Presented by Team RDFZ-China 2020
Micheal Jiang, Mingyang Li, Minjia Pan, Tianrui Yang, Yijun Zhou, Liangpu Liu, You Wang, Hang Xu, Xinran Liu, Kunpeng Zhang, Jieni Wu, Yushan Zou, Zhi Sun, Yulin Ning, Weifeng Nian
High School Affiliated of Renmin University of China, Beijing, Beijing,China, Bluepha Lab, Beijing,Beijing,China
Abstract
Depression is a common yet serious disease that affects more than 264 million people worldwide. [1] It may cause the patients to suffer greatly and harm their performances at work, at school, and in families. Severe symptoms may even lead to suicide. It has been accepted that serotonin deficiency is the major cause of depression, and efforts have been made to improve this situation. Yet patients often feel pressure and refuse to take antidepressants due to social stigmas [2].
Our project, Tea-HEE, focuses on a novel way to solve this problem. We engineered E. coli Nissle 1917 (an FDA approved probiotic) and plan to place it in patients’ intestine. It will respond to PCA, a tea metabolite derived from tea intake, and then produce 5-HTP, the precursor of serotonin that has multiple advantages for serotonin synthesis. Therefore, by ingesting tea, a common beverage, patients can get an ample supply of 5-HTP in their intestine and hence an ample supply of serotonin in their brain to fight depression without pressure.
Team:RDFZ-China/Poster
Poster: RDFZ-China
TEA-HEE: A tea induced approach to alleviate depression
Inspiration
“Apart from the side effects, the reoccurrence and inappropriate schedules of taking medicines are also contributing to the insignificant effect of the traditional antidepressant treatments.”
--From the clinical doctors
Our visits to the doctors inspire us to think about the current situation for depression. As we mentioned about the side effects of the traditional medical treatments, they explained that the main drawbacks also include the rejective emotion towards medicine and the inappropriate schedule of taking drugs. Besides, many patients refuse to admit that they have depression and therefore act passively towards the treatment. These factors show the necessity of developing a milder, more alleviative therapy for a patient.
We are inspired by multiple journals, iGEM teams and stakeholders when developing our project design.
We were inspired by an article called A green tea-triggered genetic control system for treating diabetes in mice and monkeys (3), where we learned that food can be used for genetic control systems.
We are also inspired by previous iGEM team iGEM18_UMaryland and iGEM19_UANL that uses PCA sensing circuit.
--From the clinical doctors
Our visits to the doctors inspire us to think about the current situation for depression. As we mentioned about the side effects of the traditional medical treatments, they explained that the main drawbacks also include the rejective emotion towards medicine and the inappropriate schedule of taking drugs. Besides, many patients refuse to admit that they have depression and therefore act passively towards the treatment. These factors show the necessity of developing a milder, more alleviative therapy for a patient.
We are inspired by multiple journals, iGEM teams and stakeholders when developing our project design.
We were inspired by an article called A green tea-triggered genetic control system for treating diabetes in mice and monkeys (3), where we learned that food can be used for genetic control systems.
We are also inspired by previous iGEM team iGEM18_UMaryland and iGEM19_UANL that uses PCA sensing circuit.
Introduction
Depression has serious impacts on individuals and society as a whole. According to a 2017 WHO survey, the number of people suffering from depression worldwide has reached 322 million, affecting about 4.3% of the population(4).
According to our investigations, depression is closely related to the imbalance of serotonin secretion (5, 6). Serotonin, also known as 5-HT, has complex and diverse functions. Studies have shown that low serotonin activity is one of the major causes of depression (4).
Hence, we proposed a design of engineered bacteria into the patient's intestine and to boost patient 5-HT level. Avoiding direct drug intake, the patient will ingest tea that contains unique signal molecule metabolite protocatechuic acid (PCA) that can be recognized by our engineered bacteria. Then PCA can further induce the production of 5-HTP, the precursor of 5-HT. Under our design, the amount of 5-HTP produced can be controlled by tea intake.
According to our investigations, depression is closely related to the imbalance of serotonin secretion (5, 6). Serotonin, also known as 5-HT, has complex and diverse functions. Studies have shown that low serotonin activity is one of the major causes of depression (4).
Hence, we proposed a design of engineered bacteria into the patient's intestine and to boost patient 5-HT level. Avoiding direct drug intake, the patient will ingest tea that contains unique signal molecule metabolite protocatechuic acid (PCA) that can be recognized by our engineered bacteria. Then PCA can further induce the production of 5-HTP, the precursor of 5-HT. Under our design, the amount of 5-HTP produced can be controlled by tea intake.
Inspiration
KNOWING: Knowing the scientific mechanism and treatments of depression, thus increasing general public's and researchers' awareness towards depression.
ACCEPTING: Designing a milder and more acceptable alternative therapy for depression patients, thus letting them accept their disorder and being proactive to the 'treatment'.
CARING: Calling people in the society to pay more attention to depressed patients and adding warmth and hope to their lives.
REALIZING: building and embedding engineering bacteria in patients' guts to realize our tea-induced alternative therapy.
Poster Title
Experiment & Methology
1.SensorThe intention of our project is to treat depression using drug therapy and psychotherapy. To minimize patient's 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 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.
Through literature and parts library review, we found three sensor units that can respond to PCA. Different sequence of promoters strongly influence the intensity of sensitivity. Also considering the safety problems that too much 5-HTP might cause 5-HTP syndromes (see 5-HTPsyndrome in later parts), we hope to choose a promoter with least leaking yet with high sensitivity.
Acativity test
Pcau-pPCA-GFP: excitation: 485nm, emission:515nm:
The data on the left of figure14 are tested data of Pcau-pPCA(BBa_K2825002). Two groups of our repeat data show good activation of PCA on downstream promoter. Yet according to the graph, the leakage of GFP is also very serious. This may be the result of having our reporter genes on a high copy plasmid, which increases the overall leakage, which is not what we want.
PcaU-pPCA-GFP:exicitaion: 485nm , emission:535nm:
The data in the right of figure 15 are the tested data of PcaU-pPCA-GFP under emission light 535nm. The data shows PcaU-pPCA being dynamic against the concentration of PCA.
PcauAM-YFP: excitation:485nm, emission:535nm:
The data in the right of figure 15 are the tested data of PcaUAM-YFP under emission light 525nm. The data shows both p3B5B (BBa_K3395009) and p3B5C promoter(BBa_) can be controlled by the pcauAM activator. And under the horizontal comparison between p3B5B and p3B5C promoter, p3B5C promoter have much higher sensitivity than p3B5C promoter.
PcaUAM-YFP: excition:485nm, emission:525nm:
The data in the left of figure 14 are PcaUAM-YFP under emission light 525nm varied by different concentration of PCA.
Best sensor:
Comparing the leakage and sensitivity, we found that PcauAM expressor-p3b5b downstream promoter is the best sensor system.
2. Effector :
We conducted enzymatic assay for TPH1 to examine whether it fullfill our need
Standard curve of 5-HTP
Figure 18 above show the absolute standard curve we make in two experiments. From the figure, it is clearly that under the light absorption of 330, as the concentration of 5-HTP increases, the light absorption also increases.
Cons enzymatic reaction:
pTAC-TPH1(BBa_K3395004)
In this experiment, we make the concentration gradient of enzyme from low to high and labeled them column 1/2/3/4/5/6. The line chart show the absorption in 330 nm (relate with the concentration of 5-HTP)is vary by two factors: time, and the concentration of enzyme. Because we used solution with bacteria but not pure protein solution in this experiment, the volume spontaneous fluorescence interfered with the fluorescence value. Yet we can prove our enzyme is working with this data. Because the concentration of 5-HTP first increases very fast at the beginning and then the reaction slow down after about 20min, which conforms to the reaction curve of a normal enzyme. From column 3 to 6, as the concentration of initial TPH1 increases, the initial gradient of 5-HTP also increases. This indicated that our TPH1 is functional. We also build a model to explain this occurrence. It can be found on our model page.
Idea
We measure the specific mechanism behind the expression by applying the law of action in micromolecular reactions involved in transcription, and we fit our model with the calculation of experiment data. Eventually, we successfully find the result of PCA having an induced activation on the expression of GFP. we successfully find out that the synthesis of 5-HTP will be faster when the concentration of TPH1 increases, which closely fits our experiment in trend.
In the modeling of TPH1's enzymatic reaction, we also measure the close relation between the production of 5-HTP varied by TPH1 concentration by applying the law of action throughout this enzymatic reaction. Eventually, we successfully find out that the synthesis of 5-HTP will be faster when the concentration of TPH1 increases, which closely fits our experiment in trend.
Idea
Further Goals
1. We plan to first apply our circuit to E.coli Nissle 1917, as this is the strain we hope to use in practice. Testing the sensor unit with urine hours after tea intake is a good reference for the concentration of PCA existing in the intestine after drinking tea and prove our design works in the human body for PCA sensing.
2. We want to combine the two components with the negative feedback loop and test it as an integrated pathway in terms of PCA sensitivity and TPH1 expression.
3. For PCA sensitivity we will use human urine as well as pure PCA gradient as a testing agent. We will have GFP linked downstream to TPH1 and thus determine TPH1 expression.
Modeling Improvements
1. We will collect more integrated data to improve our fitting of existing formulas. Besides, we tend to connect our two models and analyze them based on the data that is obtained from the human body.
2. We plan to simulate negative feedback that restrains the production of 5-HTP by inhibiting the expression of TPH1 if they exceed, thus making our model safer and more practical.
Next stage hardware--Capsules designs
Since many patients with mild symptoms do not receive effective treatment in the first place, our project can contribute to alleviative therapies for them, before their condition gets severe.
We designed to build a set of bacteria-based air-sensitive capsules that delivers our engineered bacteria to the intestine and release them. 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.
Safety concerns and solutions
1. To avoid serotonin syndrome as a result of having too much 5-HTP in a short period, we decided to introduce some sort of negative feedback mechanisms downstream of TPH1. To limit its secretion, we have proposed three ways of achieving so: by inhibiting PCA binding to pcaU, by inhibiting the operator region, and by interrupting TPH1 transcription.
2. To prevent leakage we also consider adding a kill switch that detects quorum, pH, and temperatures and kills off the bacteria as soon as it leaves the intestine.
1. We plan to first apply our circuit to E.coli Nissle 1917, as this is the strain we hope to use in practice. Testing the sensor unit with urine hours after tea intake is a good reference for the concentration of PCA existing in the intestine after drinking tea and prove our design works in the human body for PCA sensing.
2. We want to combine the two components with the negative feedback loop and test it as an integrated pathway in terms of PCA sensitivity and TPH1 expression.
3. For PCA sensitivity we will use human urine as well as pure PCA gradient as a testing agent. We will have GFP linked downstream to TPH1 and thus determine TPH1 expression.
Modeling Improvements
1. We will collect more integrated data to improve our fitting of existing formulas. Besides, we tend to connect our two models and analyze them based on the data that is obtained from the human body.
2. We plan to simulate negative feedback that restrains the production of 5-HTP by inhibiting the expression of TPH1 if they exceed, thus making our model safer and more practical.
Next stage hardware--Capsules designs
Since many patients with mild symptoms do not receive effective treatment in the first place, our project can contribute to alleviative therapies for them, before their condition gets severe.
We designed to build a set of bacteria-based air-sensitive capsules that delivers our engineered bacteria to the intestine and release them. 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.
Safety concerns and solutions
1. To avoid serotonin syndrome as a result of having too much 5-HTP in a short period, we decided to introduce some sort of negative feedback mechanisms downstream of TPH1. To limit its secretion, we have proposed three ways of achieving so: by inhibiting PCA binding to pcaU, by inhibiting the operator region, and by interrupting TPH1 transcription.
2. To prevent leakage we also consider adding a kill switch that detects quorum, pH, and temperatures and kills off the bacteria as soon as it leaves the intestine.
Idea
To make synthetic biology as well as general sciences more accessible for people has always been our goal. This year, we worked on two projects about science education.
Genetic circuits play an important role in synthetic biology. They show how a biological system work with simple glyphs. Thus, in order to make synthetic biology accessible for more people, we designed the Genetic Circuit Jigsaw Puzzle.
Food therapy is an important part of our project. In order to make our project more complete and benefit wider society, we’ve been thinking about ways of educating people about healthy diet. The video series’ name is “Fundamentals of Immunology,” the content of which includes important nutrients, various diet patterns and how everyday food can fight diseases.
Genetic Circuit Jigsaw Puzzle
Genetic circuits play an important role in synthetic biology. They show how a biological system work with simple glyphs. Thus, in order to make synthetic biology accessible for more people, we designed the Genetic Circuit Jigsaw Puzzle.
Fundamentals of Healthy Diet videos
Food therapy is an important part of our project. In order to make our project more complete and benefit wider society, we’ve been thinking about ways of educating people about healthy diet. The video series’ name is “Fundamentals of Immunology,” the content of which includes important nutrients, various diet patterns and how everyday food can fight diseases.
Maybe the world is not perfect, but we want to show the patients that there are still many wonderful things to explore and be passionate about.
Idea
1. Accepting
Moderate tea drinking has limited side effects and is not enough to pose a health threat. Its effect on regulating mood and even treating depression can be a good supplement to our drug. As tea plays an important role in the Eastern Asian and worldly culture, I believe our new Tea-hee therapy is a good fit for our users.
2. Caring
Moderate tea drinking has limited side effects and is not enough to pose a health threat. Its effect on regulating mood and even treating depression can be a good supplement to our drug. As tea plays an important role in the Eastern Asian and worldly culture, I believe our new Tea-hee therapy is a good fit for our users.
GeneticCircuit Jigsaw Puzzle+ Fundamentals of Healthy Diet videos
3. Realizing:Parts:
We successfully constructed 2 TPHI which are TPH1(BBa_K3395000), musTPH1（BBa_K3395001), and we constructed the PcaUAM sensor (BBa_K3395015). The BBa_K2825002 in the part library has been improved to become our new pcauam_p3B5B part. Its number is BBa_K3395015.
Experiment:
1.We test the sensor activity of PcaU-pPCA, PcaUAM-p3B5B, PcaUAM-p3B5C system under different emission light, thus finding out PcauAM expressor-p3b5b downstream promoter is the best sensor system.
2. We conducted the enzymatic activity of hTPH1 induced by IPTG, which confirmed the efficiency of our effector TPH1.
Model:
1. we successfully simulate the chemical reactions involved in this expression, and the result of PCA having an induced activation on the expression of GFP.
2. we successfully find out that the synthesis of 5-HTP will be faster when the concentration of TPH1 increases, which closely fits our experiment in trend.
Reference
1. GBD 2017 Disease and Injury Incidence and Prevalence Collaborators. (2018). Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017. The Lancet. DOI.
2.https://www.who.int/news-room/fact-sheets/detail/depression
3. Yin J, Yang L, Mou L, et al. A green tea–triggered genetic control system for treating diabetes in mice and monkeys[J]. Science translational medicine, 2019, 11(515).
4. World Health Organization. Depression and Other Common Mental Disorders Global Health Estimates[M]. World Health Organization,2017
5. Ruhé HG, Mason NS, Schene AH (April 2007). "Mood is indirectly related to serotonin, norepinephrine and dopamine levels in humans: a meta-analysis of monoamine depletion studies". Molecular Psychiatry. 12 (4): 331–59. doi:10.1038/sj.mp.4001949. PMID 17389902. （wikipedia）
6. Studies assessing the binding potential of the serotonin transporter or serotonin receptors have also been inconsistent, but generally point towards abnormal serotonin signalling. Manji & Zarate 2011, p. 112.
7. https://nutritionreview.org/2013/04/5htp-5hydroxytryptophan-prozac-ssris/
8.:Park D H, Stone D M, Kim K S, et al. Characterization of recombinant mouse tryptophan hydroxylase expressed in Escherichia coli[J]. Molecular and Cellular Neuroscience, 1994, 5(1): 87-93.
People
Yali Hao: Provide training for the experiments and support the management of the equipment
WeiXu Wang：Supervise and provide suggestions for the Math Model
YeQing Zong，ShiYuan Li：Give advice for the project design
Companies
BluephaLab：Providing the Safety Training and Lab Equipment
SnapGene：Software support for the Gene Design
MathWorks：Support for Mathematics modelling
1. GBD 2017 Disease and Injury Incidence and Prevalence Collaborators. (2018). Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017. The Lancet. DOI.
2.https://www.who.int/news-room/fact-sheets/detail/depression
3. Yin J, Yang L, Mou L, et al. A green tea–triggered genetic control system for treating diabetes in mice and monkeys[J]. Science translational medicine, 2019, 11(515).
4. World Health Organization. Depression and Other Common Mental Disorders Global Health Estimates[M]. World Health Organization,2017
5. Ruhé HG, Mason NS, Schene AH (April 2007). "Mood is indirectly related to serotonin, norepinephrine and dopamine levels in humans: a meta-analysis of monoamine depletion studies". Molecular Psychiatry. 12 (4): 331–59. doi:10.1038/sj.mp.4001949. PMID 17389902. （wikipedia）
6. Studies assessing the binding potential of the serotonin transporter or serotonin receptors have also been inconsistent, but generally point towards abnormal serotonin signalling. Manji & Zarate 2011, p. 112.
7. https://nutritionreview.org/2013/04/5htp-5hydroxytryptophan-prozac-ssris/
8.:Park D H, Stone D M, Kim K S, et al. Characterization of recombinant mouse tryptophan hydroxylase expressed in Escherichia coli[J]. Molecular and Cellular Neuroscience, 1994, 5(1): 87-93.
People
Yali Hao: Provide training for the experiments and support the management of the equipment
WeiXu Wang：Supervise and provide suggestions for the Math Model
YeQing Zong，ShiYuan Li：Give advice for the project design
Companies
BluephaLab：Providing the Safety Training and Lab Equipment
SnapGene：Software support for the Gene Design
MathWorks：Support for Mathematics modelling