August, 1st
Two colonies for E28-AGAIN and E41-AGAIN plates were picked and inoculated in LB + Cm12.5; J101-31, J101-E5 and J101-E7 plasmids were extracted:
| Plasmid |
DNA (ng/μl) |
| J101-31 |
??? |
| J101-E5 |
??? |
| J101-E7 |
??? |
These plasmids were digested with EcoRI and SpeI restriction enzymes and screened by gel-electrophoresis; a medium-size agarose gel was then prepared.
MGZ1 competent cells were prepared according to protocol. They were put in 22 100 μl test-tubes.
Gel electrophoresis was performed:
Medium size gel
Insert lengths were all correct so J101-31, J101-E5, J101-E7 were transformed from MiniPrepped DNA in MGZ1 competent cells.
E37-2, E38-1, E39-1, E40-2 and E42-1 were inoculated in 5 ml LB + Cm12.5 and grown ON for plasmid extraction.
August, 2nd
MGZ1 J101-31, J101-E5 and J101-E7 plates were all grown; one colony was picked from each of them and inoculated in LB + Cm12.5.
Plasmids harboring E28-AGAIN-1, E28-AGAIN-2, E41-AGAIN-1, E28-AGAIN-2, E37-2, E38-1, E39-1, E40-2 and E42-1 were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:
| Plasmid |
DNA (ng/μl) |
| E28AGAIN-1 |
21.4 |
| E28AGAIN-2 |
21.0 |
| E41AGAIN-1 |
33.7 |
| E41AGAIN-2 |
27.7 |
| E37-2 |
18.5 |
| E38-1 |
26.0 |
| E39-1 |
21.6 |
| E40-2 |
19.9 |
| E42-1 |
25.1 |
Digestions of E28AGAIN-1, E28AGAIN-2, E41AGAIN-1 and E42AGAIN-2 were performed for screening; a 5x mix was prepared and for each sample the volumes specified in the table below were used:
| DNA (μl) |
H2O (μl) |
Enzyme 1 (μl) |
Enzyme 2 (μl) |
Buffer H (μl) |
Final Volume (μl) |
| 5 |
16.5 |
0.5 EcoRI |
0.5 PstI |
2.5 |
25 |
A small-size agarose gel was prepared for gel electrophoresis.
TOP10 competent cells, harboring BBa_T9002 construct, were co-transformed with ENTERO4C5 to confer resistance to Cloramphenicol (12.5 mg/ml) and plated on LB agar + Amp + Cm 12.5 plates.
In the afternoon gel electrophoresis was performed:
Small size gel
Unfortunately insert length was correct only for E28-AGAIN-1 so we ligated again E41:
| Ligation Name |
Vector |
Vector volume (μl) |
Insert |
Insert volume (μl) |
Buffer (μl) |
T4 Ligase (μl) |
| E41N |
E36 (S-P) |
4 |
E3-1 (X-P) |
4 |
1 |
1 |
Glycerol stocks were prepared according to the protocol for J101-31, J101-E5 and J101-E7.
E37-2, E38-1, E39-1, E40-2, E42-1 and E28-AGAIN-1 DNA was sent to BMR genomics for sequencing.
5 μl of E37-2, E38.1, E39-1, E40-2 and ENTERO4C5 were inoculated in 1 ml M9 + Cm12.5 for a preliminary test on the autoinducer inactivation enzyme A (aiiA) enzyme.
August, 3rd
T9002-ENTERO4C5 plate was grown and a colony was picked and inoculated in LB + Amp + Cm12.5; E41N ligation was transformed in 100 μl of MGZ1 competent cells and plated on a LB + Cm12.5 plate.
34 LB agar + Cm12.5 plates were prepared according to protocols.
E37-2, E38.1, E39-1, E40-2 and ENTERO4C5 cultures were diluted 1:100 in a final volume of 5 ml of M9 + Cm12.5 for the preliminary test; a 1000 x 75 ng/μl atc stock was prepared.
After 3 hours cultures were induced with 5 μl of atc; after 4 hours 2.5 μl of 2mM 3OC6-HSL (1:2000 dilution) were added (final 3OC6-HSL concentration 1μM) and the experiment started (t = 0 h).
250 μl samples were taken at t = 0 h, t = 1 h, and t = 4 h; O.D. at 600 nm was measured:
|
E37 |
E38 |
E39 |
E40 |
ENTERO4C5 |
| t = 0 h |
0.12 |
0.11 |
0.13 |
0.10 |
0.11 |
| t = 1 h |
0.22 |
0.15 |
0.21 |
0.28 |
0.19 |
| t = 4 h |
0.47 |
0.60 |
0.53 |
0.37 |
0.53 |
Then samples were centrifuged at 13.000 rpm; finally the supernatant was collected and stored at -20°C.
Glycerol stock for T9002-ENTERO4C5 was prepared.
Two 5 μl inocula of E32, E33, E34, E35, J101-E5, J101-31, J101-E6, J101-E7 and ENTERO4C5 were prepared in 1 ml of M9 + Cm12.5 for a preliminary test on TetR repressible promoter.
August, 4th
E41N plate was grown; three colonies were picked (E41N-1, E41N-2, E41N-3) and inoculated in LB + Cm12.5.
E32-1, E33-1, E34-1, E35-1, J101-E5-1, J101-31-1, J101-E6-1, J101-E7-1, E32-2, E33-2, E34-2, E35-2, J101-E5-2, J101-31-2, J101-E6-2, J101-E7-2, ENTERO4C5-1 and ENTERO4C5-2 were diluted 1:500 in a final volume of 1 ml.
After 2 hours and 30 minutes inducible cultures were supplemented with anhydrotetracyclin (atc) at final concentrations of: 1 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml. Moreover uninduced cultures were supplemented with ddH2O.
ENTERO-RBS and T9002-ENTERO4C5 were inoculated for preliminary aiiA test (with supernatants collected on August, 3rd).
August, 5th
ENTERO-RBS and T9002-ENTERO4C5 cultures were diluted 1:500 in a final volume of 1 ml and 10 ml respectively; they were grown for six hours. Plasmid purification was performed for E41N-1, E41N-2 and E41N-3:
| Plasmid |
DNA (ng/μl) |
| E41N-1 |
62.3 |
| E41N-2 |
17.3 |
| E41N-3 |
43.9 |
Screening digestion with EcoRI and PstI restriction enzymes was carried out:
| Plasmid |
DNA (μl) |
H2O (μl) |
Enzyme 1 (μl) |
Enzyme 2 (μl) |
Buffer H (μl) |
Final Volume (μl) |
| E41N-1 |
2.5 |
19 |
0.5 EcoRI |
0.5 Pstl |
2.5 |
25 |
| E41N-2 |
8.5 |
13 |
0.5 EcoRI |
0.5 Pstl |
2.5 |
25 |
| E41N-2 |
3.5 |
18 |
0.5 EcoRI |
0.5 Pstl |
2.5 |
25 |
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.
A small-size agarose gel was prepared; in the afternoon gel electrophoresis was carried out:
Smallsize gel
Only E41N-1 showed the correct band, so we decided to keep it. E41N-2 and E41N-3 glycerol stocks were thrown.
TECAN test on E37, E38, E39, E40 and ENTERO4C5 supernatants was carried out; as supernatants were not diluted enough it was not possible to precisely measure 3OC6-HSL concentration as the biosensor (T9002-ENTERO4C5) worked in the saturation zone.
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