Coding

Part:BBa_K5184056

Designed by: Qianyue Jin, Ziyan Zhou   Group: iGEM24_GreatBay-SCIE   (2024-09-27)
Revision as of 12:12, 27 September 2024 by Leviz (Talk | contribs)


sc-t55AtCPR1

To equip our insecticide with enhanced prevention efficacy against T. urticae, we also decide to synthesize 9-hydroxy-zingiberene (9HZ) and 9-hydroxy-10,11-epoxy zingiberene (9H10epoZ), two oxidized products of the monocyclic sesquiterpene 8epiZ. However, the reductase AtCPR1 was originally found in eukaryotic organisms. They were originally immobalized on the ER membrane. However, since E. coli does not have this structure, the 55 amino acid N-terminus ER transit peptide of the reductase is truncated to enhance solubility and expression rate. Also, a SpyCathcer is added, which will form an isopeptide bond with the SpyTag, thus imitating the colocalization of the two enzymes in eukaryotes. Our usage of sc-t55AtCPR1 provide future iGEM teams with a novel method to synthesize an enzyme originally found in eukaryotes through a prokaryotic chassis.

Usage and Biology

"t55AtCPR1 is the truncated version a cytochrome P450 reductase found in Arabidopsis thaliana. It contains two domains, one with binding sites for FAD, flavin adenine dinucleotide, and NADPH, nicotinamide adenine dinucleotide; the other with binding site for FMN, flavin mononucleotide. The two cofactors, FAD and FMN are flavin proteins with multiple variable oxidation states, enabling them to control electron movement. The electron from NADPH is transferred via the two flavin proteins, FAD and FMN in order, and finally transferred to where the reductive power is required, in context of our part collection, ShZPO, a cytochrome P450 oxidase. The 55aa N-terminus ER transit peptide of the oxidase is truncated to enhance solubility and expression rate of the enzyme in E. coli. The SpyTag-SpyCather system was originally found in Streptococcus pyogenes, with its fibronectin-binding protein FbaB containing a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes is obtained."

Sequence and Features


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]


[edit]
Categories
Parameters
None