Difference between revisions of "Part:BBa K1789011"
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==Usage and Biology==
 
==Usage and Biology==
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This part is constructed with IaaH and a terminatior.
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This part is constructed with IaaH expression gene and a terminatior.
  
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IaaH is the tryptophan-2-mono-oxygenase. This enzyme catalyzes the oxidative carboxylation of L-tryptophan to indole-3-acetamide. IaaH is the indoleacetimide hydrolase. This enzyme catalyses the hydrolysis of indoleacetamide to indoleacetate and ammonia. This pathway is relevantly easy to be expressed in prokaryotic cells, and its substrate (Trp) can be synthesized by host cells.  
+
IaaH is the tryptophan-2-mono-oxygenase. This enzyme catalyzes the oxidative carboxylation of L-tryptophan to indole-3-acetamide. IaaH is the indoleacetimide hydrolase. This enzyme catalyses the hydrolysis of indoleacetamide to indoleacetate and ammonia. This pathway is relevantly easy to be expressed in prokaryotic cells, and its substrate (Trp) can be synthesized by host cells.
  
 
==Sequence and Features==
 
==Sequence and Features==
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==Experimental Validation==
 
==Experimental Validation==
  
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===Sequencing===
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This part is validated through four ways: Enzyme digestion, PCR, and Sequencing
 +
===PCR===
  
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This part is sequenced as correct after construction.
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'''Methods'''
  
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===Enzyme digestion test ===
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The PCR is performed with Premix EX Taq by Takara.
  
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A double-enzyme digestion test was implemented to verify this part.
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The PCR protocol is selected based on the Users Manuel.
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The Electrophoresis was performed on a 1％ Agarose glu.
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[[File:IAAH.jpg|250px|]]
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The length of the digested DNA fits the design.
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'''Results'''
  
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===Functional Test===
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[[File:nudt-IAAH+Ter.jpg|150px|]]
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The function of this part can be verified with in the experimental validation of BBa_K1789022.
+
  
 +
The result of the agarose electrophoresis was shown on the picture above.
 +
 +
===Enzyme digesting===
 +
 +
'''Methods'''
 +
 +
After the assembly,the plasmid was transferred into the Competent ''E. coli'' DH5α. After culturing overnight in LB,we minipreped the plasmid for digesting.
 +
The preparation of the plasmid was performed with TIANprep Mini Plasmid Kit from ''TIANGEN''. The digesting procedure was performed with EcoRI and XbaI restriction endonuclease bought from ''TAKARA''.
 +
 +
The plasmid was digested in a 20μL system at 37 ℃ for 2 hours.
 +
The Electrophoresis was performed on a 1％ Agarose glu.
 +
 +
'''Results'''
 +
 +
[[File:Iaah+ter酶切.jpg|500px|]]
 +
 +
The result of the agarose electrophoresis was shown on the picture above.
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===

Latest revision as of 01:40, 19 September 2015

IAAH+Ter

It is the combination of IAAH and terminatior.

Usage and Biology

This part is constructed with IaaH expression gene and a terminatior.

IaaH is the tryptophan-2-mono-oxygenase. This enzyme catalyzes the oxidative carboxylation of L-tryptophan to indole-3-acetamide. IaaH is the indoleacetimide hydrolase. This enzyme catalyses the hydrolysis of indoleacetamide to indoleacetate and ammonia. This pathway is relevantly easy to be expressed in prokaryotic cells, and its substrate (Trp) can be synthesized by host cells.

Sequence and Features

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1005
  • 1000
    COMPATIBLE WITH RFC[1000]

Experimental Validation

This part is validated through four ways: Enzyme digestion, PCR, and Sequencing

PCR

Methods

The PCR is performed with Premix EX Taq by Takara.

The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose glu.

Results

Nudt-IAAH+Ter.jpg

The result of the agarose electrophoresis was shown on the picture above.

Enzyme digesting

Methods

After the assembly,the plasmid was transferred into the Competent E. coli DH5α. After culturing overnight in LB,we minipreped the plasmid for digesting. The preparation of the plasmid was performed with TIANprep Mini Plasmid Kit from TIANGEN. The digesting procedure was performed with EcoRI and XbaI restriction endonuclease bought from TAKARA.

The plasmid was digested in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.

Results

Iaah+ter酶切.jpg

The result of the agarose electrophoresis was shown on the picture above. This parts functional test can be involved into k1789022.