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:
| Plasmid |
DNA (ng/μl) |
| E44 |
89.8 |
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.
The team also worked very hard on the wiki!
September, 17th
September, 18th
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