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1 Notebook : August 27
- 1.1 summary
- 1.2 lab work
  - 1.2.1 2 - Gel electrophoresis of the colony PCR products
  - 1.2.2 A - Aerobic/Anaerobic regulation system / B - PCB sensing system
    - 1.2.2.1 1 -Gel electrophoresis of parts of Gibson assembly.
      - 1.2.2.1.1 2 - Gel purification of the PSB1C3 Clean for Gibson

Notebook : August 27

summary

We got colonies after the transformation of E.coli with the ligation mix "PnirB_PSB1C3 and RBS_LacZ_Term"

promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3 "Pndh*_PSB1C3 plus RBS_LacZ+Term" "Pndh*_PSB1C3 plus RBS_AmilCP+Term" so we tested for correct ligation with PCR on colonies

We did not get any colonies for the Gibson assembly so we analyzed by gel electrophoresis the parts used for the Gibson assembly to verify their sizes and concentrations.
Digestion DnpI purification of the the PSB1C3 clean for Gibson and electrophoresis of it .

lab work

A.aero/anaerobic regulation system
- 1.Colony PCR on e.coli with FNR Promoter plus RBS_LacZ+Term_PSB1C3 or RBS_AmilCP+Term_PSB1C3 for 8 colonies of each

Xiaojing

A1-8: promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3
B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3
C1-8: promoter fnr(repressor) plus RBS_LacZ+Term_PSB1C3
D1-8: promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
Picking of 32 colonies

Preparation Master mix
- H₂O : 490µL
- dNTP : 17.5µL
- VR primer : 17.5µL
- VF2 primer : 17.5µL
- DreamTaq buffer 10x : 87.5µL
- DreamTaq enzyme : 7µL

Protocol : Colony PCR PCR Program :

2 - Gel electrophoresis of the colony PCR products

[[]]	6µL DNA Ladder 10µL sample per well Gel : 0.8%

Expected size : 3583bp

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

1 -Gel electrophoresis of parts of Gibson assembly.

[[]]

Well 1 : 6µL DNA Ladder
Well 2 : 1µL of PSB1C3 Clean
Well 3 : 3µL of RBS_BphR2 part1+1µl of 6X loading dy
Well 4 : 3µL of BphR2 part2+1µl of 6X loading dy
Well 5: 3µL of RBS_FNR part1 +1µl of 6X loading dy
Well 6: 3µL of FNR part1 +1µl of 6X loading dy
Well 7 : 3µL of FNR part2 +1µl of 6X loading dy

Gel : 1.0%

Now we do a Digestion Dnp1 purification of the  the PSB1C3 clean for Gibson.

PSB1C3 clean for Gibson	17µl
enzyme Dnp1	1µl
buffer	2µl
total	20µl

Incubate at 37°C for 1h30mins.

2 - Gel purification of the PSB1C3 Clean for Gibson

2.1 - Migration of 20µl PSB1C3 Clean for Gibson digested by Dnp1 '

[[]]	Well 1 : 6µL DNA Ladder Well 2 : 1µL of PSB1C3 Clean dig Dnp1(08/06/2013) Well 3 : 20µL of PSB1C3 Clean dig Dnp1(08/27/2013) Gel : 1.0%

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@@ Line 6: / Line 6: @@
 *We got colonies after the transformation of E.coli with the ligation mix "PnirB_PSB1C3 and RBS_LacZ_Term"
 promoter fnr(activator)nirB plus  RBS_AmilCP+Term_PSB1C3
-promoter fnr(repressor) plus  RBS_LacZ+Term_PSB1C3
+"Pndh*_PSB1C3 plus  RBS_LacZ+Term"
-promoter fnr(repressor) plus  RBS_AmilCP+Term_PSB1C3t
+"Pndh*_PSB1C3 plus  RBS_AmilCP+Term"
-so we do Colony PCR for it
+so we tested for correct ligation with PCR on colonies
-* We didn't get colonies on Gibson so we do a gel electrophoresis of  parts of Gibson assembly to verify the size
+* We did not get any colonies for the Gibson assembly so we analyzed by gel electrophoresis the parts used for the Gibson assembly to verify their sizes and concentrations.
-* Digestion Dnp1 purification of the  the PSB1C3 clean for Gibson and  electrophoresis of  it .
+* Digestion DnpI purification of the  the PSB1C3 clean for Gibson and  electrophoresis of  it .
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Team:Paris Saclay/Notebook/August/27

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