Difference between revisions of "Part:BBa K1152015:Design"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K1152015 short</partinfo> | <partinfo>BBa_K1152015 short</partinfo> | ||
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===Design Notes=== | ===Design Notes=== | ||
− | + | [[image:ccdB-CPEC.png|250px|thumb|right|'''Figure 1:''' ccdB CPEC assembly strategy for exchanging T-domains in indC]] | |
+ | 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. | ||
+ | {|class="wikitable" | ||
+ | |+ Table 1: Primer for removal of internal RFC[10] cutting sites in indC using a CPEC approach | ||
+ | |- | ||
+ | !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). | ||
+ | |||
+ | {|class="wikitable" | ||
+ | |+Table 2: CPEC assembly Cycler Parameters | ||
+ | |- | ||
+ | !Cycles!!Temperature [°C]!!Time [s] | ||
+ | |- | ||
+ | |1||98||30 | ||
+ | |- | ||
+ | |rowspan="3"|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). | ||
+ | |||
+ | [[file:ccdB_TOP10.png|200px|''Figure 1'']] | ||
+ | |||
+ | {|class="wikitable" | ||
+ | |+ Table 1: Primer for removal of internal RFC[10] cutting sites in indC using a CPEC approach | ||
+ | |- | ||
+ | !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). | ||
− | == | + | {|class="wikitable" |
+ | |+Table 2: CPEC assembly Cycler Parameters | ||
+ | |- | ||
+ | !Cycles!!Temperature [°C]!!Time [s] | ||
+ | |- | ||
+ | |1||98||30 | ||
+ | |- | ||
+ | |rowspan="3"|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. |
Revision as of 02:04, 6 October 2013
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.