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Team:Evry/Notebook/w10
From 2013.igem.org
Week 10: 19th August - 25th August
2nd Construction
Primer hybridation
After sequencing analysis we observed that our Golden Gate results were not significant for the 2nd construction.
However, our sequences of natural promoter give us good results. Then we supposed that the problem of our Golden Gate come from the hybridation of our synthetic promoters.
We made new hybridations of:
- Fur Binding Site 1 to 15 (5 µM)
- Andersen promoter J23100 (5 µM)
- RBS 0034 (5 µM)
Golden Gate
A new Golden Gate of our 2nd construction are made with:
- Fur Binding Site 1, 2 or 3 + sfGFP
- Fur Binding Site 1, 2 or 3 + LacI-LVA
5 µL of each Golden Gate product are transformed into Top10 competent cells. To controlled the quality of the transformation, a negative controle (transformation procedure without plasmid) and a positive controle (transformation procedure with plasmid 1A3) are made.
| Name | Clone | Concentration | 260/280 | 260/230 | Sequencing |
|---|---|---|---|---|---|
|
Fur Binding Site n°1
|
1 | 20.2 ng/µL | 2.07 | 1.29 | Bad |
|
Fur Binding Site n°1
|
1 | 15.2 ng/µL | 2.39 | 1.65 | No sequencing |
| 2 | 25.5 ng/µL | 2.01 | 1.84 | No sequencing | |
| 3 | 45.6 ng/µL | 1.91 | 1.90 | Good | |
| 4 | 54.7 ng/µL | 1.83 | 1.27 | Bad | |
|
Fur Binding Site n°2
|
1 | 29.4 ng/µL | 2.01 | 1.46 | Bad |
| 2 | 25.5 ng/µL | 2.07 | 2.00 | Bad | |
| 3 | 15.0 ng/µL | 2.21 | 1.72 | No sequencing | |
| 4 | 12.2 ng/µL | 2.16 | 1.28 | No sequencing | |
|
Fur Binding Site n°2
|
1 | 9.8 ng/µL | 2.53 | 4.42 | No sequencing |
| 2 | 27.7 ng/µL | 1.93 | 1.67 | No sequencing | |
| 3 | 41.8 ng/µL | 1.87 | 1.69 | Bad | |
| 4 | 31.1 ng/µL | 1.82 | 1.26 | Bad | |
|
Fur Binding Site n°3
|
1 | 37.8 ng/µL | 1.85 | 1.40 | Good |
| 2 | 78.3 ng/µL | 1.87 | 1.85 | Good | |
| 3 | 53.4 ng/µL | 1.72 | 0.98 | Good | |
| 4 | 23.3 ng/µL | 2.06 | 2.53 | No sequencing | |
|
Fur Binding Site n°3
|
2 | 43.8 ng/µL | 1.78 | 1.21 | Bad |
| 4 | 13.9 ng/µL | 2.23 | 3.79 | No sequencing |
Other
We prepared BL21 competent cells.
We launched Golden Gate 3 again.
Isolation and culture of 4 colonies of Fur 1, 2, 3 with sfGFP or LacI.
