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, E33, 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
"
