Team:Pasteur Paris/Week 10

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week 10

08/03 - 08/07




Toxicity and solubility of TPA (Terephthalic acid)


  1. Dissolution of TPA in DMSO (dimethyl sulfoxide). The best concentration obtained is 62 mg/l

  2. Dilution of TPA stock in LB Broth => TPA precipitates at a concentration of 3.9 g/l, maximum concentration in LB Broth is 0.9 mg/l

  3. Solubilization of TPA by NaOH

  4. Creation of a stock solution of soluble TPA in water at 0.5 mol/l

  5. Test of the TPA Solubilization in LB Broth

  6. Preparation of different media containing terephthalic acid (TPA) and ethylene glycol (EG):
      Media 1 to 10 are made in solid form:
    1. M9
    2. M9 + TPA (1.2 mM)
    3. M9 + TPA (6 mM)
    4. M9 + TPA (12 mM)
    5. M9 + sucrose (1.2 mM)
    6. M9 + sucrose (6 mM)
    7. M9 + sucrose (12 mM)
    8. M9 + sucrose + TPA (6 mM)
    9. M9 + sucrose + TPA (1.2 mM)
      10. Media 11 to 15 are made in both solid and liquid form:
    10. LB Broth
    11. LB + TPA (1.2 mM)
    12. LB + TPA (6 mM)
    13. LB + TPA (12 mM)
    14. LB + TPA (20 mM)

  7. Growth curve of BAP 1 in media 11 to 14 in order to assess TPA toxicity.

  8. Plating of BAP 1 bacteria on all solid media to assess TPA toxicity and the ability of TPA to be used as a carbon source.

Enzymatic Assay of NB-Esterase


  1. XbaI ans PstI enzymatic digestion of the Biobrick BBa_K808030 and pDG011 plasmid.

  2. Gel purification of the digested fragments on 0.8% agarose gel.

  3. Ligation of pDG011 plasmid and BBa_K808030 using T4 DNA ligase.

  4. Gel migration on 0.8% agarose gel.

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Figure 8: Gel for verification of the ligation of BBa_K808030 and pDG011 plasmid.
There is no band for the ligation sample.


Gibson Assembly


  1. Ligation of TphA1/, Transp_, BBa_K1392932_, using T4 DNA ligase.

  2. DNA concentration measurement using a spectrophotometer.

Biobrick

DNA Concentration (ng/µl)

OD (230 nm)

OD (260 nm)

OD (280 nm)

OD (260 nm) / OD (280 nm)

OD (260 nm) / OD (230 nm)

808010

30.00

9.33

0.75

0,30

1.87

0.06

808011

50.00

11.78

1.00

0.63

1.61

0.09

808012

17.50

10.93

0.33

0.18

0.84

0.03

808013

5.00

10.55

0.10

0.06

1.56

0.01

808014

22.5

12.13

0.47

0.30

1.54

0.04

1095000

37.50

18.48

0.78

0.43

1.83

0.04