Notebook : August 27

summary

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

"PnirB_PSB1C3 plus RBS_AmilCP+Term" "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

Expected size : 3583bp

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

1 -Gel electrophoresis of parts of Gibson assembly.

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

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 '