Contents 1 Notebook : August 13 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 - Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI 1.1.1.3 2 - Extraction of BBa_K1155004, BBa_1155005, BBa_K1155006 from DH5αstrain 1.1.1.4 3 - Digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI and by SpeI/EcoRI 1.1.1.5 4 - Electrophoresis to check the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI and by SpeI/EcoRI 1.1.1.6 5 - Liquid culture of MG1655Z1 Δfnr::Km 1.1.1.7 Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in pSB3K3 1.1.1.8 1 - Digestion of BBa_J04450 by EcoRI/PstI 1.1.1.9 2 - Electrophoresis to check the digestion of BBa_J04450 by EcoRI/PstI 1.1.2 A - Aerobic/Anaerobic regulation system / B - PCB sensor system 1.1.2.1 Objective : obtaining FNR and BphR2 proteins (Gibson assembly) 1.1.2.2 1 - PCR of FRN Part I, FNR Part II, BphR2 Part I 1.1.2.3 2 - Electrophoresis of PCR products : FRN Part I, FNR Part II, BphR2 Part I 1.1.2.4 3 - Electrophoresis of PCR product : BphR2 Part I 1.1.2.5 4 - PCR of BphR2 Part I

Notebook : August 13

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI

Nadia

Well 1 : 6µL DNA Ladder Well 2 : 5µL BBa_K1155000 digested by SpeI/PstI +1µl of 6X loading dye Gel : 0.8%

Expected sizes :

• Pndh* : 111bp
• pSB1C3 : 2070bp

We can't see any band for BBa_K1155000 digestion. The digestion failed because we used the wrong enzymes. We will do it again with the good ones.

2 - Extraction of BBa_K1155004, BBa_1155005, BBa_K1155006 from DH5αstrain

XiaoJing

Protocol : High-copy plasmid extraction

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

Anaïs, Nadia

Used quantities :

• BBa_K1155000 :
  • Buffer FD : 5µL
  • H2O : 39µL
  • DNA : 5µL
  • SpeI FD : 1µL

• BBa_K1155000 :
  • Buffer FD : 5µL
  • H2O : 38µL
  • DNA : 5µL
  • SpeI FD : 1µL
  • EcoRI FD : 1µL

• BBa_K1155004, BBa_K1155005, BBa_K1155006 :
  • Buffer FD : 2µL
  • H2O : 7µL
  • DNA : 10µL
  • SpeI FD : 1µL

• BBa_K1155004, BBa_K1155005, BBa_K1155006 :
  • Buffer FD : 2µL
  • H2O : 6µL
  • DNA : 10µL
  • SpeI FD : 1µL
  • EcoRI FD : 1µL

We incubat the digestion at 37°C during 10 minutes.

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

Anaïs, Nadia

Well 1 : 6µL DNA Ladder Well 2 : 5µL BBa_K1155000 digested by SpeI + 1µl of 6X loading dye Well 3 : 5µL BBa_K1155000 +1µl of 6X loading dye Well 4 : 5µL BBa_K1155000 digested by EcoRI/Spe I + 1µl of 6X loading dye Gel : 0.8%

Expected sizes :

• Pndh* : 111bp
• pSB1C3 : 2070 kb

We lost all our digestion product so we will do it again.

Well 0 : 6µL DNA Ladder Well 1 : 5µL BBa_K1155006 digested by SpeI + 1µl of 6X loading dye Well 2 : 5µL BBa_K1155005 digested by SpeI + 1µl of 6X loading dye Well 3 : 5µL BBa_K1155004 digested by SpeI + 1µl of 6X loading dye Well 4 : 5µL BBa_K1155006 digested by EcoRI/SpeI + 1µl of 6X loading dye Well 5 : 5µL BBa_K1155005 digested by EcoRI/SpeI + 1µl of 6X loading dye Well 6 : 5µL BBa_K1155004 digested by EcoRI/SpeI + 1µl of 6X loading dye Gel : 0.8%

Expected sizes :

• NarK, Nar G, NirB : 200kb
• pSB1C3 : 2070bp
• NarK in pSB1C3, NarG in pSB1C3, NirB in pSB1C3 : 2270bp

We lost all our double digestion product so we will do it again. We obtain BBa_K1155004, BBa_K1155005, BBa_K1155006 digested by SpeI fragments at the right size. We can purify it.

5 - Liquid culture of MG1655Z1 Δfnr::Km

XiaoJing

We picked up one colony in 5mL of LB and 5µL of kanamycine. We do it twice.

We incubated our culture at 37°C with agigation at 150rpm.

We obtain fragments at the right size.

Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in pSB3K3

1 - Digestion of BBa_J04450 by EcoRI/PstI

Anaïs

Used quantities :

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

We incubated the digestion at 37°C during 10 minutes.

2 - Electrophoresis to check the digestion of BBa_J04450 by EcoRI/PstI

Anaïs, Nadia

Well 4 : 6µL DNA Ladder Well 7 : 5µL of BBa_J04450 digested by EcoRI/PstI + 1µL of 6X loading dye Gel : 0.8%

Expected sizes :

• pSB3K3 : 2750bp
• GFP : 1069bp

We obtained a fragment at the right size.

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

Objective : obtaining FNR and BphR2 proteins (Gibson assembly)

1 - PCR of FRN Part I, FNR Part II, BphR2 Part I

Anaïs

Used quantities :

• Bphr2 Part I :
  • Oligo 54F : 1µL
  • Oligo 55R : 1µL
  • Buffer Phusion : 10µL
  • DNA of Pseudomonas pseudoalcaligenes : 1µL
  • dNTP : 1µL
  • Phusion : 0.5µL
  • H2O : 35.5µL

• FNR Part I :
  • Oligo 59F : 1µL
  • Oligo 60R : 1µL
  • Buffer Phusion : 10µL
  • DNA Escherichia coli : 1µL
  • dNTP : 1µL
  • Phusion : 0.5µL
  • H2O : 35.5µL

• FNR Part II :
  • Oligo 61F : 1µL
  • Oligo 62R : 1µL
  • Buffer Phusion : 10µL
  • DNA Escherichia coli : 1µL
  • dNTP : 1µL
  • Phusion : 0.5µL
  • H2O : 35.5µL

PCR Program :

• BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I :

• FNR Part I, FNR Part II, RBS-FNR Part I :

2 - Electrophoresis of PCR products : FRN Part I, FNR Part II, BphR2 Part I

Damir

Well 1 : 5µL FNR Part II + 1µl of 6X loading dye Well 2 : 5µL BphR2 Part I + 1µl of 6X loading dye Well 3 : 5µL FNR Part I + 1µl of 6X loading dye Well 4 : 6µL DNA Ladder Gel : 1%

Well 1 : 5µL FNR Part II + 1µl of 6X loading dye Well 2 : 5µL BphR2 Part I + 1µl of 6X loading dye Well 3 : 5µL FNR Part I + 1µl of 6X loading dye Well 4 : 6µL DNA Ladder Gel : 1%

Expected sizes :

• FNR Part I : 597 kb
• FNR Part II : 200 kb
• BphR2 Part I : 178 kb

It's impossible to read the first gel. We do it again. In the second gel, we obtain FNR Part I and FNR Part II fragments at the right size. We can purify it. We also obtain BphR2 Part I frangment at the right size but it was mix with other DNA frangments. We will try to make a gel purification of it.

3 - Electrophoresis of PCR product : BphR2 Part I

Anaïs, Damir, Nadia

Well 1 : 6µL DNA Ladder Well 2 : 40µL of BphR2 Part I + 8µL of 6X loading dye Gel : 1%

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

Even if the two lightest stripes are overlapping, aims to do a second gel purification with these two stripes, we did the gel purification . After argumentation, we decided to do the PCR of BphR2 Part I again thanks to new PCR program and new quantities.

4 - PCR of BphR2 Part I

Anaïs

Used quantities :

• Oligo 54F : 1µL
• Oligo 55R : 1µL
• DNA : 2µL
• Buffer Phusion : 10µL
• dNTP : 1µL
• Phusion : 1µL
• H2O : 33.5µL

PCR Program :

5 - Gel purification of PCR product : FNR Part I, FNR Part II, RBS-FNR Part I, BphR2 Part II, RBS-BphR2 Part I'

XiaoJing

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

Nanodrop :

• FNR Part I : 152.7ng/µL
• FNR Part II : 137.2ng/µL
• RBS-FNR Part I : 153.7ng/µL
• BphR2 Part II : 129.9ng/µL
• RBS-BphR2 Part I : 158.1ng/µL

The purification was good. We will do the Gibson assembly.

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