Difference between revisions of "Part:BBa K4012010"
 
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GbCHS is a sequence code for Chalcone synthase in Anthurium andraeanum. The primary product of this enzyme is 4,2',4',6'-tetrahydroxychalcone (also termed naringenin-chalcone or chalcone) which can under specific conditions spontaneously isomerize into naringenin. It is a Acyltransferase involved in Flavonoid biosynthesis. The catalytic activity:
 
GbCHS is a sequence code for Chalcone synthase in Anthurium andraeanum. The primary product of this enzyme is 4,2',4',6'-tetrahydroxychalcone (also termed naringenin-chalcone or chalcone) which can under specific conditions spontaneously isomerize into naringenin. It is a Acyltransferase involved in Flavonoid biosynthesis. The catalytic activity:
 
4-coumaroyl-CoA + 2 H+ + 3 malonyl-CoA = 2',4,4',6'-tetrahydroxychalcone + 3 CO2 + 4 CoA
 
4-coumaroyl-CoA + 2 H+ + 3 malonyl-CoA = 2',4,4',6'-tetrahydroxychalcone + 3 CO2 + 4 CoA
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[[Image:CHS reaction.jpg|thumbnail|900px|center|'''Figure 1:'''
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[https://parts.igem.org/Part:BBa_K4012010] The schematic of catalytic activity ]]
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<partinfo>BBa_K4012010 parameters</partinfo>
 
<partinfo>BBa_K4012010 parameters</partinfo>
 
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===GbCHS in Level1 plasmid assembly===
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[[Image:CHS.jpg|thumbnail|750px|center|'''Figure 2:'''
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[https://parts.igem.org/Part:BBa_K4012010] A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence GbCHS]]
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The construction schematic of GbCHS sequence demonstrated as Fig .2. The initiation of the GbCHS sequence is done by promoter pTEF1, with termination done by tADH1. The sequence ConL2 and ConR3 are connector sequences within the Level 1 plasmid assembly. Furthermore, typr9-KVF and type9-VR are imposed to enact selection through colonies PCR, results shown in Fig.2 The band length 2500bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.
  
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===GbCHS in Level2 plasmid assembly===
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===Obtaining the pTEF1 fragment and BsaI digested verification===
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[[Image:yeast PCR.jpg|thumbnail|750px|center|'''Figure 3:'''
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<div style="text-align: center" img style="max-width: 40%;" src==https://2021.igem.org/wiki/images/8/83/T--AISSU_Union--Fig.1_Results_of_yeast_toolkit_plasmids_enzyme-digested_verification.jpg>
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[https://parts.igem.org/Part:BBa_K4012010] A: The results of colony PCR of upstream transformant; B: The results of colony PCR of downstream transformant; C: Construction of catechin metabolic pathway; D：Construction of naringenin metabolic pathway]]
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          <p>Fig.1 Results of yeast toolkit plasmids enzyme-digested verification</p>
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GbCHS is successfully inserted into the vector type9 C-mTurquoise-Ura.

Latest revision as of 11:53, 21 October 2021


GbCHS

GbCHS is a sequence code for Chalcone synthase in Anthurium andraeanum. The primary product of this enzyme is 4,2',4',6'-tetrahydroxychalcone (also termed naringenin-chalcone or chalcone) which can under specific conditions spontaneously isomerize into naringenin. It is a Acyltransferase involved in Flavonoid biosynthesis. The catalytic activity: 4-coumaroyl-CoA + 2 H+ + 3 malonyl-CoA = 2',4,4',6'-tetrahydroxychalcone + 3 CO2 + 4 CoA

Figure 1: [1] The schematic of catalytic activity


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 891
    Illegal SapI.rc site found at 840


GbCHS in Level1 plasmid assembly

Figure 2: [2] A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence GbCHS

The construction schematic of GbCHS sequence demonstrated as Fig .2. The initiation of the GbCHS sequence is done by promoter pTEF1, with termination done by tADH1. The sequence ConL2 and ConR3 are connector sequences within the Level 1 plasmid assembly. Furthermore, typr9-KVF and type9-VR are imposed to enact selection through colonies PCR, results shown in Fig.2 The band length 2500bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

GbCHS in Level2 plasmid assembly

Figure 3: [3] A: The results of colony PCR of upstream transformant; B: The results of colony PCR of downstream transformant; C: Construction of catechin metabolic pathway; D：Construction of naringenin metabolic pathway

GbCHS is successfully inserted into the vector type9 C-mTurquoise-Ura.