well 1 : 6µL of DNA Ladder Well 2 : 20µL of BBa_K1155003 digested by XBaI/PstI + 4µL of 6X loading dye Well 3 : 20µL of BBa_K1155003 digested by XBaI/PstI + 4µL of 6X loading dye Well 4 : 20µL of BBa_K1155007 digested by XBaI/PstI + 4µL of 6X loading dye Well 5 : 20µL of BBa_K1155007 digested by XBaI/PstI + 4µL of 6X loading dye

Expected sizes :

• RBS_LacZ-Term : 3500bp
• RBS_AmilCP-Term : 824bp

We obtained fragments at the right size. We will purify it.

9 - Gel purification of the digestion of BBa_K1155003 and BBa_K1155007 by XBaI/PstI

Nadia

Nanodrop :

• RSB-LacZ-Term : 59.6ng/µL
• RBS-AmilCP-Term : 28.7ng/µL

We obtained low quantities of plasmid that's why we will make an Ethanol precipitation to concentrate it.

10 - Glycerol stock of MG1655Z1 Δfnr::Km

XioaJing

Used quantities :

• Glycerol : 500 µL
• MG1655Z1 Δfnr::Km: 1 mL

We stored the bacteria at -20°C.

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

Objective : obtaining FNR and BphR2 proteins

1 - Gibson assembly

Used quantities :

• RBS-BphR2 :
  • pSB1C3 : 3µL
  • BphR2 Part I : 1µL
  • BphR2 Part II : 1µL
  • Gibson mix : 15µL

• FNR :
  • pSB1C3 : 3µL
  • FNR Part I : 1µL
  • FNR Part II : 1µL
  • Gisbon mix : 15µL

• RBS-FNR :
  • pSB1C3 : 3µL
  • RBS-FNR Part I : 1µL
  • FNR Part II : 1µL
  • Gibson mix : 15µL

We incubate these mix at 50°C during 1h inside PCR machine. Then we keep these mix at 4°C during the week end.

2 - Electrophoresis of PCR products : BphR2 Part I

Damir

Well 1 : 6µL DNA Ladder Well 2 : 5µL of BphR2 Part I+1µL of 6X loading dye

Well 1 : 6µL DNA Ladder Well 2 : 5µL of BphR2 Part I + 1µL of 6X loading dye Well 3 : 5µL of BphR2 Part I + 1µL of 6X loading dye

Expected sizes :

• BphR2 Part I : 178kb

On the first gel, all deposits disappear so we did the electrophoresis again. On the second gel, we didn't obtain stripes at the good size. We do the PCR again using new quantities and a new PCR program.

3 - PCR of BphR2 Part I

Damir

Used quantities :

• Oligo 54F : 2µL
• Oligo 55R : 2µL
• DNA : 1µL
• Buffer Phusion : 10µL
• dNTP : 1µL
• Phusion : 1µL
• DMS0 : 2µL
• H2O : 31µL

PCR program :

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