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, 20th to test AiiA enzyme efficiency.
August, 23rd
T9002-ENTERO and ENTERO-RBS were diluted 1:500 in a final volume of 1 ml of M9 + 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.
T9002-ENTERO and ENTERO-RBS were inoculated in 1 ml of M9 + Amp + Cm12.5 to test teh supernatants collected on August, 20th and test AiiA enzyme efficiency.
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