Difference between revisions of "Part:BBa K5184004"
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===Characterization===
 
===Characterization===
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After communication with Dr. Su from Earlham Institute, we opted for the yeast <i>S. cerevisiae</i> (strain CEN.PK2-1C).[figure 1] As an eukaryote with ER membranes, <i>S. cerevisiae</i> enables co-localization of the oxidase and reductases while also ensures efficient enzyme expression, reducing the chance of the proteins folding incorrectly.
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After communication with Dr. Su from Earlham Institute, we opted for the yeast <i>S. cerevisiae</i> (strain CEN.PK2-1C).[figure 1] As an eukaryote with ER membranes, <i>S. cerevisiae</i> enables co-localization of the oxidase and reductases while also ensures efficient enzyme expression.
  
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<center><html><img src="https://static.igem.wiki/teams/5184/parts/scie812.svg" width="600"/></html></center>
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<center><html><img src="https://static.igem.wiki/teams/5184/parts/img-7739.jpeg" width="600"/></html></center>
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<center><b>Figure 1: Integration of the two cytochrome P450 enzymes coding sequences into S. cerevisae genome: the pCRCT plasmid, encoding the endonuclease Cas9 and sgRNA for His integration locus leads to restriction of the His locus, of which, after a series of homologous recombination between the yeast genome and insert fragments, lead to integration of the cytochrome P450 enzymes into the <i>S. cerevisae</i> genome</b></center>
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<center><b>Figure 1: (A) Biosynthesis pathway of 9HZ and 9H10epoZ, starting from IPP and DMAPP that are products of the MVA pathway (B) Integration of the two cytochrome P450 enzymes coding sequences into <i>S. cerevisae</i> genome: the pCRCT plasmid, encoding the endonuclease Cas9 and sgRNA for His integration locus leads to restriction at the His locus, of which, after a series of homologous recombination between the yeast genome and insert fragments, leading to integration of the cytochrome P450 enzyme genes into the <i>S. cerevisae</i> genome</b></center>
  
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We inserted DNA fragments to site His of CEN.PK2-1C using lithium acetate transformation. Afterwards, yeast colony PCR was conducted, which shows the target strands were integrated into the genome successfully. The sequencing result also shows that the fragments are integrated into the yeast genome with no mutation. The constructed strains are named ShZPO-SlCPR2 and ShZPO-AtCPR1 respectively.[figure2A&B]
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We inserted DNA fragments to site His of CEN.PK2-1C using lithium acetate transformation. Afterwards, yeast colony PCR was conducted, which shows the target strands were integrated into the genome successfully. The sequencing result also shows that the fragments are integrated into the yeast genome with no mutation. The constructed strains are named ShZPO-SlCPR2 and ShZPO-AtCPR1 respectively.[figure 2]
  
 
<center><html><img src="https://static.igem.wiki/teams/5184/parts/scie813.webp" width="600"/></html></center>
 
<center><html><img src="https://static.igem.wiki/teams/5184/parts/scie813.webp" width="600"/></html></center>
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<center><b>Figure 2: A. Colony PCR results of ShZPO-SlCPR2A in His locus, ShZPO-SlCPR2B in His locus, ShZPO-tCPR1A in His locus, and ShZPO-AtCPR1B in His locus in <i>S. cerevisae</i> B. Alignments of sequencing results of colony CPR products against designed locus</b></center>
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<center><b>Figure 2: (A) Colony PCR results of ShZPO-SlCPR2A in His locus, ShZPO-SlCPR2B in His locus, ShZPO-tCPR1A in His locus, and ShZPO-AtCPR1B in His locus in <i>S. cerevisae</i> (B) Alignments of sequencing results of colony CPR products against designed locus</b></center>
  
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ShZPO-SlCPR2 and ShZPO-AtCPR1 were cocultured with pW1-ZIS-NPPS-Mvan4662+pMVA in <i>E. coli</i> strain DH5α respectively. Fermentation of the coculture was carried out, which is induced by IPTG and lasted 48 hours at 28°C 200 rpm using dodecane as solvent. After the products were collected and underwent GC-MS analysis, 9HZ, a mid-product of the redox reaction of 7epiZ to 9H10epoZ was detected from the co-culture using ShZPO-SlCPR2 only.[figure 3A&B&C]
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ShZPO-SlCPR2 and ShZPO-AtCPR1 were cocultured with pW1-ZIS-NPPS-Mvan4662+pMVA in <i>E. coli</i> strain DH5α respectively. Fermentation of the coculture was carried out, which is induced by IPTG and lasted 48 hours at 28°C 200 rpm using dodecane as solvent. After the products were collected and underwent GC-MS analysis, 9HZ, a mid-product of the redox reaction of 7epiZ to 9H10epoZ was detected from the co-culture using ShZPO-SlCPR2 only.[figure 3]
  
 
<center><html><img src="https://static.igem.wiki/teams/5184/parts/scie814a.webp" width="600"/></html></center>
 
<center><html><img src="https://static.igem.wiki/teams/5184/parts/scie814a.webp" width="600"/></html></center>
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<center><b>Figure 2: A. Gas-phase chromatography results for culture of ShZPO-SlCPR2 in His locus with dodecane as solvent B. Mass spectrometry and structure elucidation results of the sample</b></center>
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<center><b>Figure 3: (A) Gas-phase chromatography results for culture of ShZPO-SlCPR2 in His locus with dodecane as solvent (B) Mass spectrometry and structure elucidation results of the sample</b></center>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
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Latest revision as of 22:05, 1 October 2024


slCPR2

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. In order to make the oxidase ShZPO funciton efficiently, we plan to incorporate the NADPH-cytochrome P450 reductase SlCPR2 as its redox partner and electron supplier. Our exploration of the reductase provide future iGEM team with a novel way of generating sesquiterpenes from a monocyclic sesquiterpene through oxidation carried out by the collaboration of an oxidase and a reductase.

This part belongs to a collection used for the synthesis of 9HZ and 9H10epoZ, two sesquiterpenes with strong repellent effects towards spider mites. This part collection includes SltNPPS, Mvan4662, ShZIS, ShZPO, SlCPR2 and AtCPR1.

Essential Information

Sequences

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]

Usage and Biology

SlCPR2 is a NADPH-cytochrome P450 reductase found in Solanum lycopersicum. Analysis of the reductase revealed that their is a membrane anchor domain at the N-terminus of the amino acid sequence of SlCPR2. The enzyme was found to be predominantly localized on the ER membrane. The co-localization of reductase SlCPR2 and oxidase ShZPO on the ER membrane ensures efficient electron transfer. 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. We constructed a novel sesquiterpene synthesis pathway in E. coli. Using glucose as our raw material, we introduce the MVA pathway, which transforms glucose into dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). Afterwards, SltNPPS, a neryl diphosphate synthase catalyze the production of NPP from IPP and DMAPP. Mvan4662 is then introduced to catalyze the formation of Z,Z-FPP. Then, ShZIS transforms Z,Z-FPP into 7epiZ. In the end, ShZPO works collaboratively with SlCPR2 or AtCPR1.

Characterization

After communication with Dr. Su from Earlham Institute, we opted for the yeast S. cerevisiae (strain CEN.PK2-1C).[figure 1] As an eukaryote with ER membranes, S. cerevisiae enables co-localization of the oxidase and reductases while also ensures efficient enzyme expression.

Figure 1: (A) Biosynthesis pathway of 9HZ and 9H10epoZ, starting from IPP and DMAPP that are products of the MVA pathway (B) Integration of the two cytochrome P450 enzymes coding sequences into S. cerevisae genome: the pCRCT plasmid, encoding the endonuclease Cas9 and sgRNA for His integration locus leads to restriction at the His locus, of which, after a series of homologous recombination between the yeast genome and insert fragments, leading to integration of the cytochrome P450 enzyme genes into the S. cerevisae genome

We inserted DNA fragments to site His of CEN.PK2-1C using lithium acetate transformation. Afterwards, yeast colony PCR was conducted, which shows the target strands were integrated into the genome successfully. The sequencing result also shows that the fragments are integrated into the yeast genome with no mutation. The constructed strains are named ShZPO-SlCPR2 and ShZPO-AtCPR1 respectively.[figure 2]

Figure 2: (A) Colony PCR results of ShZPO-SlCPR2A in His locus, ShZPO-SlCPR2B in His locus, ShZPO-tCPR1A in His locus, and ShZPO-AtCPR1B in His locus in S. cerevisae (B) Alignments of sequencing results of colony CPR products against designed locus

ShZPO-SlCPR2 and ShZPO-AtCPR1 were cocultured with pW1-ZIS-NPPS-Mvan4662+pMVA in E. coli strain DH5α respectively. Fermentation of the coculture was carried out, which is induced by IPTG and lasted 48 hours at 28°C 200 rpm using dodecane as solvent. After the products were collected and underwent GC-MS analysis, 9HZ, a mid-product of the redox reaction of 7epiZ to 9H10epoZ was detected from the co-culture using ShZPO-SlCPR2 only.[figure 3]

Figure 3: (A) Gas-phase chromatography results for culture of ShZPO-SlCPR2 in His locus with dodecane as solvent (B) Mass spectrometry and structure elucidation results of the sample