Difference between revisions of "Part:BBa K1152008:Design"
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− | |+Table 1: Primer for removal of internal RFC[10] cutting sites in indC using a CPEC approach | + | |+ Table 1: Primer for removal of internal RFC[10] cutting sites in indC using a CPEC approach |
|- | |- | ||
!Name!!Primer Sequence 5' - 3' | !Name!!Primer Sequence 5' - 3' | ||
|- | |- | ||
− | |1_indC_fw|CATACTAGAG AAAGAGGAGAAA GGTACC ATGTTAGAAAATAATATTACACAATG | + | |1_indC_fw||CATACTAGAG AAAGAGGAGAAA GGTACC ATGTTAGAAAATAATATTACACAATG |
|- | |- | ||
− | |2_EcoRI_rv|CAATACCCACC GAAcTC TTTGAGCT AATTTCTGACAGACAATACC | + | |2_EcoRI_rv||CAATACCCACC GAAcTC TTTGAGCT AATTTCTGACAGACAATACC |
|- | |- | ||
− | |3_EcoRI_fw|AGCTCAAA GAgTTC GGTGGGTATTG GGCTTTTTTGTGATC | + | |3_EcoRI_fw||AGCTCAAA GAgTTC GGTGGGTATTG GGCTTTTTTGTGATC |
|- | |- | ||
− | |4_SpeI_rv|ATAAGCCAG ACTcGT GGGTTTAGGAACTTG GAACTTGAACTGTG | + | |4_SpeI_rv||ATAAGCCAG ACTcGT GGGTTTAGGAACTTG GAACTTGAACTGTG |
|- | |- | ||
− | |5_SpeI_fw|CAAGTTCCTAAACCC ACgAGT CTGGCTTAT ATTATTTATACCTCTGGTAGCAC | + | |5_SpeI_fw||CAAGTTCCTAAACCC ACgAGT CTGGCTTAT ATTATTTATACCTCTGGTAGCAC |
|- | |- | ||
− | |6_indC_rv|CAGCGTTATTA GCTAGC TCA TTAGATTATTTTCTCAATCTCAG | + | |6_indC_rv||CAGCGTTATTA GCTAGC TCA TTAGATTATTTTCTCAATCTCAG |
|- | |- | ||
− | |7_backbone_fw|TAATGA GCTAGC TAATAACGCTGATAGTGCTAGTG | + | |7_backbone_fw||TAATGA GCTAGC TAATAACGCTGATAGTGCTAGTG |
|- | |- | ||
− | |8_backbone_rv|CAT GGTACC TTTCTCCTCTTT CTCTAGTATGTGTG | + | |8_backbone_rv||CAT GGTACC TTTCTCCTCTTT CTCTAGTATGTGTG |
|- | |- | ||
|} | |} | ||
+ | The following experimental procedures were performed to [http://hdl.handle.net/1721.1/81332| RFC 99]: | ||
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). | 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). | ||
Latest revision as of 15:45, 6 October 2013
IndC Indigoidine Synthetase (cds)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1239
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2502
Design Notes
The native indC contains an internal EcoRI and SpeI cutting site which was mutated using a CPEC approach. We amplified indC in three fragments from P. luminescens genomic DNA:
- start codon to EcoRI cutting site (Primer 1/2; Table 1)
- EcoRI cutting site to SpeI cutting site (Primer 3/4; Table 1)
- Spe cutting site to stop codon (Primer 5/6; Table 1).
The primers contain point mutation (indicated with small letters in Table 1) to remove the cutting sites. The backbone was amplified with the primers 7/8. Basically every backbone which contains the part J04450 can be used.
Name | Primer Sequence 5' - 3' |
---|---|
1_indC_fw | CATACTAGAG AAAGAGGAGAAA GGTACC ATGTTAGAAAATAATATTACACAATG |
2_EcoRI_rv | CAATACCCACC GAAcTC TTTGAGCT AATTTCTGACAGACAATACC |
3_EcoRI_fw | AGCTCAAA GAgTTC GGTGGGTATTG GGCTTTTTTGTGATC |
4_SpeI_rv | ATAAGCCAG ACTcGT GGGTTTAGGAACTTG GAACTTGAACTGTG |
5_SpeI_fw | CAAGTTCCTAAACCC ACgAGT CTGGCTTAT ATTATTTATACCTCTGGTAGCAC |
6_indC_rv | CAGCGTTATTA GCTAGC TCA TTAGATTATTTTCTCAATCTCAG |
7_backbone_fw | TAATGA GCTAGC TAATAACGCTGATAGTGCTAGTG |
8_backbone_rv | CAT GGTACC TTTCTCCTCTTT CTCTAGTATGTGTG |
The following experimental procedures were performed to [http://hdl.handle.net/1721.1/81332| RFC 99]: 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 resulting plasmid is K1152008, from which we subsequently amplified the coding sequence with primers that introduce the RFC[10] prefix and suffix for standard BioBrick assembly of the indC cds on pSB1C3.
Source
The coding sequence was amplified from Photorhabdus luminescens laumondii TT01 DSM15139.
References
- Brachmann AO, Kirchner F, Kegler C, Kinski SC, Schmitt I, Bode HB (2012) Triggering the production of the cryptic blue pigment indigoidine from Photorhabdus luminescens. J Biotechnol 157: 96-99.
- Quan J, Tian J (2009) Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One 4: e6441.