Notebook : August 9

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining Δ fnr E. coli strain by transduction to test our biobricks

Abdou, Anais, Damir, Nadia, XiaoJing

We do the exprience again because the lysis made on wensday didn't happen. It's probably because the phage strain was too old ( 2001)

Protocol : transduction

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.

Now we can do a gel purification of the highest band which contains the PSB3K3 plasmid.

2.2 - Electroelution of the highest band to extract the PSB3K3 plasmid from the gel

Nadia

Protocol : Electroelution

We let the plasmid precipitate during the night.

Obtaining RBS_LacZ+Term_PSB1C3

1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies

Anaïs

• Colony counting :
  • Low concentration petri dish : 47 colonies
  • High concentration petri dish : 145 colonies

• Picking of 25 colonies

• Preparation of 700µL of Master mix
  • H₂O : 590µL
  • dNTP : 28µL
  • VF2 primer : 3.5µL
  • VR primer : 3.5µL
  • DreamTaq buffer 10x : 70µL
  • DreamTaq enzyme : 5µL

Protocol : Colony PCR

PCR Program :
 [IMAGE]

2 - Gel electrophoresis of the colony PCR products

Anaïs, Damir

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

Expected size : 3583bp

Colonies 10, 14, 15 exhibit plasmids with the right length.