Paris/July 18

From IGEM07

yesterday -- tomorrow

Contents

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Transduction of w121 strain

Titration of the stock of phage from 12.7.7. : ~109 phages/ml : OK

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FtsZTS strain screening

We want to test these strains :

  • 121.4
  • 121.5
  • 121.6
  • 129
  • 1.129
  • DCR 14.1
  • DCR 14.2
  • DCR 14.3

I made glycerol stock of these strains, stored in the freezer. Test :

  • Spread these strains on two plates (preheated before spreading) :
    • One at 30°C
    • One at 42°C

The results tomorrow.

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Minipreps

We have 2 clones for each plasmid :

  • E0422
  • E0241
  • E0840
  • B0030
  • J61047

Elution within 50µL H2O
DNA is within the Miniprep box in the -20°C fridge.

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Digestion products

Photos !!!

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DAP solution contamination test

Results :

  • 50µL of H20 (control) OK
  • 50µL of aliquot DAP 50mM (in use) OK
  • 50µL of DAP 1mM CONTAMINATED
  • 50µL of DAP 0.2 mM OK
  • 50µL of DAP 50mM OK
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PCRs

PCR : Assembly PCR Lox71-Fts1-FTsZ1 + FtsZ2
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 2580
50, 55, 60, 65°C dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 3 Lox71-FtsA-F 2.5µL NO
3m00' Oligo R 10µM 2 FtsZ-R 2.5µL Image (click to enlarge)
Number cycles Water 30µL [[Image:|30px]]
35 Polymerase Phusion 0.5µL Band (0=ladder)
DNA Lox71-FtsA-FtsZ1 2µL + FtsZ2 2µL
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E.coli pKS::DGAT

  • After overnight culture (see July 17), we do not observe significant triglyceride synthesis.
  • We decide to test in solid culture and wait some days. E.coli transformed by pKS::DGAT and the negative control (E.coli transformed by part B0015) were spread on LB medium +- oleate 2mM +- IPTG (0.4mM).
  • We try in M9 Minimum Medium (see Protocols) replacing lactose/galactose by 2mM oleate, too.
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