Part:BBa_K5009001
High-efficiency TPH originates from Zebrafish
Usage
5-Hydroxytryptamine (5-HT), also known as serotonin, often referred to as the "feel-good" neurotransmitter, is involved in regulating mood, sleep, appetite, and a range of other physiological functions. Imbalances in serotonin levels, particularly a deficiency, are implicated in the pathophysiology of depression.
Therefore, we hope to produce serotonin through synthetic biology methods to treat depression. Through literature research and an analysis of previous team projects, we have found that expressing serotonin using symbiotic bacteria is a promising approach. The rate-limiting step in the biosynthesis pathway of serotonin involves tryptophan hydroxylase 1 (TPH1) catalyzing tryptophan to form 5-hydroxytryptophan, which is then converted to serotonin by tryptophan decarboxylase (TDC). For more project background visit: https://2023.igem.wiki/squirrel-chn-ii/description
Biology: Zerba fish source TPH expression in Ecoli
In our project, the tryptophan hydroxylases (TPH) from human, mice, zebrafish, and rabbits have been selected and analyzed for their catalytic efficiency in the rate-limiting step of producing serotonin. Therefore, the CDs from four types of TPH have been codon optimized and synthesized by our sponsor- ATANTARES biotech, and constructed onto the Ecoli expressing vector pET-28a using Gibson assembly. (Download the plasmid file at: https://2023.igem.wiki/squirrel-chn-ii/experiments ) The expressing vector was then transformed into Ecoli strain BL21(DE3). After that, serious of experiments has been performed to adjust the optimum expressing condition. (Detailed protocols can be downloaded at: https://2023.igem.wiki/squirrel-chn-ii/experiments.) The optimum expression condition in a 500ml flask is shown below.
We ran an SDS-PAGE gel for the final product of our concentrated purification product. Four bands were clearly observed on the gel. Showing that the protein has been successfully expressed in Ecoli cells.
Characterization: Compare activity with TPH from other sources
1. in silico binding affinity prediction: The full-length 3D structures of the four candidate 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.
2. 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.
3. In vitro enzyme activity testing: To measure the HTP activity, we followed the protocol listed in part ( BBa_K1598002, https://parts.igem.org/Part:BBa_K1598002) 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.
4. 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 submitted cDNA for the TPH origin from zebrafish as this new basic part.
More about our design, visit: https://2023.igem.wiki/squirrel-chn-ii/engineering.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 610
Illegal XbaI site found at 1423 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 610
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 610
Illegal BglII site found at 299
Illegal BglII site found at 498 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 610
Illegal XbaI site found at 1423 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 610
Illegal XbaI site found at 1423 - 1000COMPATIBLE WITH RFC[1000]
| None |