Team:UNIPV-Pavia/Calendar/September/week3

September, 12th

E31-1, E41N-1, E42-1, E43-3 and ENTERO4C5 were diluted 1:100 in 13 falcon tubes (4ml M9 + Cm12.5 pH = 6.0). After two hours they were induced with aTc (6 ng/ml, 8 ng/ml and 100 ng/ml). Supernatants were taken at the induction time, after 1 hour, 2 hours and 4 hours.
E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 were inoculated in 1 ml M9 + Cm12.5 and grown ON.

September, 13th

Supernatants collected on September, 12th were tested, measuring 3OC₆-HSL concentration with T9002-ENTERO in two differents tests.
1:100 dilution of E37-2, E38-1, E39-1, E40-2 and ENTERO4C5 in 4ml M9 + Cm12.5 and two hours growth, after which cultures were induced with aTc; addition of 3OC₆-HSL 100 nM after one hour and collection of supernatants at t = 0h, t = 1 h, t = 2 h and t = 4 h (time refers to 3OC₆-HSL addition).
Inoculum of E44 for plasmid purification in 5 ml LB + Cm34; inoculum of T9002-ENTERO and ENTERO-RBS for 3OC₆-HSL concentration measurement; ENTERO4C5 was inoculated in M9 + Cm12.5 at pH = 6.0 and pH = 7.0 in order to detect 3OC₆-HSL degradation in cultures not expressing AiiA.

September, 14th

T9002-ENTERO, ENTERO-RBS were diluted 1:100 in M9 + Amp + Cm12.5 respectively in 18 ml and 1 ml. After two hours a test was performed on supernatants collected on September, 13th.
ENTERO4C5 was diluted in M9 + Cm12.5 at the proper pH; after growing for two hours at 37°C, 220 rpm, cultures were supplemented with 100 nM 3OC₆-HSL and supernatants were taken at t = 0 h, t = 1 h, t = 2 h, t = 4 h and t = 8 h.
E44 was plasmid purified:

As we realized that T9002-ENTERO did not produce the desired output (saturation of known induction curve occured at 5 nM, even if we expected that it was in the linear working zone of the biosensor) we decided to use BBa_T9002 for our measurements, so we inoculated it together with RBS in M9 + Amp, in order to perform tests on supernatants from cultures expressing AiiA.

September, 15th

BBa_T9002 and RBS cultures were diluted 1:100 in M9 + Amp; after two hours the test on supernatants of cultures expressing AiiA enzyme at various aTc inductions started. In the afternoon the over night liquid cultures were diluted 1:100 again in order to test the remaining supernatants collected.
Parts were sent to the Registry at MIT.

September, 16th

BBa_T9002 and RBS cultures were diluted 1:100 in M9 + Amp; after two hours the test on supernatants of cultures not expressing AiiA enzyme and M9 without cells at pH = 6.0 and pH = 7.0 started (SN collected on September, 14th).
The team also worked very hard on the wiki!

September, 17th

Wiki update and definition of results.

September, 18th

Wiki update and definition of results.