Difference between revisions of "Part:BBa K1110003:Design"
(→‎Design Notes)
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===Design Notes===
 
===Design Notes===
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For the design, we used assembly standard 25 and removed a start codon from the sequence. Using the pGAPZα expression system, the GSU team has been working to express the protein in Pichia Pastoris. The pGAPZα vector includes an alpha secretion, MYC epitome, and 6xHis tag region.   This sequence is optimized for this expression system.
+
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, consist 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===
 
===Source===

Revision as of 15:06, 1 September 2015

Mambalgin-1 cDNA


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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, consist 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.