Difference between revisions of "Part:BBa K1598001"

Latest revision as of 15:15, 9 October 2022

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. This is the intermediate construct consisting of the human TPH1 gene alone. This construct was used in the assembly of BBa_K1598002.

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

Functional Parameters

You can access the characterization data for this part cloned into Lac inducible expression casette here

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.

Added by LZU-HS-Pro-A

In our project, after completing the construction of the transgenic strain, we needed to verify that the strain could successfully express the desired substances, i.e. 5-HTP.

  We first observed the strain fluorescently transfected under a fluorescent microscope.

  To further accurately detect the expression amount, we use ELISA (immunoassay (IA)) technique. ELISA technique is performed by solid phase of antigen or antibody and enzymatic labeling of antigen or antibody, and after adding the substrate of enzyme reaction, the substrate is catalyzed by enzyme to become colored product, and the amount of product is observed for qualitative or quantitative analysis. Therefore, we choose this technique for further detection of expression.

Figure 1 Successful expression of 5-HTP in nissle 1917 (5-HTP is given in mg/L units, and to verify expression, both post-cleavage strains and pre-cleavage strains were used for comparison)

 The experimental results demonstrated that the engineered strain we constructed could effectively produce 5-HTP, but because most of the substances were not secreted outside the bacterium, resulting in far more fragmented organisms than the non-fragmented group. The production of GABA was much higher than that of 5-HTP. We speculate that this may be due to the long synthesis pathway of 5-HTP or the fact that the culture environment provided was not the optimal environment.