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 :

• Bba_K1155000 :
  • Buffer : 5µL
  • H2O : 38µL
  • Pfnr : 5µL
  • SpeI : 1µL
  • PstI : 1µL

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

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

| style="width:350px;border:1px solid black;" | | style="width:350px;border:1px solid black;vertical-align:top;" | Picture: lysed cells comparison.

• 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.
• 50µl phage: the petri dish is clear, bacteria are lysed by phages.
• We used a wild type strain to keep a stock and a Δ fnr E. coli strain to obtain phages that might have encapsidated a Δfnr::Km fragment.

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10µl,50µl and 100µl petri dishes are clear so phages are multiplied.

We let the antibiotic over night to select the right strain.

2 - ObtaµLining RBS_lacZ_ter.

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