Part:BBa_K1598002:Experience
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Applications of BBa_K1598002
Squirrel-CHN-II 2023
To better understand the TPH activity in the intestine, we decided to use a hybrid of in silico and in vivo experiments to investigate the TPH activity. The tryptophan hydroxylases (TPH) from human, as well as from mice, zebrafish, and rabbits have been selected and analyzed for their catalytic efficiency in the rate-limiting step of producing serotonin. By predicting the binding affinity of the TPHs to the tryptophan molecule we could predict the catalytic effectiveness of the enzyme selected.
In silico binding affinity prediction: The full-length 3D structures of the four TPHs from human, mouse, zebrafish, and rabbit are acquired as the PDB files from the uniport database under the following entry numbers: P17752; P09810; Q6PBX4; P17290 for each host organism respectively. For entry numbers P09810, Q6PBX4, and P17290, the known x-ray/NMR structures of the proteins are missing. Instead, the Alphafold predicted full-length structure of the proteins was selected for further analysis.
Results for in silico binding affinity prediction:
The zebrafish TPH would hypothetically mediate the highest efficiency of tryptophan hydrolysis among the four selected TPHs. Our wet lab experiments and results would, furthermore, help us identify the most potent TPH enzyme to be produced in E. coli expression and used for the final product.
In vitro enzyme activity testing: To measure the HTP activity, we followed the protocol listed in this part by team UCL in 2015. In the protocol, a continuous fluorometric assay for HTP activity based on the different spectral characteristics of tryptophan and 5hydroxytryptophan is presented.
Results for in vitro enzyme activity testing:
Our wet lab experiments and results fit our in silico prediction of HTP activity and show that the zebrafish TPH has the highest efficiency of tryptophan hydrolysis among the four selected TPHs. Therefore, we will choose this strain of HTP in our final product.
Hence, we also submitted a new basic part ( BBa_K5009001 ) for the TPH origin from zebrafish.
More about our design, visit: https://2023.igem.wiki/squirrel-chn-ii/engineering
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