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Week 11
08/10 - 08/14
Yeast Assembly
The Bacteria was first sent to the iGEM Toulouse and could have suffered from the travel so we ordered it again.
- Reception of a STAB culture of the Biobrick BBa_J61047.
- Spreading of BBa_J61047 on Petri dishes containing LB medium and LB + Cm 34 µg/ml.
No colonies were observed on Petri dishes containing LB + Cm 34 µg/ml but the bacteria grew on LB media.
- Bacterial growth of the bacteria in liquid medium (5 ml of LB + Cm 34 µg/ml) and on a Petri dish (LB + Cm 34 µg/ml) and overnight incubation at 37°C.
- Storage at 4°C.
- Bacterial growth of the Bacteria in LB: incubation at 37°C for 1h30 at 140rpm.
- 100 µl of bacteria in 5 ml of LB
- 100 µl of bacteria in 5 ml of LB + Cm 34 µg/ml
- 5 ml of LB without bacteria as negative control
=> No bacterial growth was observed.
Enzymatic Assay of NB-Esterase
- XbaI ans PstI enzymatic digestion of the Biobrick BBa_K808030 and pDG011 plasmid.
- Gel purification of the digested fragments on 0.8% agarose gel.
- Ligation of pDG011 plasmid and BBa_K808030 using T4 DNA ligase.
- Gel migration on 0.8% agarose gel.
Figure 8: Gel for verification of the ligation of BBa_K808030 and pDG011 plasmid.
There is no band for the ligation sample.
Gibson Assembly
- Ligation of TphA1/, Transp_, BBa_K1392932_, using T4 DNA ligase.
- DNA concentration measurement using a spectrophotometer.
Biobrick |
DNA Concentration (ng/µl) |
OD (230 nm) |
OD (260 nm) |
OD (280 nm) |
OD (260 nm) / OD (280 nm) |
OD (260 nm) / OD (230 nm) |
808010 |
30.00 |
9.33 |
0.75 |
0,30 |
1.87 |
0.06 |
808011 |
50.00 |
11.78 |
1.00 |
0.63 |
1.61 |
0.09 |
808012 |
17.50 |
10.93 |
0.33 |
0.18 |
0.84 |
0.03 |
808013 |
5.00 |
10.55 |
0.10 |
0.06 |
1.56 |
0.01 |
808014 |
22.5 |
12.13 |
0.47 |
0.30 |
1.54 |
0.04 |
1095000 |
37.50 |
18.48 |
0.78 |
0.43 |
1.83 |
0.04 |
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