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Week 10
08/03 - 08/07
Toxicity and solubility of TPA (Terephthalic acid)
- Dissolution of TPA in DMSO (dimethyl sulfoxide). The best concentration obtained is 62 mg/l
- 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
- Solubilization of TPA by NaOH
- Creation of a stock solution of soluble TPA in water at 0.5 mol/l
- Test of the TPA Solubilization in LB Broth
- Preparation of different media containing terephthalic acid (TPA) and ethylene glycol (EG):
Media 1 to 10 are made in solid form:
- M9
- M9 + TPA (1.2 mM)
- M9 + TPA (6.0 mM)
- M9 + TPA (12.0 mM)
- M9 + sucrose (1.2 mM)
- M9 + sucrose (6.0 mM)
- M9 + sucrose (12.0 mM)
- M9 + sucrose + TPA (6.0 mM)
- M9 + sucrose + TPA (1.2 mM)
Media 11 to 15 are made in both solid and liquid form:
- LB Broth
- LB + TPA (1.2 mM)
- LB + TPA (6.0 mM)
- LB + TPA (12.0 mM)
- LB + TPA (20.0 mM)
- Growth curve of BAP 1 (E. coli, kindly provided by Blaine Pfeifer, U. Buffalo)in media 11 to 14 in order to assess TPA toxicity.
- 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
- XbaI ans PstI enzymatic digestion of the Biobrick BBa_K808030 and pDG011 plasmid.
- Gel purification of the digested fragments on 0.8% agarose gel.
- Ligation of pDG011 plasmid and BBa_K808030 using T4 DNA ligase.
- Gel migration on 0.8% agarose gel.
Figure 8: Gel for verification of the ligation of BBa_K808030 and pDG011 plasmid.
There is no band for the ligation sample.
Gibson Assembly
- Ligation of TphA1/, Transp_, BBa_K1392932_, using T4 DNA ligase.
- 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 |
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