Part:BBa_K1110003:Design
Mambalgin-1 cDNA
- 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
For the design, we took the coding sequence for mambalgin, removed the TAG stop codon, and added RFC 25 prefix and suffix in Snapgene software to visualize. RFC 25 was used because we were anticipating the use of this part in creating fusion proteins. Additional nucleotides were added before the biobrick prefix and after the biobrick suffix to enable more efficient restriction enzyme cutting at EcoRI and PstI sites. The mambalgin construct was then ordered from IDT and ligated into pSB1C3 backbone. Using the pGAPzα expression system provided by ThermoFisher Scientific, the GSU team has been working to express the protein in Pichia Pastoris. The pGAPZα vector includes the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, an alpha secretory signal peptide, and Myc and 6x His epitopes. The MCS, between the alpha secretion signal and epitopes, consists of restriction sites which will enable insertion of mambalgin. In-frame insertion into the MCS of pGAPza will be possible after using PCR to modify the construct - removing the RFC 25 prefix and suffix and adding restriction sites and nucleotides.
Source
Dendroaspis polylepis Synthesized by IDT
References
Mambalgin peptide coding sequence was retrieved from supplementary materials in Diochot, Sylvie, et al. "Black mamba venom peptides target acid-sensing ion channels to abolish pain." Nature 490.7421 (2012): 552-555.