Team:UNIPV-Pavia/Calendar/September/week1

September, 2nd

E3-1C3-2 was purified in order to screen the part length.

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.
2 μl of the purified plasmidic DNA of E3-1C3-2 were digested with 0.5 μl EcoRI and PstI restriction enzymes in a 25 μl final volume; a small size agarose gel was prepared.

Small size gel

The sample did not show the correct band.
500 ml M9 and 1 l LB were prepared.

September, 4th

R0040 in J61002-1C3-1, R0040 in J61002-1C3-2, J101-31-1C3-1 and J101-31-1C3-2 were glycerol stocked and then plasmidic DNA was purified with MiniPrep commercial kit:

2.5 μl of each sample were digested with 0.5 μl EcoRI and PstI resctriction endonucleases in a 25 μl final volume. A small size agarose gel was prepared. In the afternoon electrophoresis was carried out; all samples showed the correct insert.

Small size gel

Inoculum of T9002-ENTERO and ENTERO-RBS to measure 3OC₆-HSL concentration in supernatants collected on August, 31st.
Inoculum of E31-1, E41N-1, E42-1, E43-2, E43-3 and ENTERO-4C5 to collect the supernatants and measure the 3OC₆-HSL produced by luxI enzyme.