Coding

Part:BBa_K1789000

Designed by: Xinyuan Qiu   Group: iGEM15_NUDT_CHINA   (2015-09-12)

IaaM

This part is the coding sequence of tryptophan-2-mono-oxygenase.

Usage and Biology

Indole-3-acetic acid (IAA), also known as auxin, is a plant hormone, which can promote growth and protect the soil from erosion. IAA can be synthesized through IAM pathway, originated from Pseudomonas savastanoi. Two important enzymes are contained in this pathway, AKA IaaM and IaaH.

IaaPathway.jpg

IaaM 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
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 402
    Illegal BamHI site found at 1347
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 109
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental Validation

This part is validated through four ways: Enzyme cutting, PCR, Sequence, and functional testing

PCR

Methods

The PCR is performed with Premix EX Taq by Takara.

F-Prime: 5'-GAACCTCTTACGTGCCCGATCAA-3'

R-Prime: 5'-CGCCGCAGCCGAACGAC-3'

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

Results

NUDT-IAAM-PCR.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 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

NUDT-GFP2-cut.jpg

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

Functional Test

This part is tested together with the part BBa_K1789001, in the device BBa_K1789022.

The result shows that this part can work as expected to generate IAA together with IAAH.

[edit]
Categories
Parameters
None