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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.
Gibson Assembly
- DNA concentration of TphA1/ annealing using a spectrophotometer.
Biobrick |
DNA Concentration (ng/µl) |
OD (230 nm) |
OD (260 nm) |
OD (280 nm) |
OD (260 nm) / OD (230 nm) |
OD (260 nm) / OD (280 nm) |
TphA1/ |
875 |
7.53 |
17.50 |
5.85 |
2.33 |
2.99 |
- DNA dissolution of the ordered oligonucleotides in DNAse, RNAse free water.
- Gibson Assembly of Cluster 2A.
- Gibson Assembly of Cluster 2B.
- Gibson Assembly of Cluster 2 (cluster 2A + Cluster 2B).
- PCR Amplification of Cluster 2 Gibson Assembly.
- Gel migration on 0,8% agarose gel.
Figure 9: Gel of the migration of the PCR product with control.
Stain |
1 |
2 |
3 |
4 |
5 |
6 |
Tube number |
_ |
1 |
2 |
3 |
4 |
5 |
Contents |
Ladder |
Mix+ DNA |
Mix +primer for + DNA |
Mix + primer rev + DNA |
Mix + primers + DNA |
Mix + primers |
- BBa_K808007 and BBa_K808030 PCR amplification.
- Gel migration on 0.8% Agarose gel.
Figure 10: Gel of the migration of the Biobricks BBa_K808007 and BBa_K808030.
Stain |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
Tube number |
1 |
2 |
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5 |
6 |
7 |
8 |
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10 |
Contents |
Mix + 808007
DNA |
Mix + primer for + 808007
DNA |
Mix + primers + 808007 |
Mix + primer rev + 808007
DNA |
Mix+ primers |
Mix + 808030 DNA |
Mix + primer for + 808030 DNA |
Mix + primers + 808030 DNA |
Mix + primer rev + 808030 DNA |
Mix + primers |
- Enzymatic digestion of received oligonucleotides BBa_K936011 and BBa_K1392932.
- Dilution of the received oligonucleotides BBa_K316003/BBa_K1095000, BBa_K1095000/BBa_K1392931, /936011, BBa_K1095000 in 30 µl of DNAse RNAse free water.
- Primers dilution in TE buffer (1X) according to the company’s guidelines.
- PCR of BBa_K808014, BBa_K1095000, BBa_K1392931, BBa_K316003.
- Storage at 4°C.
- Gibson assembly of cluster 2A.
Gibson assembly of cluster 2C.
Gibson assembly of cluster 2 (cluster 2A + cluster 2B and 2C together).
=> No results on the gel.
- PCR amplification of Biobricks BBa_K316003, BBa_K808014, BBa_K1392932, BBa_K936011, BBa_K195000, BBa_K1392931 by gel migration.
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DNA ladder |
BBa_K808014 |
BBa_K109500 |
BBa_K1392931 |
BBa_K1392932 |
BBa_K316003 |
Primer for |
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0 |
1 |
0 |
1 |
0 |
1 |
0 |
1 |
0 |
1 |
0 |
1 |
0 |
1 |
0 |
1 |
0 |
1 |
1 |
Primer rev |
0 |
0 |
1 |
1 |
0 |
0 |
1 |
1 |
0 |
0 |
1 |
1 |
0 |
0 |
1 |
1 |
0 |
0 |
1 |
Well |
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20 |
Figure 11: Gel of the migration of the Biobricks BBa_K316003, BBa_K808014, BBa_K1392932, BBa_K936011, BBa_K195000, BBa_K1392931.
- The PCR amplification did not work for BBa_K808014 so we re-do amplification by PCR of the Biobrick BBa_K808014 using TaKaRa Ex Taq polymerase.
- PCR amplification of Inter-sequences (1392931/1392932 and pNB Est/Trans) for the Gibson assembly using TaKaRa Ex Taq Polymerase.
- DNA purification of cluster 2 assembly (Macherey-Nigel Kit).
- PCR amplification of the assembled cluster 2 using TaKaRa Ex Taq Polymerase.
pNP assay - Enzymatic assay
- Ligation of BBa_808030 and pDG011 plasmid using T4 DNA ligase.
- Electro-competent BAP 1 cells.
- Enzymatic digest of BBa_K808030 and pDG011 plasmid using XbaI and PstI.
- Transformation of pDG011 plasmid in chemically competent DH5-α cells.
- Digestion of BBa_K808030 and pDG011 plasmid with restriction enzyme XbaI and PstI.
- Gel migration of BBa_K808030 and pDG011 plasmid.
- Gel purification.
- DNA concentration assessed with a NanoDrop:
- BBa_808030 = 4.9 ng/µL
- pDG011 = 5.5 ng/µL
- MiniPrep of pDG011 plasmid.
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