Difference between revisions of "Part:BBa K5184055"

Revision as of 07:59, 30 September 2024

st-t25ShZPO

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 zingiberene oxidase ShZPO was originally found in eukaryotic organisms. They were originally immobilized on the ER membrane. However, since E. coli does not have this structure, the 25 amino acid N-terminus ER transit peptide of the oxidase 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 sc-t25ShZPO 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

Usage and Biology

t25ShZPO is the truncated version of the plant P450 monooxidase, ShZPO, which is responsible for the two sequential oxidations of 7epiZ to produce 9H10,11epoZ via first a hydroxylation at C9 and then a epoxidation and C10 and C11. Like other enzymes of the P450 superfamily, t25ShZPO has a heme group as cofactor to facilitate necessary electron transfers associated with oxidation of its substrate. The oxidase also requires cooperation with a P450 reductase, in context of our part collection SlCPR1 or AtCPR2, to supply the electrons required for its catalytic activities. The 25aa 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, plasmid pW1-ZIS-NPPS-Mvan4662-st t25ShZPO-sc t76SlCPR2, aiming to test whether the enzymes can maintain their functions after expression and whether the SpyTag can be linked to the SpyCatcher [figure 9B]. Also, we incorporated 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 pW1-ZIS-NPPS-Mvan4662-st t25ShZPO-sc t76SlCPR2, 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]

Fermentation of pW1-ZIS-NPPS-Mvan4662-st t25ShZPO-sc t76SlCPR2 in DH5α was induced by IPTG and lasted 48 hours using dodecane as solvent. After the products were collected and underwent GC-MS analysis, we discovered that still, 9HZ and 9H10epoZ were not detected. Instead, only 7epiZ was produced.[figure 3A&B] 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 4]