Difference between revisions of "Part:BBa K5184054"
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<partinfo>BBa_K5184054 short</partinfo> | <partinfo>BBa_K5184054 short</partinfo> | ||
− | To equip our insecticide with enhanced prevention efficacy against | + | 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 immobalized on the ER membrane. However, since <i>E. coli</i> 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 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-t25ShZPO provide future iGEM teams with a novel method to synthesize an enzyme originally found in eukaryotes through a prokaryotic chassis. |
==Essential Information== | ==Essential Information== | ||
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===Usage and Biology=== | ===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. | 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 25aa N-terminus ER transit peptide of the oxidase is truncated to enhance solubility and expression rate of the enzyme in <i>E. coli</i>. |
− | 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. | + | The SpyTag-SpyCather system was originally found in <i>Streptococcus pyogenes</i>, 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=== | ===Characterization=== | ||
− | + | To increase the expression level and functionality of oxidase and reductases in <i>E. coli</i>, 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 <i>E. coli</i>, we incorporated the plasmids pET28a-sc t25ShZPO, pET28a-st t76SlCPR2 and pET28a-sc t55AtCPR1 to test whether the enzymes can be successfully expressed in <i>E. coli</i>.[figure 1C] | ||
− | <center><html><img src="https://static.igem.wiki/teams/5184/parts/ | + | <center><html><img src="https://static.igem.wiki/teams/5184/parts/scie88.webp" width="600"/></html></center> |
− | <center><b>Figure 1: A. | + | <center><b>Figure 1: A. Attachment of SpyTag and SpyCatcher to two cytochrome P450 enzymes; the formation of isopeptide bond between lysine on SpyCatcher and aspartic acid on SpyTag can link the two enzymes together, this proximity allows electron transfer and thus catalysis of the CYP (t25ShZPO) enzyme to occur B. Plasmid construct of pW1-p-ZIS-Mvan4662-NPPS-T-P-SpyTag-t25ShZPO-SpyCatcher-t76SlCPR2-t C. Plasmid constructs of pET28a-SpyTag-t76SlCPR2, pET28a-SpyCatcher-t25ShZPO, and pET28a-SpyCatcher-t55AtCPR1 </b></center> |
− | We | + | We employed GoldenGate Assembly to build the plasmid pET28a-sc t25ShZPO, pET28a-st t76SlCPR2, pET28a-sc t55AtCPR1[figure 2A&B] and then transformed the built plasmids into the <i>E. coli</i> strain DH5α. The colony PCR results and sequencing results show that the plasmids are successfully constructed with no mutations.[figure2C&D] |
− | <center><html><img src="https://static.igem.wiki/teams/5184/parts/ | + | <center><html><img src="https://static.igem.wiki/teams/5184/parts/scie89.webp" width="600"/></html></center> |
− | <center><b>Figure | + | <center><b>Figure 2: A. Colony PCR results of pET28a-sc t55AtCPR1, pET28a-st t76SlCPR2, and pET28a-sc t25ShZPO B. Alignment of the sequencing results in Fig10A against designed plasmids C. Colony PCR results of pW1-ZIS-Mvan4662-NPPS-st t25ShZPO-sc t76SlCPR2; the insert region is amplified using two sets of primers and their products designated SCIE22E and SCIE22F D. Alignment of the sequencing results of colony PCR products against designed plasmids</b></center> |
− | + | Plasmids pET28a-sc t25ShZPO, pET28a-st t76SlCPR2, pET28a-sc t55AtCPR1 were transformed into <i>E.coli</i> strain 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] | |
− | <center><html><img src="https://static.igem.wiki/teams/5184/parts/ | + | <center><html><img src="https://static.igem.wiki/teams/5184/parts/scie810.webp" width="600"/></html></center> |
− | <center><b>Figure 3: | + | <center><b>Figure 3: Liquid culture of pET28a-CYP/CPR in BL21(DE3) was harvested, supernatant and precipitate was collected after centrifugation. S: Supernatant, P: Precipitate</b></center> |
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<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display |
Latest revision as of 22:21, 1 October 2024
sc-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 immobalized 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 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-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
- 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
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 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 plasmid 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 strain 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]