Team:RDFZ-China/Design

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Design

Overview

The ultimate goal of our project is to build an external control system that is considered as a supplement to the treatment of depression. To reach that target, we need to build a system in E.coil Nissle 1917 which can be activated by the molecule PCA (protocatechuic acid) to produce TPH1 (tryptophan hydroxylase) when PCA's concentration increases. TPH1 enzyme then can help 5-HTP synthesis in the gut. The whole system is fixed in the human intestine as living bacteria. Thus, for safety considerations, hardware container, kill switch, and the selection of bacteria strain will be included in the design section.

Sensor

The original intention of our project is to treat depression using drug therapy and psychotherapy. To minimize patients' psychological burdens, we adapted the diet therapy and designed a corresponding induced expression control system based on the tea metabolite protocatechuic acid (PCA). Inspired by the previous iGEM team who constructed the pcau-p3b5c complex in pGLO plasmid (obtained from 2018 UMaryLand team in iGEM's Parts library). The activity of the regulatory protein PcaU(BBa_K3317005) can be enhanced by the metabolite PCA. With PCA, PcaU shows an increased ability to trigger the downstream inducible P3b5C promoter, thus inducing the expression of downstream sequence.

With further literature review, we found another activator PcaUAM with one downstream promoter and p3b5b. We planned to build the pathway with different activators and promoters in order to find the most efficient combination.

Figure 5 shows the original sensor in the article

This is the sensor we found in the iGEM parts library. It was designed by UANL team in 2019. We planned to use it as an experimental control group. This sensor plasmid is constructed on pUC57 plasmid and reported using eGFP. The sensitivity of this sensor can be represented by GFP (green fluorescent protein) expression for both qualitative and quantitative data.

We also chose the low copied p15A plasmid as another gene carrier for our PcauAMsensor (both p3b5b and p3b5c)

Figure 6 shows the PcauAm repressor combine with p3b5b downstream promoter

According to our research, different sequences of promoters strongly influence the intensity of sensitivity. Also, considering the safety problem that too much 5-HTP might cause serotonin syndrome (see 5-HTP syndrome in later parts), we hope to choose a promoter with the least leakage and possibility to be activated. Those characteristics will help us build negative feedback systems in the future. Since the promoter p3B5b only has one different base pair compared with the p3B5c promoter, we also conducted a point mutation to mutate that single base pair on p3b5c and construct p3b5b. We designed two PCR primers: RDFZ-p3b5c-F / RDFZ-p3b5c-R that can still attach to p3b5b promoter with just one single base change when annealing. Thus, during the extension period, with one base pair change, we can successfully obtain the p3b5c promoter on BN056 plasmid.

Figure 7 shows the PcauAM repressor combine with p3b5c promote

TPH1 & musTPH1:

TPH1 (Tryptophan hydroxylase) is an enzyme that can hydroxylate tryptophan with 6-MePH4 to produce 5-HTP. Tryptophan is one of the most common amino acids in gut and 6-MePH4 also can be found in gut with certain concentration.

We found the human sourced TPH1 in the iGEM parts library. We also found tryptophan hydroxylase in mice. To prove they meet our standard, we obtained the sequence of both TPH1 and musTPH1, and we constructed, expressed and conduced enzyme activity assay on TPH1.

Figure 1 and 2 show the composite parts of Pet28b-TPH1 and pet28b-musTPH1op

We chose pET-28b plasmid because pET-28b(BBa_K1444028) is an expression vector commonly used in E.coil also a high copy-number plasmid. This can make our reaction happen quickly and efficiently.

We verified the enzyme activity of TPH1 by using the fluorescence property of 5-HTP derived from the 2015 UCL team.

Also, we will do some experiments on mice TPH1 next year as a supplement to human-source TPH1.

Based on “A continuous fluorescence assay for Tryptophan Hydroxylase” Moran and Fitzpatrick, AnalyticalBiochemistry 266, 148–152 (1999), TPH enzyme can hydroxylate tryptophan quickly when 600um 6-MePH4 and other compounds are added as catalase.

Strain selection

We plan to use a safer food-grade engineered bacteria — E.coli Nissle 1917 — to maximize the control of adverse effects that bacteria may have on the human body. As E.coli BL21 is very similar to and is much more accessible than E.coli Nissle 1917, we decided to use E.coli BL21 as a substitution for experiment. The sub-module verification result on BL21 is very likely to be valid for E.coli Nissle 1917.

Hardware

Considering the way we send our bacteria into the gut and make them survive, we intend to use an embedded intestinal capsule that can send our bacteria to a specific part of the gut with a basic nutrition supply. The intestinal capsule hardware design has been explored based on scientific research and advice from experts. Eventually, we found a kind of system that uses a gas sensor to localize the capsule that contains bacteria. However, most of them depend on expensive microscale or even nanoscale electronic devices, so our project aspires to build a set of bacteria-based air-sensitive capsules, and we already found some team making gas-sensing systems such as the 2019 Edinburgh Team in which their PyeaR promoter (BBa_K216005) can response to nitrate, nitrite and nitric oxide.

When the oxygen or other gases in the surrounding environment reach a certain concentration after the capsule reaches the intestine, the outer skin of the capsule will dissolve and the internal engineering bacteria will become exposed. They will then secrete adhesion molecules to attach to the lumen, and will fall off after a long period and commit suicide as soon as they leave the intestine. Considering the time and limits in the experiments this year, we hope to achieve the hardware design mostly in our second-phase. The figure below refers to the major pathways we planned to construct.

Negative feedback

According to some experts and doctors' opinions, too much 5-HT will cause serotonin syndrome. Thus, in order to prevent this from happening, we designed a set of negative feedback systems that can repress 5-HTP production. The first solution is to build a dual plasmid system with models to calculate the ideal amount of 5-HTP. When the 5-HTP concentration reaches the maximum amount we want, we can then activate another downstream system.

Figure 9 shows 3 possible ways to repress TPH1 enzyme from over generating.

Several ways are proposed to construct the system. The first way is to repress TPH1 sequence directly, the second way is to find an enzyme aimed at Pcau protein, and the third way is to prevent PCA from attaching to Pcau protein. However, we still cannot find a similar sequence or protein that works as we expected. Thus, the design of our control mechanism is also an important part of our project design next year.

Reference

1.Adam J. Meyer, Thomas H. Segall-Shapiro, Emerson Glassey, Jing Zhang & Christopher A. Voigt Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensorshttps://www.nature.com/articles/s41589-018-0168-3
2.Kourosh kalantar-ZadehA human pilot trial of ingestible electronic capsules capable of sensing different gases in the gut https://www.nature.com/articles/s41928-017-0004-x
3. Nakatani Y, Sato-Suzuki I, Tsujino N, Nakasato A, Seki Y, Fumoto M, Arita H (May 2008). "Augmented brain 5-HT crosses the blood-brain barrier through the 5-HT transporter in rat". The European Journal of Neuroscience. 27 (9): 2466–72. doi:10.1111/j.1460-9568.2008.06201.x. PMID 18445233.
4.Gijsman HJ, van Gerven JM, de Kam ML, Schoemaker RC, Pieters MS, Weemaes M, de Rijk R, van der Post J, Cohen AF (April 2002). "Placebo-controlled comparison of three dose-regimens of 5-hydroxytryptophan challenge test in healthy volunteers". Journal of Clinical Psychopharmacology. 22 (2): 183–9. doi:10.1097/00004714-200204000-00012. PMID 11910264.
5. Gilbert, N. (2019). The science of tea’s mood-altering magic. Nature, 566(7742), S8–S9. doi:10.1038/d41586-019-00398-1
6. P. G. Pietta, P. Simonetti, C. Gardana, A. Brusamolino, P. Morazzoni, E. Bombardelli, Catechin metabolites after intake of green tea infusions. Biofactors 8, 111–118 (1998).
7. “A continuous fluorescence assay for Tryptophan Hydroxylase” Moran and Fitzpatrick, AnalyticalBiochemistry 266, 148–152 (1999)