This sequence was used with E. Coli cells.
This is the full sequence for the spider silk actuator. It includes the bicistonic sequence, FOS, a linker, the silk hexamer, another linker, JUN, a stop codon, and a transcription terminator. It is triggered by PoPS.
There were no complications when creating the full sequence. However, some parts, such as the hexamer, took special considerations which are detailed on their basic part pages.
The monomer amino acid sequence was taken from 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.
This is a spider silk hexamer protein. The monomer amino acid sequence was taken from Xia et al. and codon optimized for expression in E. coli. Repeats above 10 base pairs were also minimized to allow for ease of synthesis.
Needed to minimize repeats and allow for ideal mRNA forming without too many loops while still maintaining codon optimization for E. coli.
Dominique Dabija, Christopher Jackson, Debha Amatya
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.
Fos-p1n is a protein domain that contains a leucine zipper that covalently links to Jun-p1n. Fos-p1n is useful in creating heterodimerized parts that self-assemble into tightly linked chains.
Design was modified to codon optimized for expression in E. coli bacterium.
Jun-p1n is a protein domain that contains a leucine zipper that covalently bonds to Fos-p1n. Jun-p1n is useful for creating heterodimerized proteins that self-assemble into tightly linked chains.
This part was codon optimized for expression in the E. coli bacterium.
Transcription Terminator (99% efficient)
The part comes from the E. Coli chassis.
This is a terminator sequence to stop transcription. Put it at the very end of your sequence.
This is a bicistonic sequence which includes a ribosome binding site, a leader, another ribosome binding site, and the start codon ATG. This goes at the beginning of the actuator to help the ribosome bind and start translation.
This sequence is originally from BIOFAB. It is part BD12, apFAB535.
This is a stop codon for E.coli. The sequence is TAA
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, 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. The sequence was codon optimized for E. coli. Fos-Jun leucine zipper sequences were taken from . 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. They were also codon optimized for E.coli. Our system was placed into a Rhamnex 67K Xbrane plasmid and tested using varying concentrations of rhamnose.
This is a Spider Silk Generating Device that uses Fos and Jun leucine zipper sequences flanking a spider silk protein hexamer. The leucine zippers are linked to the spider silk hexamer by 8 AA linkers. RBS+Leader+ATG are part of the start of the gene, and the terminator is at the end.
Our overall design had a base pair limit of 3000. This pointed to usage of Fos and Jun leucine zippers to attempt to get the spider silk hexamers to link up head to tail via covalent bonds in the E. coli cells. We were not able to spin the spider silk protein out, isolate it, or analyze its structural properties.
Conversion of the iGEM parts registry to SBOL2.1