![](https://parts.igem.org/images/partbypart/icon_composite.png)
Part:BBa_K1152015:Design
IndC Indigoidine Synthetase device with T-domain of plu2642
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 4087
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1467
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2730
Design Notes
We amplified the plasmid K1152008 without the T-domain with primers 1/2 and the ccdB cassette from pDONR with primers 3/4. In a second CPEC assembly, the indC-ccdB part was created and transformed into OneShot ccdB survival cells.
Name | Primer Sequence 5' - 3' |
---|---|
1_K1152013_fw | AAGTGGATTGAACAGACAGACTCTAAAAC |
2_K1152013_rv | AGTATCTGTATGTAATGGCACCAATAGACGC |
3_ccdB_fw | TGCCATTACATACAGATACT ACTGGCTGTGTATAAGGGAGCCTGAC |
4_ccdB_rv | AGAGTCTGTCTGTTCAATCCACTT CGCGTGGATCCGGCTTAC |
In a 6 ul PCR Master Mix (NEB Phusion High-Fidelity in High Fidelity buffer), all fragments were put together in an equimolar ratio. CPEC assembly was performed using an optimized protocol (Table 2).
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 30 |
5 | 98 | 5 |
53 | 15 | |
72 | 60 | |
1 | 72 | 180 |
Transformation was performed with 5 ul of the CPEC reaction product.
The prepped plasmid was then transformed into both E. coli TOP10 and OneShot cells, outlining that there are no background colonies (Figure 1).
Name | Primer Sequence 5' - 3' |
---|---|
1_K1152013_fw | AAGTGGATTGAACAGACAGACTCTAAAAC |
2_K1152013_rv | AGTATCTGTATGTAATGGCACCAATAGACGC |
3_ccdB_fw | TGCCATTACATACAGATACT ACTGGCTGTGTATAAGGGAGCCTGAC |
4_ccdB_rv | AGAGTCTGTCTGTTCAATCCACTT CGCGTGGATCCGGCTTAC |
In a 6 ul PCR Master Mix (NEB Phusion High-Fidelity in High Fidelity buffer), all fragments were put together in an equimolar ratio. CPEC assembly was performed using an optimized protocol (Table 2).
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 30 |
5 | 98 | 5 |
53 | 15 | |
72 | 60 | |
1 | 72 | 180 |
Transformation was performed with 5 ul of the CPEC reaction product.
Using the same strategy as for introducing the ccdB gene, we replaced the ccdB gene with the novel T-domain. Using this strategy, every colony on the plate with transformed cells contains the new T-domain.