Notebook : August 12

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining Pfnr_RBS-LacZ-Term in PSB1C3

1 - Digestion of Bba_K1155000, Bba_K1155007 and Bba_K1155003

Anaïs, Nadia, XiaoJing

Protocol : digestion

Used quantities :

• Pfnr in PSB1C3 (Bba_K1155000) :
  • Buffer : 5µL
  • H2O : 38µL
  • Pfnr : 5µL
  • SpeI : 1µL
  • PstI : 1µL

• RBS-LacZ-Term in PSB1C3 (Bba_K1155007) :
  • Buffer : 5µL
  • H2O : 23µL
  • RBS-LacZ-Term : 20µL
  • XBal : 1µL
  • PstI : 1µL

• RBS-AmilCP-Term in PSB1C3 (Bba_K1155003) :
  • Buffer : 5µL
  • H2O : 33µL
  • RBS-AmilCP-Term : 10µL
  • XBal : 1µL
  • PstI : 1µL

2 - Electrophoresis of digestion pieces

We gonna check if the digestion was good by control the size of digestion pieces.

Well 1 : 6µL DNA Ladder Well 2 : 5µL of digested Bba_K1155000+1µl of 6X loading dy Well 3 : 5µL of digested Bba_K1155007+1µl of 6X loading dy Well 4 : 5µL of digested Bba_K1155003+1µl of 6X loading dy Gel : 0.8%

Expected sizes :

• RBS_LacZ_Term : 3500 kb
• RBS_AmilCP_Term :
• PSB1C3 :

We can't see any band for Bba_K1155000 digestion. We just do it again and let it in the fridge over night. 

...

1 - Digestion of Bba_K1155000, Bba_K11

Abdou

Clone 10,14 and 15 plamid extraction using nucleospin kit.

Protocol : hight copy plamid extraction

Results: concentration measured by nanodrop

• clone 10: C=38ng/µl 260/280= 1.78
• clone 14: C=48.5ng/µl 260/280=1.90
• clone 15: C=52 ng/µl 260/280=1.78

We still have to sequence plasmids in order to verify our results.

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

1 - Gibson assembly.

August 1st PCR purification to be sure about the experiment. BphR2 part1, BphR2 part2 and RBS_BphR2_part1, FNR part1, FNR part2 and RBS_FNR_part1.

Protocol : PCR_clean_up

Results:

we have no fragment so we must do again these PCRs