Part:BBa_K5184056
sc-t55AtCPR1
To equip our insecticide with enhanced prevention efficacy against spider mites, we also decide to synthesize 9-hydroxy-zingiberene (9HZ) and 9-hydroxy-10,11-epoxy zingiberene (9H10epoZ), two oxidized products of the monocyclic sesquiterpene 7epiZ. However, the reductase AtCPR1 was originally found in eukaryotic organisms. They were originally immobilized 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.
Essential Information
Sequences
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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."
Characterization
To increase the expression level and functionality of oxidase and reductases in E. coli, we introduced the spytag-spycatcher system in addition to truncating the N-terminal anchor regions [figure 9A]. Through the formation of an isopeptide bond between the tag and the catcher, introduction of this system links the oxidase and reductases together, thus facilitating efficient electron transfer. To test whether the enzymes can be expressed successfully in E. coli, we constructed the plasmids pET28a-sc t25ShZPO, pET28a-st t76SlCPR2 and pET28a-sc t55AtCPR1 to test whether the enzymes can be successfully expressed in E. coli.[figure 1C]
We employed GoldenGate Assembly to build the plasmids pET28a-sc t25ShZPO, pET28a-st t76SlCPR2, pET28a-sc t55AtCPR1[figure 2A&B] and then transformed the built plasmids into the E. coli strain DH5α. The colony PCR results and sequencing results show that the plasmids are successfully constructed with no mutations.[figure2C&D]
Plasmids pET28a-sc t25ShZPO, pET28a-st t76SlCPR2, pET28a-sc t55AtCPR1 were transformed into E.coli strand BL21(DE3), fermented and induced by IPTG. The SDS-PAGE result shows that only reductases linked with the SpyTag or SpyCatcher could be expressed successfully, and the oxidase was expressed in inclusion bodies.[figure 3]
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