Difference between revisions of "Part:BBa K1598002"

Latest revision as of 01:40, 12 October 2023

Lac induced expression cassette + TPH1 (tryptophan hydroxylase 1)

With the aim to address serotonin deficiency and dysregulation of gut-brain communication in patients with mental health disorders and we have developed and characterized the biobrick that overexpresses human tryptophan hydroxylase for 5-HTP production.

Usage and Biology

Serotonin is an essential metabolite, key neurotransmitter in mood regulation and a target of vast majority of antidepressant drugs. The majority of serotonin in the body is produced by enterochromaffin cells located in the gut [1]. Metabolomics study has revealed that serotonin levels in blood plasma are 2.8 fold higher in conventional as opposed to germ-free mice [2], suggesting that the interactions between host and gut microflora play crucial role in regulating the production of serotonin and functioning of serotonergic system. The rate-limiting step of synthesis of serotonin is catalyzed by Tryptophan hydroxylase, TPH, which converts tryptophan, an essential amino acid, into 5-hydroxytryptophan (5-HTP) [3]. It was shown that germ-free mice exhibit decreased expression of tryptophan hydroxylase, the rate-limiting enzyme in serotonin synthesis, in enterochromaffin cells and decreased level of serotonin in the colon and the blood and that both recolonization of the gut and supplementation with 5-HTP, a product of TPH, restore the serotonin levels [4].

Sequence and Features

Characterization

Expression of IPTG-inducible TPH1 (BBa_K1598002)

Instability and insolubility of human TPH1 expressed in E. coli has been previously reported [5]. In order to overcome the stability issues of our TPH enzyme, the induced bacteria were cultured overnight at room temperature, as suggested by previous research [5]. On the following morning cells were lysed, and protein expression was confirmed and quantified using Bradford assay with Bovine Serum Albumin as a standard.


Fig. 1. Standard curve for Bradford Assay. The absorbance of 10-fold diluted samples of induced lysate and uninduced control is indicated on the graph.
Fig. 2. Protein concentration in induced lysate and uninduced control quantified using Bradford Assay


Enzyme activity of IPTG-inducible TPH1 (BBa_K1598002)

In order to characterize our biobrick, we have adapted the fluorometric assay developed by Moran and Fitzpatrick, which exploit difference in spectral properties of tryptophan and 5-HTP [6]. According to the authors, tryptophan exhibits negligible fluorescence above 300 nm, and using excitation wavelength of 300 nm results in 5-hydroxytryptophan being almost exclusively excited. The emission is measured at 330 nm. As the assay was reported to be suitable for unpurified samples, we have performed fluorescence measurements using crude cell lysates. The detailed characterization protocol is available here: pdf or in Protocols section of our Team's wiki.

In order to demonstrate the TPH1 activity, samples containing range of crude lysate concentrations were added to the appropriate assay buffer and the reactions were initiated by adding 120 uM tryptophan solution. For blank measurements, uninduced control was used instead of induced sample.




Fig. 3. Increase in fluorescence over time indicates 5-HTP production. The reported fluorescence values are shown as means of 8 replicates +/- SD. All measurements are expressed as ratio of fluorescence of the sample to maximum fluorescence intensity observed .

References

[1] Manocha, M. and Khan, W. Serotonin and GI Disorders: An Update on Clinical and Experimental Studies. Clinical and Translational Gastroenterology, 2012, 3(4), p.e13
[2] Wikoff, W., Anfora, A., Liu, J., Schultz, P., Lesley, S., Peters, E. and Siuzdak, G. Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. Proceedings of the National Academy of Sciences, 2009, 106(10), pp.3698-3703
[3] O’Mahony, S., Clarke, G., Borre, Y., Dinan, T. and Cryan, J. Serotonin, tryptophan metabolism and the brain-gut-microbiome axis. Behavioural Brain Research, 2015, 277, pp.32-48.
[4] Yano, J., Yu, K., Donaldson, G., Shastri, G., Ann, P., Ma, L., Nagler, C., Ismagilov, R., Mazmanian, S. and Hsiao, E. Indigenous Bacteria from the Gut Microbiota Regulate Host Serotonin Biosynthesis. Cell, 2015, 161(2), pp.264-276.
[5] Kowlessur, D. and Kaufman, S. (1999). Cloning and expression of recombinant human pineal tryptophan hydroxylase in Escherichia coli: purification and characterization of the cloned enzyme. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1434(2), pp.317-330.
[6] Moran, G. and Fitzpatrick, P. (1999). A Continuous Fluorescence Assay for Tryptophan Hydroxylase. Analytical Biochemistry, 266(1), pp.148-152.

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