September, 2nd
E3-1C3-2 was purified in order to screen the part length.
| Plasmid |
DNA (ng/μl) |
| E2-1C3-2 |
82.1 |
R0040 in J61002-1C3, E3N-1C3 and J101-31N-1C3 were transformed in 200 μl of TOP10 competent cells.
Inoculum of E24-2, E25-1, E26-2, E27-2 and RBS32 in M9 + Amp to collect supernatant after 3OC6-HSL supplementation.
Inoculum in M9 + Cm12.5 of T9002-ENTERO and ENTERO-RBS to measure 3OC6-HSL concentration in supernatants at different pHs collected on August, 31st.
September, 3rd
All the cultures were diluted 1:200 in M9 with the proper antibiotic. After two hours T9002-ENTERO and ENTERO-RBS were aliquoted in 96-well microplate and induced with known concentrations of 3OC6-HSL and with the supernatants collected on August, 31st.
E24-2, E25-1, E26-2, E27-2, RBS32 and sterile M9 were supplemented with 100 nM 3OC6-HSL. Supernatants were collected at t = 0 h, t = 7 h and t = 24 h from all of these tubes.
R0040 in J61002-1C3 and J101-31N-1C3 showed colonies while E3N-1C3 did not grow; two colonies were picked for each plate and inoculated in Lb + Cm34, 5 ml.
E3-1C3-2 was purified and quantified:
2 μl of the purified plasmidic DNA were digested with 0.5 μl EcoRI and PstI restriction enzymes in a 25 μl final volume; a small size agarose gel was prepared.
The gel did not show the correct band.
500 ml M9 and 1 l LB were prepared.
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