Coding

Part:BBa_K5184059

Designed by: Ziyan Zhou   Group: iGEM24_GreatBay-SCIE   (2024-09-27)
Revision as of 12:16, 27 September 2024 by Leviz (Talk | contribs)


st-t76SlCPR2

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 9epiZ. However, since the reductase SlCPR2 was originally found in eukaryotic organisms. They were originally immobalized on the ER membrane. However, since E. coli does not have this structure, the 76 amino acid N-terminus ER transit peptide of the reductase is truncated to enhance solubility and expression rate. Also, a SpyTag is added, which will form an isopeptide bond with the SpyCatcher, thus imitating the colocalization of the two enzymes in eukaryotes. Our usage of st-t76SlCPR2 provide future iGEM teams with a novel method to synthesize an enzyme originally found in eukaryotes through a prokaryotic chassis.

Usage and Biology

t76SlCPR2 is the truncated version a cytochrome P450 reductase found in Solanum habrochaites. Consisting of two domains, one with a binding site for flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) and another with a binding site for flavin mononucleotide (FMN), SlCPR2 functions through transferring electrons to cytochrome P450 oxidases, in our context ShZPO. To perform its functions, SlCPR2 requires the presence of NADPH and the cofactors FAD and FMN, which are two falvin priteins existing in various redox forms and able to control electron movement. The electrons provided by NADPH are transferred to FAD and FMN in order, and finally, the electrons required for the reduction reaction are transferred. 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]


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Categories
Parameters
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