Difference between revisions of "Part:BBa M36394:Design"
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===Source=== | ===Source=== | ||
| − | RBS Leader and ATG came from BioFAB. Terminator comes from BioFAB. Spider silk monomer sequence was repeated six times, and the monomer AA sequence comes from Xia et al. The sequence was codon optimized for E. coli. Fos-Jun leucine zipper sequences were taken from O'Shea et al. They were also codon optimized for E.coli. | + | RBS Leader and ATG came from BioFAB (part number BD12). BD12 is a medium strength bicistronic RBS sequence, which we chose assuming we did not want a lot of spider silk being produced to kill the cell or too little to detect. Terminator comes from BioFAB (part number pFAB391). It has 99% termination efficiency. Spider silk monomer sequence was repeated six times, and the monomer AA sequence comes from Xia et al. Xia et al. designed the sequence from the natural gene N. clavipes spidroin I. The sequence was codon optimized for E. coli using. Fos-Jun leucine zipper sequences were taken from O'Shea et al. They were also codon optimized for E.coli. |
===References=== | ===References=== | ||
Revision as of 00:08, 9 December 2011
Spider Silk Actuator
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 109
Illegal PstI site found at 151
Illegal PstI site found at 510 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 109
Illegal PstI site found at 151
Illegal PstI site found at 510
Illegal NotI site found at 302 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 109
Illegal PstI site found at 151
Illegal PstI site found at 510 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 109
Illegal PstI site found at 151
Illegal PstI site found at 510 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 663
Design Notes
Our overall design had a base pair limit of 3000. The point of using of Fos and Jun leucine zippers was to attempt to get the spider silk hexamers to link up head to tail via covalent bonds in the E. coli cells. Thus, we hoped to generate extremely long chains of spider silk without coding for a huge monomer in the DNA. We were not able to spin the spider silk protein out, isolate it, or analyze its structural properties.
Source
RBS Leader and ATG came from BioFAB (part number BD12). BD12 is a medium strength bicistronic RBS sequence, which we chose assuming we did not want a lot of spider silk being produced to kill the cell or too little to detect. Terminator comes from BioFAB (part number pFAB391). It has 99% termination efficiency. Spider silk monomer sequence was repeated six times, and the monomer AA sequence comes from Xia et al. Xia et al. designed the sequence from the natural gene N. clavipes spidroin I. The sequence was codon optimized for E. coli using. Fos-Jun leucine zipper sequences were taken from O'Shea et al. They were also codon optimized for E.coli.
References
Xia, X.-X., Z.-G. Qian, C. S. Ki, Y. H. Park, D. L. Kaplan, and S. Y. Lee. "Native-sized Recombinant Spider Silk Protein Produced in Metabolically Engineered Escherichia Coli Results in a Strong Fiber." Proceedings of the National Academy of Sciences 107.32 (2010): 14059-4063. Print.
O'Shea, E., R. Rutkowski, W. Stafford, and P. Kim. "Preferential Heterodimer Formation by Isolated Leucine Zippers from Fos and Jun." Science 245.4918 (1989): 646-48. Print.