August, 16th
Inoculum of T9002-ENTERO4C5 and ENTERO-RBS (1 ml of M9 + Cm12.5) to test again diluted supernatants of E37, E38, E39, E40 and ENTERO4C5.
Inoculum of E41N-1 and J101-4C5 for plasmid purification 6 ml of LB + Cm12.5.
Streak of E32-2, E33-2, J101-31, J101-E5 and ENTERO4C5 on an LB-agar plate + Cm12.5 to test and characterize PtetR promoter with different Ribosome Binding Sites (BBa_B0030, BBa_B0031).
August, 17th
E41N-1 and J101-4C5 plasmids were purified with mini Prep kit:
| Plasmid |
DNA (ng/μl) |
| E41N-1 |
33.7 |
| J101-4C5 |
25.4 |
T9002-ENTERO4C5 and ENTERO-RBS were diluted 1:500 in a final volume and 1 ml of 12 ml M9 + Cm12.5 respectively and grown for 6 hours.
3OC6-HSL was diluted to known concentrations in order to induce T9002-ENTERO4C5 biosensor and obtain a reference curve for the measurements on E37, E38, E39, E40 and ENTERO4C5 supernatants.
Supernatants were diluted in M9 + Amp + Cm12.5 in order to obtain 1:500 and 1:200 final dilutions in the 200 μl-well of TECAN microplate.
In the afternoon supernatants were tested; this time results were more precise, even if negative control seemed to inactivate 3OC6-HSL as fast as other cultures.
1.5 μl of J101-4C5 purified DNA was transformed in 100 μl of MGZ1 competent cells.
E32, E33, J101-31, J101-E5 and ENTERO4C5 plate was grown so two colonies for each strain were picked and inoculated in 1 ml of M9 + Cm12.5 for PtetR characterization.
BBa_K300005 was inoculated in 6 ml of LB + Cm34 in order to extract pSB1C3 standard shipping vector.
August, 18th
J101-4C5 plate was grown and a colony was picked and inoculated in 750 μl LB + Cm12.5; in the late afternoon 250 μl glycerol 80% were added in order to prepare a glycerol stock of this culture.
Cultures involved in PtetR characterization were diluted 1:500 in a final volume of 1 ml M9 + Cm12.5 and grown for 2.5 hours. Then they were induced with 10 different final concentrations of anhydrotetracycline (atc) (0 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 8 ng/ml, 10 ng/ml50 ng/ml and 100 ng/ml).
Plasmid purification was performed for
BBa_K300005:
Then the plasmid was digested with EcoRI and PstI endonucleases:
| Plasmid |
Kind |
DNA (μl) |
H2O (μl) |
Enzyme 1 (μl) |
Enzyme 2 (μl) |
Buffer H (μl) |
Final Volume (μl) |
| BBa_K300005 |
Vector |
4 |
16.5 |
1 EcoRl |
1 Pstl |
2.5 |
25 |
Gel electrophoresis was carried out:
Small size gel
After gel extraction, digested DNA was quantified:
Digested DNA (pSB1C3) was stored at -20°C, in order to ligate the parts to send to the Registry.
Streak of E34-1, E35-2, J101-E7, J101-4C5 and ENTERO4C5 on an LB-agar plate + Cm12.5 to test and characterize PtetR promoter with different Ribosome Binding Sites (BBa_B0032, BBa_B0034).
Inoculum of E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 in 1 ml M9 + Cm12.5 to test the efficiency of AiiA enzyme.
August, 19th
E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 were diluted 1:500 in 6 ml M9 + Cm12.5. After 3 hours they were induced with 75 ng/ml atc; after 1 hour from induction 100 nM 3OC6-HSL was added (t = 0 h); this time also 3OC6-HSL in M9 + Cm12.5 without cells was measured. Supernatants were collected as on August,3rd.
250 μl samples were taken at t = 0 h, t = 1 h, t = 4 h and t = 21 h; O.D.600 and pH (which was constant at about 7) were measured:
|
E37 |
E38 |
E39 |
E40 |
ENTERO4C5 |
M9 |
| t = 0 h |
0.0068 |
0.0078 |
0.0094 |
0.0060 |
0.0077 |
### |
| t = 2 h |
0.031 |
0.026 |
0.032 |
0.028 |
0.036 |
### |
| t = 4 h |
0.105 |
0.096 |
0.109 |
0.098 |
0.112 |
### |
| t = 21 h |
0.474 |
0.463 |
0.496 |
0.485 |
0.450 |
### |
Supernatants were stored at -20°C.
Inoculum of E24-1, E25-2, E26-2 and E27-2 in LB + Amp for plasmid purification.
Inoculum of E34-1, E35-2, J101-E7, J101-4C5 and ENTERO4C5 (three colonies for part) in M9 + Cm12.5, for TECAN test on PtetR promoter.
August, 20th
1:500 dilution of test cultures in 1 ml (final volume).
After 2 hours and 30 minutes PtetR cultures were induced with atc (0 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 8 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml); after two hours and 30 minutes 200 μl of every culture were aliquoted in TECAN Infinite 200.
Plasmid purification for E24-1, E25-2, E26-2 and E27-2 was carried out:
| Plasmid |
DNA (ng/μl) |
| E24-1 |
82.3 |
| E25-2 |
66.4 |
| E26-2 |
87.0 |
| E27-2 |
79.2 |
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