August, 22nd
Streak of E17-2, E18-2, J101-31, J101-E5 and ENTERO-4C5 on LB agar + Cm12.5 plate to test Plux promoter with different RBSs.
E24-1, E25-2, E26-1, E27-2 and E41N-1 purified DNA samples were sent to BMR Genomics for sequencing.
250 ml of M9 glycerol supplemented minimal medium were prepared.
Inoculum of T9002-ENTERO and ENTERO-RBS to test supernatants collected on August, 19th to test AiiA enzyme efficiency.
August, 23rd
T9002-ENTERO and ENTERO-RBS were diluted 1:500 in a final volume of 13 ml and 1 ml respectively of M9 + Amp + Cm12.5.
Three colonies of E17-2, E18-2, J101-31, J101-E5 and ENTERO-4C5 were picked and inoculated in 1 ml of M9 + Cm12.5.
E19-2, E 20-2, J101-E7, J101-4C5 and ENTEOR-4C5 were streaked on a LB agar + Cm12.5 plate.
Unfortunately TECAN test could not be performed as the instrument did not work!
31 LB agar + Cm34 plates were prepared.
Last week sequencing were ready: E28-1 has not the desired sequence, while E37-2, E38-1, E39-1, E40-2 and E42-1 were correct.
Ptet-RBS30-luxI ligation was done again from E36 and E2:
| Ligation Name |
Vector |
Vector volume (μl) |
Insert |
Insert volume (μl) |
Buffer (μl) |
T4 Ligase (μl) |
| E43 |
E36 (S-P) |
3.5 |
E2 (X-P) |
4.5 |
1 |
1 |
August, 24th
E43 ligation was transformed in 100 μl of MGZ1 competent cells.
E20-2 were streaked again.
E17-2, E18-2, J101-31, J101-E5 and ENTERO-4C5 were diluted 1:500 in 1 ml of M9 + Cm12.5; after three hours growth E17 and E18 cultures were induced with final concentrations of 3OC6-HSL of: 0 nM, 0.1 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, 10 nM and 100 nM. After three hours 200 μl of every culture were aliquoted in TECAN microplate (this time TECAN Infinite F200 worked!).
Three colonies of E19-2, E 20-2, J101-E7, J101-4C5 and ENTEOR-4C5 were inoculated in 1 ml of M9 + Cm12.5
E2-2, E3-1, E4-2, E5-2, E6-1, E7-2, E9-2, E10-1, E11-2 were inoculated in 5 ml of LB + Amp while E36, J101-E5, J101-31 and J101-E7 were inoculated in 5 ml of LB + Cm12.5; these cultures were grown in order to purify plasmids and transfer parts in pSB1C3 standard shipping plasmid.
August, 25th
Three colonies were picked from E43 plate.
Plasmid purification was carried out:
| Plasmid |
DNA (ng/μl) |
| E2-2 |
87.7 |
| E3-1 |
83.9 |
| E4-2 |
93.4 |
| E5-2 |
89.7 |
| E6-1 |
80.6 |
| E7-2 |
128.8 |
| E9-2 |
88.1 |
| E10-1 |
68.5 |
| E11-1 |
87.7 |
| E36 |
51 |
| J101-E5 |
25.7 |
| J101-31 |
56.1 |
| J101-E7 |
15.9 |
Every plasmid was digested with EcoRI and PstI resctriction endonucleases. A medium size and a small size agarose-gel were prepared and, after three hours digestion, gel-electrophoresis was carried out:
Medium size gel
Small size gel
As showed in images E36 did not show the correct insert band while J101-31, J101-E5 and J101-E7 showed light intensity bands as they were in a low copy number plasmid. The correct parts were gel-extracted and DNA was quantified:
| Part |
DNA (ng/μl) |
| E2-2 (E-P) |
4.4 |
| E3-1 (E-P) |
5.9 |
| E4-2 (E-P) |
4.2 |
| E5-2 (E-P) |
7.5 |
| E6-1 (E-P) |
3.1 |
| E7-2 (E-P) |
10.3 |
| E9-2 (E-P) |
8.8 |
| E10-1 (E-P) |
2.9 |
| E11-1 (E-P) |
8.2 |
| J101-31 (E-P) |
3.0 |
| J101-E5 (E-P) |
2.6 |
| J101 (E-P) |
0.7 |
BBa_R0040 in J61002 was inoculated in 5 ml LB + Amp while J101-31, J101-E5 and J101-E7 were again inoculated in 8 ml of LB + Cm12.5 for plasmid purification.
Glycerol stocks were prepared for E43-1 and E43-2 while E43-3 was grown ON; these tubes were refilled with LB + Cm12.5 in order to have a final volume of 8 ml, to increase plasmid purification efficiency.
T9002-ENTERO and ENTERO-RBS were inoculated in 1 ml of M9 + Amp + Cm12.5 to test the supernatants collected on August, 19th and test AiiA enzyme efficiency.
August, 26th
Glycerol stock for E43-3 was prepared according to protocols.
T9002-ENTERO and ENTERO-RBS were diluted in a final volume of 13 ml and 1 ml of M9 + Amp + Cm12.5. After growing them for 6 hours, a test was performed, measuring 3OC6-HSL concentration in supernatants collected on August, 19th.
Plasmid purification was carried out:
| Plasmid |
DNA (ng/μl) |
| BBa_R0040 in J61002 |
97.1 |
| J101-31 |
34.9 |
| J101-E5 |
12.1 |
| J101-E7 |
10.2 |
| E43-1 |
21.9 |
| E43-2 |
29.2 |
| E43-3 |
12.9 |
At this point, probably E43-2 and E43-3 tubes were confused! Anyway these plasmids were digested:
| Plasmid |
Kind |
DNA (μl) |
H2O (μl) |
Enzyme 1 (μl) |
Enzyme 2 (μl) |
Buffer H (μl) |
Final Volume (μl) |
| BBa_R0040 in J61002 |
Insert |
19 |
1.5 |
1 EcoRI |
1 PstI |
2.5 |
25 |
| J101-31 |
Insert |
20.5 |
0 |
1 EcoRI |
1 PstI |
2.5 |
25 |
| J101-E5 |
Insert |
20.5 |
0 |
1 EcoRI |
1 PstI |
2.5 |
25 |
| J101-E7 |
Insert |
20.5 |
0 |
1 EcoRI |
1 PstI |
2.5 |
25 |
| E43-1 |
Insert |
20.5 |
0 |
1 EcoRI |
1 PstI |
2.5 |
25 |
| E43-2 |
Insert |
20.5 |
0 |
1 EcoRI |
1 PstI |
2.5 |
25 |
| E43-3 |
Insert |
20.5 |
0 |
1 EcoRI |
1 PstI |
2.5 |
25 |
A medium size agarose-gel was prepared. BBa_R0040 in J61002 resulted incorrect, while other bands had a light intensity but we decided however to gel-extract and ligate them:
| Ligation Name |
Vector |
Vector volume (μl) |
Insert |
Insert volume (μl) |
Buffer (μl) |
T4 Ligase (μl) |
| E2 in pSB1C3 |
pSB1C3 (E-P) |
1 |
E2-2 (E-P) |
7 |
1 |
1 |
| E3 in pSB1C3 |
pSB1C3 (E-P) |
1.5 |
E3-1 (E-P) |
6.5 |
1 |
1 |
| E4 in pSB1C3 |
pSB1C3 (E-P) |
1.5 |
E4-2 (E-P) |
6.5 |
1 |
1 |
| E5 in pSB1C3 |
pSB1C3 (E-P) |
1.5 |
E5-2 (E-P) |
6.5 |
1 |
1 |
| E6 in pSB1C3 |
pSB1C3 (E-P) |
1 |
E6-1 (E-P) |
7 |
1 |
1 |
| E7 in pSB1C3 |
pSB1C3 (E-P) |
2 |
E7-2 (E-P) |
6 |
1 |
1 |
| E9 in pSB1C3 |
pSB1C3 (E-P) |
1.5 |
E9-2 (E-P) |
6.5 |
1 |
1 |
| E10 in pSB1C3 |
pSB1C3 (E-P) |
1 |
E10-1 (E-P) |
7 |
1 |
1 |
| E11 in pSB1C3 |
pSB1C3 (E-P) |
1.5 |
E11-1 (E-P) |
6.5 |
1 |
1 |
| J101-31 in pSB1C3 |
pSB1C3 (E-P) |
1 |
J101-31 (E-P) |
7 |
1 |
1 |
| J101-E5 in pSB1C3 |
pSB1C3 (E-P) |
1 |
J101-E5 (E-P) |
7 |
1 |
1 |
| J101-E7 in pSB1C3 |
pSB1C3 (E-P) |
1 |
J101-E7 (E-P) |
7 |
1 |
1 |
E24-2, E25-1, E26-2, E27-2 and RBS32 were inoculated in 1 ml of M9 + Amp to test AiiA efficiency in high copy number plasmids, taking cultures supernatants with a protocol similar to the one used for low copy number vectors.
August, 27th
E24-2, E25-1, E26-2, E27-2 and RBS32 were diluted 1:500 in a final volume of 6 ml of M9 + Amp; a tube with 6 ml of M9 + Amp medium was also prepared. These six tubes were grown for 2 hours; then 3OC6-HSL was added at a final concentration of 100 nM. Supernatants were taken at the moment of supplementation (t = 0 h), after 1 hour, after 2 hours and after 4 hours; then they were stored at -20°C.
E2-1C3, E3-1C3, E4-1C3, E5-1C3, E6-1C3, E7-1C3, E9-1C3, E10-1C3, E11-1C3, J101-31-1C3, J101-E5-1C3 and J101-E7-1C3 were transformed in 100 μl of TOP10 competent cells; plates were grown for 2 days at about 28°C.
E43-3 was again plasmid purified to screen its length (11.4 ng/μl).
August, 28th
Inoculum of T9002-ENTERO and ENTERO-RBS in 1 ml of M9 + Amp + Cm12.5 to test AiiA efficiency in high copy number plasmids with the suprnatants collected on August, 27th.
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