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
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