Team:Manchester-Graz/Collaborations/UCL

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iGEM Manchester - UCL

UCL

In corporation with the iGEM Team UCL we modeled several of their pathways in our expression system:

Serotonin

Although serotonin is well known as a brain neurotransmitter, it is estimated that 90 percent of the body's serotonin is made in the digestive tract. In fact, altered levels of this peripheral serotonin have been linked to diseases such as irritable bowel syndrome, cardiovascular disease, and osteoporosis [1]. Serotonin is converted in two steps from tryptophan to serotonin (Fig. 1). For simulating the expression of Serotonin in the iGEM Manchester-Graz expression system (see our Model) two enzymes had to be introduced into the expression system: the Tryptophan hydroxylase and the Amino acid decarboxylase.

Manchester-Graz_UCL_Fig1
Figure 1 Serotonin Pathway. Tryptophan is converted to Serotonin in two steps. Starting from Tryptophan over 5-Hydroxytryptophan (5HTP) to Serotonine. AADC = Amino acid decarboxylase.

The desired genes were introduced into the Manchester-Graz expression system model by adjusting the transcription and translation rates to proper values and by changing some species (Fig. 2).

Manchester-Graz_UCL_Fig2
Figure 2 : Simbiology overview of the model. The expression of the two enzymes (Tryptophan hydroxylase and AADC) is under control of the two quorum sensing systems. Serotonin gets through the cell membrane by diffusion.

In the simulation the species in the cell reach a relatively low level, whereas the amount of Serotonin in the environment increases constantly.
The simulation of the expression model gives an amount of molecules over time (Fig. 3). This can be recalculated into concentration values using the Avogadro constant and the molar mass of serotonin.

serotonin%20graph
Figure 3 Serotonin in the environment. Serotonin gets exported from the cell via diffusion and increases over time.

In the simulation we see that Serotonin is expressed constantly by the cells and exported to the environment (Fig. 3). The rate of serotonin production is: 415.14 μmol/h/L or 73.16 mg/h/L.

Serotonin is unlikely to pass the blood brain barrier, so in collaboration both team developed the idea of producing 5HTP (5-Hydroxytryptophan), which can afterwards be converted to Serotonin by enzymes in the human body.

The model was changed in a way only one enzyme, the Tryptophan Hydroxylase gets expressed (Fig. 4)

5HTP%20overview
Figure 4 Simbiology overview of the changed model. Only Tryptophan Hydroxylase is expressed

If the model is simulated, the rate of 5HTP production is 424.58 µmol/h/L or 93.5 mg/h/L (Fig. 5).

5HTP%20sim
Figure 5 Simulation of 5HTP (5-Hydroxytryptophan) production over time

Dopamine

Dopamine is another important neurotransmitter which, produced by gut bacteria has some positive effects neurological diseases such as Parkinson’s disease. The Modeling of the dopamine/L-Dopa pathway (Fig. 6) was done by iGEM Manchester-Graz 2015.

Look into: http://2015.igem.org/Team:Manchester-Graz/Modeling

Manchester-Graz_UCL_Fig10
Figure 6 Overview of the Dopamine pathway. Tyrosine is converted in two steps to Dopamine either via Tyramine or L-DOPA. AADC= Amino acid decarboxylase, CYP2D6= Cytochrome P450 enzyme D6

iGEM team Manchester-Graz modeled the pathway also in two ways. In one model, all three enzymes were introduced, in the second model only Tyrosine Hydroxylase was introduced, because Dopamine has positive effects when expressed in the gut, but only L-DOPA can pass the blood brain barrier.

GABA (Gamma amino butyric acid)

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the body and hence GABA-mediated neurotransmission regulates many physiological functions, including those in the gastrointestinal (GI) tract. GABA is located throughout the GI tract and is found in enteric nerves as well as in endocrine-like cells, implicating GABA as both a neurotransmitter and an endocrine mediator influencing GI function. GABA mediates its effects via GABA receptors which are either ionotropic GABA(A) or metabotropic GABA(B). The latter which respond to the agonist baclofen have been least characterized, however accumulating data suggest that they play a key role in GI function in health and disease [2].
GABA is produced by a two-step pathway from Glutamine (Fig. 7). Glutamine is converted to glutamic acid by the enzyme glutaminase and the glutamic acid decarboxylase (GADC) converts it further to GABA.

Manchester-Graz_UCL_Fig4
Figure 7 Glutamine is converted in two steps to GABA (gamma amino butyric acid). GADC (Glutamic acid decarboxylase) converts glutamic acid to GABA.

For the simulation of the GABA production in the Manchester-Graz expression system model the setup had to be adjusted in a way the transcription and translation rates fit to the proper length of the genes (Fig. 8).

Manchester-Graz_UCL_Fig5
Figure 8 Overview of the Simbiology model with introduced GABA pathway.

The simulation showed an increasing amount of GABA vs. time (Fig. 9). By recalculating the amounts of molecules to concentration amounts the simulation showed a GABA production of 296.53 μmol/h/L or 30.58 mg/h/L

Manchester-Graz_UCL_Fig6
Figure 9 Simulation of the GABA pathway controlled by the iGEM Manchester-Graz expression system.

Acetylcholine

Acetylcholine is one of many neurotransmitters in the autonomic nervous system (ANS). It acts on both the peripheral nervous system (PNS) and central nervous system (CNS) and is the only neurotransmitter used in the motor division of the somatic nervous system. Acetylcholine has different effects on several tissues controlled by the nervous system [3]. Choline is the precursor molecule for the neurotransmitter acetylcholine and gets absorbed in the gut. Choline gets converted to Acetylcholine by the choline-acetyl-transferase which links Acetyl CoA and Choline (Fig. 10).

Manchester-Graz_UCL_Fig7
Figure 10 Acetylcholine is produced by the Choline-acetyl-transferase.

For modeling the production of acetylcholine the choline-acetyl-transferase had to be introduced to the model. AcetylCoA is constantly produced by the cells whereas Choline is taken up from the environment (Fig. 11).

Manchester-Graz_UCL_Fig8
Figure 11 Overview of the Simbiology model with introduced Choline-acetyl-transferase.

Acetylcholine is constantly produced by the cells by a rate of 431.11 μmol/h/L or 62.99 mg/h/L (Fig. 12).

Manchester-Graz_UCL_Fig9
Figure 12 Simulation of Acetylcholine production under the control of the Manchester-Graz expression system.
[1] Caltech (2015). Microbes Help Produce Serotonin in Gut. [online] Available at: http://www.caltech.edu/news/microbes-help-produce-serotonin-gut-46495#sthash.2C72lcWM.dpuf [Accessed 25 Aug. 2015]
[2] Hyland, P. and Cryan, F. (2010) A Gut Feeling about GABA: Focus on GABA(B) Receptors. Front Pharmacol. 1(124)
[3] Neuroscience online (2015). Chapter 11: Acetylcholine Neurotransmission. [online] Available at: http://neuroscience.uth.tmc.edu/s1/chapter11.html [Accessed: 27 Aug. 2015]