Difference between revisions of "Part:BBa K3711046"

 
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===Functional Parameters===
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<h1>'''Description'''</h1>
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<partinfo>BBa_K3711046 parameters</partinfo>
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This is a composite component for expressing 4CL outside the cell. 4CL is transcribed and translated into 4-Coumarate:Coenzyme A Ligase，which is the key enzyme in the synthesis of lycopene. It participates in the transformation from ferulic acid to p-Coumaroyl-CoA
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. AOX1 promoter is a strong promoter induced by methanol. Under the condition of methanol induction, with the help of α factor, 4CL is translated and excreted from the cell.
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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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Plasmid with target gene is transformed into E.coli. From them, we acquire large amount of target gene using as raw material for further operation.
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**tu**
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Using E.coli for amplification, we extract and digest them with Bgl I or Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting lost while being frozen. Then, concentration of linear plasmid is also applied to achieve higher copy number and higher expression level. Several rounds of electroporation later, we successfully get all the plasmid with AOX1 as promoter into yeast.
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**tu**
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<h1>'''SDS-PAGE'''</h1>
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After confirmation from colony PCR and sequencing, we using the successfully integrated yeast for expression. At first, we try to detect our target protein in the supernatant since there is signal peptide.
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**tu**

Revision as of 09:07, 21 October 2021


AOX1-α factor-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 1187
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2021
    Illegal BsaI.rc site found at 2156