September, 5th
T9002-ENTERO and ENTERO-4C5 were diluted 1:200 and 1:800 to perform two different tests on supernatants (collected on August, 31st, from cultures and media at different pHs, and on September, 3rd, from cultures expressing AiiA enzyme in high copy number plasmids).
Samples of dry DNA were prepared for R0040 in J61002-1C3-1 and J101-31-1C3-1 and sent to BMR genomics for sequencing.
E31-1, E41N-1, E42-1, E43-2, E43-3 were washed in 1 ml of M9 + Cm12.5 in order to remove 3OC6-HSL due to PtetR leakage; then they were diluted 1:100 in 4 ml volume (falcon tube). After two hours they were induced with a final concentration of 100 ng/ml atc. Supernatants were collected at the moment of induction, named t = 0 h and then at t = 2 h, t = 4 h and t = 6 h (also from two negative controls, ENTERO4C5 and M9).
E3N-1C3 was again transformed in 200 μl of TOP10 competent cells.
T9002-ENTERO and ENTERO-RBS were again inoculated to test the supernatants collected from E31-1, E41N-1, E42-1, E43-2, E43-3, ENTERO4C5 and M9.
E37-,2 E38-1, E39-1, E40-2 and ENTERO4C5 were inoculated in 1 ml of M9 + Cm12.5 ath pH 5.9 in order to test AiiA efficiency.
In the evening we met to plan the work of the last weeks!
September, 6th
T9002-ENTERO and ENTERO-RBS were diluted 1:200 in a final volume of 1 ml and 18 ml respectively. After two hours 190 μl were aliquoted in 96-well microplate and induced with 10 μl of supernatants and 3OC6-HSL known concentrations.
E37-,2 E38-1, E39-1, E40-2 and ENTERO4C5 were diluted 1:200 in a final volume of 4 ml M9 + Cm12.5 at pH 5.9.
After two hours they were induced with 100 ng/ml atc and after one hour 3OC6-HSL was added at a final concentration of 100 nM. At this time the first sample was collected from each tube; samples were also collected after three hours, five hours and twenty-four hours.
E3N-1C3 plate was grown; eight colonies from this plate were picked while three colonies were picked from a older plate. All of these colonies were PCR-screened and grown in LB + Cm34. The electrophoresis showed that only two colonies, picked from the oldest plate, had the correct length part.
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Medium size gel
17 plates of LB + Amp were prepared.
E24-2, E25-1, E26-2 and E27-2 were transformed in 100 μl of MGZ1 competent cells and plated on LB agar plates + Amp.
T9002-ENTERO and ENTERO-RBS were inoculated to test the supernatants collected today from cultures producing AiiA enzyme (in LC number plasmid pSB4C5).
September, 7th
September, 8th
September, 9th
September, 10th
September, 11th
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