Team:Evry/Notebook/w12

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Iron coli project

Week 12: 2nd September - 8th September

Creation of a E. Coli ΔFur

02/09/13
04/09/13
We picked colonies from our four plates, that we grew overnight at 42°C, to plate each of them on one plate with kanamycin

TECAN

02/09/13
To further improve the interpretability of the plate reading, we need to transfer our construction from a BL21 to a TOP10 strain. As a start, we transformed the following constructions:

- AceB with LacI
- FecA with GFP
- FepA with GFP
- FepA with LacI
- Fes with GFP
- Fes with LacI
- YbiL with GFP
- YbiL with LacI
- YncE with GFP
- YncE with LacI
- control positive with pSB1A3
- control negative

We did not have anymore AceB with GFP plasmid available to realize the transformation. We pre-cultured a remaining glycerol stock at 37°C overnight:

10 mL LB
10 µL Carbenicillin 1000X
50 µL cells from glycerol stock

03/09/13

The transformations succeeded and made liquid cultures for glycerol conservation.
Also, we isolated the plasmid AceB-GFP from the pre-culture of the 03/09/13. As a consequence, we transformed our last plasmid in a TOP10 strain.

04/09/13

To anticipate the TECAN for the evening, we started a pre-culture of the following constructions we want to characterize:

TOP10 (growth control)
TOP10 PL-LacO (GFP control)
TOP10 YbiL-GFP
TOP10 YncE-GFP


Also, we prepared the iron solutions from 10^-3 to 10^-10 M that will be used to prepare all the different media and completed the plate.

TECAN GRID

05/09/13

To anticipate the TECAN for the evening, we started a pre-culture of the following constructions we want to characterize:

TOP10 (growth control)
TOP10 PL-LacO (GFP control)
TOP10 AceB-GFP
TOP10 Fes-GFP


Plasmid 3

We launch another PC to get Enterobactins A, B, C, D, E and F genes. Add the recquired primers in each tube:
  1. EntA: primers 065 and 066
  2. EntB: primers 067 and 068
  3. EntC: primers 069 and 070
  4. EntD: primers 071 and 072
  5. EntE: primers 073 and 074
  6. EntF: primers 075 and 076
  7. Positive controle: primers 009 and 010
  8. Negative controle: primers 009 and 010
We use pEntC as our controle.
For more details about our primers, see the corresponding page.
We then prepared a master mix 1 with:
  • 9 x 27,5 = 247,5 µL of distilled water
  • 9 x 1 = 9 µL of dNTPs
  • 9 x 10 = 90 µL of Q5 Buffer
  • 9 x 0,5 = 4,5 µL of Q5 For the negative controle, we added 1 µL of distilled water in tube 8 and 39 µL of master mix 1.
    For other tubes (6 genes and positive controle), we added 8 µL of genomic DNA in the master mix 1. Then add 40 µL of master mix 1' in each tube.

    BioBricking

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