Difference between revisions of "Part:BBa K117002:Design"
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Molecular cloning of a gene encoding the thermoactive levansucrase from Rahnella aquatilis and its growth phase-dependent expression in Escherichia coli Jeong-Woo Seo, Ki-Bang Song, Ki-Hyo Jang, Chul-Ho Kim, Bong-Hyun Jung, Sang-Ki Rhee * Microbial and Bioprocess Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon 305 -600, South Korea | Molecular cloning of a gene encoding the thermoactive levansucrase from Rahnella aquatilis and its growth phase-dependent expression in Escherichia coli Jeong-Woo Seo, Ki-Bang Song, Ki-Hyo Jang, Chul-Ho Kim, Bong-Hyun Jung, Sang-Ki Rhee * Microbial and Bioprocess Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon 305 -600, South Korea | ||
==Pitfalls== | ==Pitfalls== | ||
| − | Wild type K12 E coli contains LsrA | + | Wild type K12 E coli contains identical LsrA gene sequence. Extraction of plasmids using Qiagen plasmid extraction kit produced high quality pure plasmids. However, PCR with designed oligo fragments were unable to produce the desired base pair length of DNA. Other attempts of PCR using Platinum PCR mix did not yield any results. |
===References=== | ===References=== | ||
Revision as of 05:01, 29 October 2008
LsrA promoter (indirectly activated by AI-2)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
LsrA is initially commercially synthesized with RBS BBa_B0034 by ATIbiotech in a blue script cloning vector. LsrA is then extracted using PCR with oligo fragments of DNA sequence 5'-GAATTATGTATACATAATGGCA-3' & 5'-GGTAAATATCCTTTAATTAAA-3' with Biobricks prefix and suffix respectively.
Source of Origin Sequence
Molecular cloning of a gene encoding the thermoactive levansucrase from Rahnella aquatilis and its growth phase-dependent expression in Escherichia coli Jeong-Woo Seo, Ki-Bang Song, Ki-Hyo Jang, Chul-Ho Kim, Bong-Hyun Jung, Sang-Ki Rhee * Microbial and Bioprocess Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon 305 -600, South Korea
Pitfalls
Wild type K12 E coli contains identical LsrA gene sequence. Extraction of plasmids using Qiagen plasmid extraction kit produced high quality pure plasmids. However, PCR with designed oligo fragments were unable to produce the desired base pair length of DNA. Other attempts of PCR using Platinum PCR mix did not yield any results.
References
Molecular cloning of a gene encoding the thermoactive levansucrase from Rahnella aquatilis and its growth phase-dependent expression in Escherichia coli Jeong-Woo Seo, Ki-Bang Song, Ki-Hyo Jang, Chul-Ho Kim, Bong-Hyun Jung, Sang-Ki Rhee *Microbial and Bioprocess Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon 305 -600, South Korea
Regulation of Uptake and Processing of the Quorum-Sensing Autoinducer AI-2 in Escherichia coli Karina B. Xavier and Bonnie L. Bassler* Department of Molecular Biology, Princeton University, Princeton, New Jersey