Part:BBa_K1152016:Design
IndC Indigoidine Synthetase device synT1 (engineered)
- 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).
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.