Difference between revisions of "Part:BBa K3843002:Design"

Latest revision as of 18:01, 19 October 2021

1UTM-HRP (fusion)

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1113
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1077
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1249

Design Notes

1UTM was translationally fused to horseradish peroxidase (HRP). Before use in the fusion, 1UTM's signal peptide was removed using the SignalP 5.0 server, and the last 142 bp were removed to produce the mature 1UTM polypeptide. HRP's signal peptide was removed using the SignalP 5.0 server, and introns were removed from the genomic DNA sequence to yield the properly functioning protein. A 6-His tag followed by a TEV protease linker was added at the N-terminus to allow for purification by Ni-NTA affinity chromatography. At the C-terminus, an Avi-tag was appended, allowing for biotinylation. A flexible, moderately long (13 AA), and polar linker (consisting of Ser and Thr) separated 1UTM and HRP, allowing for proper functioning and stability of the fusion protein in aqueous solution. The overall fusion protein sequence was codon optimized for E. coli K12, and illegal restriction sites for RFC[25] were removed.

Source

The sequence of 1UTM was obtained from GenBank: https://www.ncbi.nlm.nih.gov/nuccore/X70071.

The genomic sequence of HRP was obtained from GenBank: https://www.ncbi.nlm.nih.gov/nuccore/M37156.

The sequence of the Ser-Thr linker was arbitrarily designed and obtained by back-translation of the desired amino acid sequence.

The His-tag and TEV linker sequence was retrieved from the His-tag and TEV linker that occurs in the bacterial expression vector pMCSG7: https://plasmid.med.harvard.edu/PlasmidRepository/file/sequence/pMCSG7.gb

The Avi-Tag sequence was adapted from Cull & Schatz (2000): https://doi.org/10.1016/s0076-6879(00)26068-0

References

Cull, M. G., & Schatz, P. J. (2000). Biotinylation of proteins in vivo and in vitro using Small Peptide tags. Methods in Enzymology, (326), 430–440. https://doi.org/10.1016/s0076-6879(00)26068-0

Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C. & Ferrin, T. E. UCSF Chimera--a visualization system for exploratory research and analysis (Version 1.15). J Comput Chem. 2004; 25(13): 1605-1612. https://www.ncbi.nlm.nih.gov/pubmed/15264254

@@ Line 7: / Line 7: @@
 ===Design Notes===
-UTM was translationally fused to horseradish peroxidase (HRP). Before use in the fusion, 1UTM's signal peptide was removed using the SignalP 5.0 server, and the last 142 bp were removed to produce the mature 1UTM polypeptide. HRP's signal peptide was removed using the SignalP 5.0 server, and introns were removed from the genomic DNA sequence to yield the properly functioning protein. A 6-His tag followed by a TEV protease linker was added at the N-terminus to allow for purification by Ni-NTA affinity chromatography. At the C-terminus, an Avi-tag was appended, allowing for biotinylation. A flexible, moderately long (13 AA), and polar linker (consisting of Ser and Thr) separated 1UTM and HRP, allowing for proper functioning and stability of the fusion protein in aqueous solution. The overall fusion protein sequence was codon optimized for E. coli K12, illegal restriction sites for RFC[25] were removed.
+UTM was translationally fused to horseradish peroxidase (HRP). Before use in the fusion, 1UTM's signal peptide was removed using the SignalP 5.0 server, and the last 142 bp were removed to produce the mature 1UTM polypeptide. HRP's signal peptide was removed using the SignalP 5.0 server, and introns were removed from the genomic DNA sequence to yield the properly functioning protein. A 6-His tag followed by a TEV protease linker was added at the N-terminus to allow for purification by Ni-NTA affinity chromatography. At the C-terminus, an Avi-tag was appended, allowing for biotinylation. A flexible, moderately long (13 AA), and polar linker (consisting of Ser and Thr) separated 1UTM and HRP, allowing for proper functioning and stability of the fusion protein in aqueous solution. The overall fusion protein sequence was codon optimized for <i>E. coli K12</i>, and illegal restriction sites for RFC[25] were removed.
 ===Source===