Difference between revisions of "Part:BBa K3711074"

 
 
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<partinfo>BBa_K3711074 parameters</partinfo>
 
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<h1>'''Description'''</h1>
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This is a composite part for intracellular expression of 4CL. Panb1 is a constitutive promoter in yeast, which is expressed under anaerobic conditions, while under aerobic conditions, Panb1, as a repression target of ROX1, is inhibited. When Panb1 initiates the expression, 4CL is expressed and participates in the production from ferulic acid to p-Coumaroyl-CoA.
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<h1>'''Usage and Biology'''</h1>
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In the curcumin biosynthesis pathway, 4CL is located in the upstream of the metabolic pathway and plays a key role in the synthesis of phenylpropane derivatives. 4CL is the branching enzyme that connects the lignin synthesis pathway and flavonoid pathway, controls the metabolic synthesis direction of phenylpropane derivatives, and is the key enzyme in the phenylpropane synthesis pathway. 4CL acts on different substrates to produce acyl CoA thiolipids for subsequent reactions. Through the synthesis of these phenylpropane derivatives CoA lipids (e.g. p-gumaroyl CoA, feruloyl CoA, p-coumaryl CoA), downstream enzymes use them as substrates to form different phenylpropane metabolites. Therefore, 4CL enzyme plays a switching role in the biosynthesis of curcumin. In the curcumin biosynthesis pathway, the role of 4CL in dipeptide-CoA synthase DCS/ curcumin synthase CURS is to catalyze cinnamic acid to produce cinnamyl-CoA and make the reaction to the direction of curcumin production. In the curcumin synthase CUS pathway, ferulic acid is used to catalyze the formation of gumaroyl-CoA in order to facilitate the following reaction.
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<h1>'''Molecular cloning'''</h1>
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Not quite to what we expect, after repeated transfection to the yeast, only a few products are expressed inside of eukaryotic system. Because of the large molecular weight and various types of some of our protein, we suspect that the common signal peptide we use, α-factor, is not enough to bring our protein out of the cell. While there is some of the genes without detectable products and we are hoping to get higher expression level, new primers for PCR are designed to ignore α-factor from our target gene in PCR. Then, likewise, we reconstruct this series of plasmid without α-factor through similar double-enzyme digestion and reconnection which insert our target genes right behind Panb1 promoter.
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[[File:T--HUST-China--74-1.png|400px|thumb|center|Figure1:Plasmid construction and colony PCR results of Panb1-CUS-AOX1 Terminator, Panb1-ACC-AOX1 Terminator, Panb1-4CL-AOX1 Terminator and Panb1-crtI-AOX1 Terminator transformed E.coli]]
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The bands of Panb1-CUS-AOX1 Terminator (2000+bp), Panb1-ACC-AOX1 Terminator (3000bp), Panb1-4CL-AOX1 Terminator (2500+bp) and Panb1-crtI-AOX1 Terminator (2500bp) from colony PCR are identical to the theoretical lengths of 2158bp, 2832bp, 2688bp and 2437bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid are successfully constructed.
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<br>

Latest revision as of 10:32, 21 October 2021


Panb1-4CL-AOX1 Terminator


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 124
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1210
    Illegal BsaI.rc site found at 1345


Description

This is a composite part for intracellular expression of 4CL. Panb1 is a constitutive promoter in yeast, which is expressed under anaerobic conditions, while under aerobic conditions, Panb1, as a repression target of ROX1, is inhibited. When Panb1 initiates the expression, 4CL is expressed and participates in the production from ferulic acid to p-Coumaroyl-CoA.

Usage and Biology

In the curcumin biosynthesis pathway, 4CL is located in the upstream of the metabolic pathway and plays a key role in the synthesis of phenylpropane derivatives. 4CL is the branching enzyme that connects the lignin synthesis pathway and flavonoid pathway, controls the metabolic synthesis direction of phenylpropane derivatives, and is the key enzyme in the phenylpropane synthesis pathway. 4CL acts on different substrates to produce acyl CoA thiolipids for subsequent reactions. Through the synthesis of these phenylpropane derivatives CoA lipids (e.g. p-gumaroyl CoA, feruloyl CoA, p-coumaryl CoA), downstream enzymes use them as substrates to form different phenylpropane metabolites. Therefore, 4CL enzyme plays a switching role in the biosynthesis of curcumin. In the curcumin biosynthesis pathway, the role of 4CL in dipeptide-CoA synthase DCS/ curcumin synthase CURS is to catalyze cinnamic acid to produce cinnamyl-CoA and make the reaction to the direction of curcumin production. In the curcumin synthase CUS pathway, ferulic acid is used to catalyze the formation of gumaroyl-CoA in order to facilitate the following reaction.

Molecular cloning

Not quite to what we expect, after repeated transfection to the yeast, only a few products are expressed inside of eukaryotic system. Because of the large molecular weight and various types of some of our protein, we suspect that the common signal peptide we use, α-factor, is not enough to bring our protein out of the cell. While there is some of the genes without detectable products and we are hoping to get higher expression level, new primers for PCR are designed to ignore α-factor from our target gene in PCR. Then, likewise, we reconstruct this series of plasmid without α-factor through similar double-enzyme digestion and reconnection which insert our target genes right behind Panb1 promoter.

Figure1:Plasmid construction and colony PCR results of Panb1-CUS-AOX1 Terminator, Panb1-ACC-AOX1 Terminator, Panb1-4CL-AOX1 Terminator and Panb1-crtI-AOX1 Terminator transformed E.coli

The bands of Panb1-CUS-AOX1 Terminator (2000+bp), Panb1-ACC-AOX1 Terminator (3000bp), Panb1-4CL-AOX1 Terminator (2500+bp) and Panb1-crtI-AOX1 Terminator (2500bp) from colony PCR are identical to the theoretical lengths of 2158bp, 2832bp, 2688bp and 2437bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid are successfully constructed.