Difference between revisions of "Part:BBa K1789011"
(→Enzyme digesting) |
|||
| (8 intermediate revisions by 2 users not shown) | |||
| Line 5: | Line 5: | ||
==Usage and Biology== | ==Usage and Biology== | ||
| − | This part is constructed with IaaH and a terminatior. | + | 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. | + | 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== | ||
| Line 16: | Line 16: | ||
==Experimental Validation== | ==Experimental Validation== | ||
| − | This part is validated through four ways: Enzyme | + | This part is validated through four ways: Enzyme digestion, PCR, and Sequencing |
===PCR=== | ===PCR=== | ||
| Line 28: | Line 28: | ||
'''Results''' | '''Results''' | ||
| − | [[File: | + | [[File:nudt-IAAH+Ter.jpg|150px|]] |
The result of the agarose electrophoresis was shown on the picture above. | The result of the agarose electrophoresis was shown on the picture above. | ||
| − | ===Enzyme | + | ===Enzyme digesting=== |
'''Methods''' | '''Methods''' | ||
| − | After the assembly ,the plasmid was transferred into the Competent ''E. coli'' DH5α | + | 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 | + | 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 | + | The plasmid was digested in a 20μL system at 37 ℃ for 2 hours. |
The Electrophoresis was performed on a 1％ Agarose glu. | The Electrophoresis was performed on a 1％ Agarose glu. | ||
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
- 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 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
The result of the agarose electrophoresis was shown on the picture above. This parts functional test can be involved into k1789022.