Part:BBa_K1789011
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1005
- 1000COMPATIBLE 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
The result of the agarose electrophoresis was shown on the picture above.
Enzyme cutting
Methods
After the assembly ,the plasmid was transferred into the Competent E. coli DH5α). After culturing overnight in LB,we minipreped the plasmid for cutting. The preparation of the plasmid was performed with TIANprep Mini Plasmid Kit from TIANGEN. The cutting procedure was performed with EcoRI and XbaI restriction endonuclease bought from TAKARA.
The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.
Results
The result of the agarose electrophoresis was shown on the picture above. This parts functional test can be involved into k1789022.
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