Team:CPU CHINA/Poster

Poster: CPU_CHINA

Inspiration

At the very beginning of the project, when we first held the spear of synthetic biology and thought about where our arrow shall point, the most important traditional Chinese festival, the Spring Festival, come in time. People sat together with family and friends (a much smaller scale than usual, of course, due to COVID-19), had their hearty drink to celebrate this festival. Yet at the same time many tragedies caused by drinking had frequently come to our sights, with social discussions around the notorious social norm known as wine table culture reaching its peak as well. Statistics witness a sharp increase in the number of cases sent to the emergency department due to alcoholic liver disease (ALD) at this very period, which drew our concern.

We started to initiate researches to investigate the current actuality, and what we got was disquieting. At the welcome party for new employees, these newcomes are compelled to drink by the company leader. If they refused to drink, this action would be regarded as disrespect to the boss. In one case, a new employee actually got slapped in the face by a program leader after refusing to drink, though the refusal is due to physical reasons. In another case, a college tutor abused his power, demanded the student to drink and eventually led to the student being hospitalized due to excessive drinking. There are also many merchants whose business was affected by the pandemic, and people who lost their jobs facing financial difficulties, taking excessive drinking as a way to relieve stress and depression. Some of these people gradually grew dependent on alcohol and eventually got addicted to it, finally diagnosed with alcoholic liver diseases.

We were sad to find that there had been countless similar cases in the past few decades: people being forced to drink due to social norms and people getting alcohol addiction for different reasons, their health are seriously threatened by ALD.

Prevention methods of alcoholic liver disease by inflammation control and intestinal flora regulation

Presented by Team CPU_CHINA 2020

Weixuan Lu, Roujin An, Yilin Li, Ziyan Zhu, Yanfeng Liu, Hongfa Qian, Jingjing Huang, Yue He, Jie Cheng, Yu Gao, Min Hai, Junlin Shi, Zhiyang Dou, Boyang Lin, Tong Lyu, Xiaoying Li, Xin Liu

Abstract

We engineered a synthetic organism capable of expressing and secreting anti-inflammatory cytokines IL-22, which will get to liver first due to First Pass Effect after its absorption into bloodstream and perform anti-inflammation function to protect the injured liver part from further inflammation damage. We also endowed the chasis with anti-E.faecalis function controlled by quorum sensing system. We chose bacteriocins JM79, plwα and plwβ with high specificity towards E.faecalis, the bacteriocins will function in situ to kill the over-reproduced E.faecalis to balance the intestinal flora, thus protect the liver from further immunoreaction at immunogen level. The chasis applied is E.coli Nissle 1917, which is a type of probiotic that can be taken orally in capsule. The system is expected to protect the liver with alcohol-induced inflammation from further pathological progression and is possible to be used as an adjuvant therapy in the future.

Introduction

China is already the country with the most deaths from drinking in the world, with 709,000 deaths caused by drinking every year, while people still perform disproportionate indifference to the harm of drinking and the prevention of ALD, thus ALD develops quickly into later stages such as liver steatosis, liver cirrhosis even liver cancer, at which point the damage done to liver can hardly get healed. What’s worse, the current therapy even medication for ALD are not working well. That’s why we focus on the early stage of ALD, hoping to bring up a new therapy to intervene the development of ALD at the early stage and prevent further liver damage.

After browing through huge amount of papers and reports, we found that almost half of the Chinese have a congenital deficiency in alcohol metabolism, which is an important cause of ALD as the incompletely metabolized acetaldehyde accumulates excessively in liver then causes hepatotoxicity. Another important and direct pathogenesis is the inflammation induced by the damaged liver, which is way more harmful.

IL-22 was reported by an up to date review to be a promising molecule that exhibits high anti-inflammation activity and is currently under clinical trials for ALD treatment.

Scholars made breaking discovery that Enterococcus faecalis, which has always been regarded as an important kind of probiotic in human intestines, was found to be a main contributor of liver inflammation. The density of E.faecalis in intestine would increase aberrantly in the presence of alcohol, disturbing the balance of intestinal flora. More importantly, the over-reproduced E.faecalis would secrete a kind of toxic protein, which would enter the bloodstream then reach the liver and cause the lysis of hepatocyte. High concentration of alcohol would also produce wounds on the intestinal wall, make it possible for even E.faecalis to enter the bloodstream. E.faecalis would get to liver first due to First Pass Effect and colonized some part of liver, thus create wounds on liver and at the same time induce local inflammation on liver, further exacerbate the damage.

Based on these researches, we shaped our project, taking E.faecalis density control and balancing intestinal flora as our new goal, and secretion of anti-inflammation cytokine IL-22 as another aim function to achieve.

Killing Enterococcus faecalis in the intestine: quorum sensing and bacteriocin

Methodology

Characterization

Bacteriocin:
ELISA for flag-tag
groups:
1. plw α-plw β-Hiracin JM79 with different duration of incubation, control: BL21 (DE3)
​2. Hiracin-JM79, BL21 (DE3)

Functional verification

Measurement of OD600 of Enterococcus faecalis
groups: Supernatant of Hiracin JM79, Hiracin JM79 of BL21 (DE3)

Results

1.Successful expression and secretion of the bacteriocins

2.Functional verification of antibacterial effect

Inhibiting local inflammation in liver: PalcA and the expression of IL-22

Methodology

Characterization

PalcA:
GFP Fluorescence Intensity of E. coli cultures with varying amounts of ethanol

IL-22:
ELISA for IL-22
groups: 1.0M IPTG, 0.5M IPTG, no IPTG

Functional verification

ELISA for TNF-α (IL-22 can inhibit the secretion of TNF-α from THP-1 when stimulated with LPS)
groups: IL-22 standard samples, the culture supernatant of our engineered bacteria, BL21(DE3) without plasmid transformation.

Results

1.The ethanol promoter, PalcA, is functional in the presence of a specific concentration of ethanol, but is quite inefficient.

2.Successful expression and secretion of the IL-22

3.Functional verification of anti-inflammatory effect

Killing Enterococcus faecalis in the intestine: quorum sensing and bacteriocin

Design

Quorum sensing system of Enterococcus faecalis, a main contributor to liver inflammation, is applied to sense GBAP, aiming to sense the density of E.faecalis. Three bacteriocins, Hiricin 79, plwα and plwβ are utilize to regulate the density of E.faecalis.

Inhibiting local inflammation in liver: PalcA and the expression of IL-22

Design

Our engineered bacteria is designed to sense ethanol and release IL-22, where PalcA (ethanol-induced promoter) is the sensor and T7 transcription system is applied to amplify the upstream signal of PalcA. This system enables the engineered bacteria to release appropriate amount of anti-inflammatory cytokine only when the patient drinks.

Bacteriocins Expression Model

Methodology

Secretion and antibacterial effect are simulated based on 3 different bacteria concentration ratio of E. faecalis to E. coli Nissle 1917. The results of this model included the expression amount of bacteriocin over time, further described the antibacterial effect towards E. faecalis in normal people.

Results

With this, we can calculate the antibacterial effect towards E. faecalis of normal people.

1.When the ratio is 1:10^-4, 99.9999575% of E. faecalis left.

2.When the ratio is 1:1, 99.58% of E. faecalis left.

3.When the ratio reaches 1:10², 65.43% of E. faecalis left.

The above results show the sterilization of normal people under different dosages.

Alcohol Disposal Model

Methodology

In this model, alcohol disposal is divided into three main stages according to the time and space process of in vivo metabolism.

• Pre-enteral stage ：Alcohol absorption
• Enteral stage ： Alcohol absorption and metabolism
• Metabolic elimination stage ： Alcohol metabolism

We equate the process of alcohol disposal in vivo with that of oral drugs and use the single compartment model to simulate the human body. Based on the stage division of alcohol disposal , We establish the differential equation and then solve it.

Results

This model gets the function on the alcohol concentration in the blood and the alcohol concentration in the digestive tract with time. The result is presented by the figure drawn by the MATLAB software.

As a small molecule, ethanol has its absorption and metabolism characteristics. For the all small molecules similiar to ethanol, we promote the model. It can be used to calculate the similiar molecules disposal in vivo , especially for the concentration in the blood and the digestive tract with time. We publish the equations and codes in MATLAB language used in the calculations in order that the later iGEMers use our promotion model to calculate.

IL-22 Expression Model

Methodology

Model to simulate IL-22 secretion has been established based on pathways. Two conditions (antisense system was/wasn't applied) were simulated separately, further achieved the goal of confirming the non-necessity of antisense system.

Results

Further we obtained the result to be 4.57 ng/ml (without applying antisense system), which is consistent with wet experiment and can reach the therapeutic goal, yet the simulation of applying antisense system was designed and finished, the result is way lower, to be 0.914ng/ml.

Several kinetic equations were combined to established a generalized template for simulating protein secretion. Michaelis-Menten equation has a good simulation outcome of enzymatic reactions, thus can be applied to simulate proteins' transcription. Meanwhile translation procedure can be simulated by mRNAs length divided by translation rate assuming abundant ribosomes can be observed in bacterium. Degradation of mRNAs and proteins can be simulated by first-order kinetic equation based on half-life.

Integrated Human Practices

Expert visits

Academic interviews

We obtained thought-provoking feedback from 6 experts, professors of 3 universities or institutions, and made corresponding adjustments to our projects.

Doctor interviews

We received valuable suggestions from 3 chief physicians in different hospitals in regard to experimental design and other considerations.

Psychological research

We did give priority to alcohol addiction problems all the time and conducted sufficient literature research and profound expert interviews.

Enterprise interaction

Eddingpharm Group Company Limited and Others.

Government Association

Through the government service platform, we consulted the procedural and legal precautions when applying for product registration.

Education & Public Engagement

Public survey and patient interviews

We designed four-part questions, which included general information, drinking habits, disease awareness and gene therapy cognition. We made it to collect 157 valid questionnaires.

Social group interaction

Alcoholic Anonymous

Due to the COVID-19 pandemic, we could only participate in online events organized by AA based on a thorough background research.

Raising awareness

Card game

An edutainment card game was created to publicize the dangers of drinking. Simple and interesting gameplay and an electronic version that can be downloaded and printed for play are conducive to the promotion of the game.

Education

Entrepreneurship

Our product can directly target the causes of alcoholic liver disease instead of non-differentiated liver care drugs.

Product

Taken before drinking | sensing ethanol concentration and quorum sensing signals separately | small package powder | microbial intestinal colonization

Market

Global financing for cell & gene therapy increased more than doubled y/y in 2018

Competitor

Yishanfu (polyene phosphatidylcholine capsules, by Sanofi)：targeted at liver injury of various causes

Market Plan

East Asian market (mainly China), drinker aged 15-49(mainly men) | Hospital procurement

Attribution

Team Members

• Team leader: Weixuan Lu, Roujin An
• Wet lab: Weixuan Lu, Roujin An, Yilin Li, Ziyan Zhu, Yanfeng Liu, Hongfa Qian, Jingjing Huang
• HP: Yue He, Jie Cheng, Yu Gao, Min Hai, Junlin Shi
• Designer: Tong Lyu, Xiaoying Li, Xin Liu
• Dry lab: Zhiyang Dou, Boyang Lin
• Instructor: Huandi Xu
• Advisor: Bangyan Kong, Qiming Luo, Hui Huang

Lab support

Dr. Hanmei Xu, Dr. Ming Ding

Human practices support

Dr. Weihua Chu, Dr. Lirui Wang, Dr. Hanmei Xu, Dr. Libin Wei, Dr. Yu Liu, Shijie Yu, Lidong Tang, Dr. Hongwen Zhou, Dr. Jie Meng, patients, people who participated in our public questionnaire, head nurse Wang, Mr. Ding Chaozhong, Wu Licheng, Wen Cao, Wang Liang, Dr. Wu Dongdong, Hou Jun

Other support

Ms. Leyi Liang, Ms. Mengdi Liu, Lingfeng Guo

Special Thanks

NFLS, NJMU, BIT-China, SZPT-CHINA, HZAU-China, Fudan, NWU-CHINA-A, NWU-CHINA-B

Sponsors

MATLAB, Snapgene

References

[1].Hemmati, H., Transcriptional analyses of an ethanol inducible promoter in Escherichia coliand tobacco for production of cellulase and green fluorescent protein. Biotechnology & Biotechnological Equipment& Basu, C. (2015)., 29(6), 1043–1052
[2].Ali, L., Goraya, M. U., Arafat, Y., Ajmal, M., Chen, J. L., & Yu, D. (2017). Molecular Mechanism of Quorum-Sensing in Enterococcus faecalis: Its Role in Virulence and Therapeutic Approaches. Int J Mol Sci, 18(5).
[3].Gowhari A., Shabgah, Gholizadeh J, et al. Interleukin-22 in human inflammatory diseases and viral infections. [J]. Autoimmunity reviews, 2017.
[4].Kong, X., Feng, D., Mathews, S. & Gao, B. Hepatoprotective and anti- fibrotic functions of interleukin-22: therapeutic potential for the treatment of alcoholic liver disease. [J]. Gastroenterol. Hepatol. 28, 56–60 (2013).
[5].Park O, Wang H, Weng H, et al. In vivo consequences of liver specific interleukin-22 expression in mice: Implications for human liver disease progression. [J]. Hepatology, 2011 54(1): 252-261.
[6].Holo, H., Jeknic, Z., Daeschel, M., Stevanovic, S., & Nes, I. F. (2001). Plantaricin W from Lactobacillus plantarum belongs to a new family of two-peptide lantibiotics. Microbiology (Reading, England), 147(Pt 3), 643–651
[7].Sánchez, J., Diep, D. B., Herranz, C., Nes, I. F., Cintas, L. M., & Hernández, P. E. (2007). Amino acid and nucleotide sequence, adjacent genes, and heterologous expression of hiracin JM79, a sec-dependent bacteriocin produced by Enterococcus hirae DCH5, isolated from Mallard ducks (Anas platyrhynchos). FEMS microbiology letters, 270(2), 227–236.
[8].Zhang, G., Brokx, S., & Weiner, J. H. (2005). Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli. Nature Biotechnology, 24(1), 100–104.
[9].Munoz-Morris MA, Heitz F, Divita G, et al. The peptide carrier Pep-1 forms biologically efficient nanoparticle complexes [J]. Biochem Biophys Res Commun, 2007, 355(4)877-882
[10]https://2018.igem.org/Team:TAS_Taipei/Experiments
[11].Wang, W., Li, Y., Wang, Y., Shi, C., Li, C., Li, Q., & Linhardt, R. J. (2018). Bacteriophage T7 transcription system: an enabling tool in synthetic biology. Biotechnology Advances.
[12].M K, HM S. A portable expression resource for engineering cross-species genetic circuits and pathways. Nature communications. 2015;6:7832.
[13].Liang, X., et al. (2018). "Integrating T7 RNA Polymerase and Its Cognate Transcriptional Units for a Host-Independent and Stable Expression System in Single Plasmid." ACS Synth Biol 7(5): 1424-1435.
[14].Ryan KJ, Ray CG, eds. (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill. pp. 294–295. ISBN 0-8385-8529-9.
[15].Sánchez, J., Diep, D. B., Herranz, C., Nes, I. F., Cintas, L. M., & Hernández, P. E. (2007). Amino acid and nucleotide sequence, adjacent genes, and heterologous expression of hiracin JM79, a sec-dependent bacteriocin produced by Enterococcus hirae DCH5, isolated from Mallard ducks (Anas platyrhynchos). FEMS microbiology letters, 270(2), 227–236.
[16].Holo, H., Jeknic, Z., Daeschel, M., Stevanovic, S., & Nes, I. F. (2001). Plantaricin W from Lactobacillus plantarum belongs to a new family of two-peptide lantibiotics. Microbiology (Reading, England), 147(Pt 3), 643–651.
[17].Geldart, K. G., Kommineni, S., Forbes, M., Hayward, M., Dunny, G. M., Salzman, N. H., & Kaznessis, Y. N. (2018). Engineered E. coli Nissle 1917 for the reduction of vancomycin-resistant Enterococcus in the intestinal tract. Bioengineering & translational medicine, 3(3), 197–208.
[18].Zhang, G., Brokx, S., & Weiner, J. H. (2006). Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli. Nature biotechnology, 24(1), 100–104.
[19].Cederbaum, A. I. (2012). Alcohol metabolism. Clin Liver Dis, 16(4), 667-685.
[20].Leeman, R. F., Corbin, W. R., Nogueira, C., Krishnan-Sarin, S., Potenza, M. N., & O'Malley, S. S. (2013). A human alcohol self-administration paradigm to model individual differences in impaired control over alcohol use. Exp Clin Psychopharmacol, 21(4), 303-314.
[21].Lieber, C. S. (2005). Metabolism of alcohol. Clin Liver Dis, 9(1), 1-35.
[22].Plawecki, M. H., & Crabb, D. W. (2014). Metabolism. Handb Clin Neurol, 125, 55-69.
[23].Singh, J. (2020). Analysis of fractional blood alcohol model with composite fractional derivative. Chaos, Solitons & Fractals, 140.
[24].Hu, B. W., Lai, J. H. (1996). STUDY OF ALCOHOL PHARMACOKINETICS IN HUMAN BODY. Chin J Forensic Med (01),1-5.
[25].Qiao, L. . (2010). Simulating bioreaction processes based on simbiology. Computer Applications & Software.
[26].Ali, L., Goraya, M. U., Arafat, Y., Ajmal, M., Chen, J. L., & Yu, D. (2017). Molecular Mechanism of Quorum-Sensing in Enterococcus faecalis: Its Role in Virulence and Therapeutic Approaches. Int J Mol Sci, 18(5).
[27].Del Papa, M. F., & Perego, M. (2011). Enterococcus faecalis virulence regulator FsrA binding to target promoters. J Bacteriol, 193(7), 1527-1532.
[28].Nakayama, J., Cao, Y., Horii, T., Sakuda, S., & Nagasawa, H. (2001). Chemical synthesis and biological activity of the gelatinase biosynthesis-activating pheromone of Enterococcus faecalis and its analogs. Biosci Biotechnol Biochem, 65(10), 2322-2325.
[29].Rutherford, S. T., & Bassler, B. L. (2012). Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med, 2(11).
[30].Hämäläinen, M. (2020). The importance of drug-target binding kinetics for drug efficacy – Without on you are off!. Retrieved 21 October 2020, from https://www.cytivalifesciences.co.jp/contact/pdf/L11_32-1351-12AA.pdf
[31].Hussain, R., & Siligardi, G. (2016). Characterisation of Conformational and Ligand Binding Properties of Membrane Proteins Using Synchrotron Radiation Circular Dichroism (SRCD). *Advances in experimental medicine and biology*, *922*, 43–59.
[32].Pul, Ü., Lux, B., Wurm, R., & Wagner, R. (2008). Effect of upstream curvature and transcription factors H-NS and LRP on the efficiency of Escherichia coli rRNA promoters P1 and P2 - a phasing analysis. Microbiology (Reading, England), 154(Pt 9), 2546–2558.
[33].Kotzsch, A., Vernet, E., Hammarström, M., Berthelsen, J., Weigelt, J., Gräslund, S., & Sundström, M. (2011). A secretory system for bacterial production of high-profile protein targets. *Protein science : a publication of the Protein Society*, *20*(3), 597–609.
[34].Patching, S. G., Edara, S., Ma, P., Nakayama, J., Hussain, R., Siligardi, G., & Phillips-Jones, M. K. (2012). Interactions of the intact FsrC membrane histidine kinase with its pheromone ligand GBAP revealed through synchrotron radiation circular dichroism. Biochimica et biophysica acta, 1818(7), 1595–1602.
[35].Pérez-Ortín JE, Alepuz P, Chávez S, Choder M. Eukaryotic mRNA decay: methodologies, pathways, and links to other stages of gene expression. J Mol Biol. 2013 Oct 23;425(20):3750-75.
[36].Rieu, A., Weidmann, S., Garmyn, D., Piveteau, P., & Guzzo, J. (2007). Agr system of Listeria monocytogenes EGD-e: role in adherence and differential expression pattern. *Applied and environmental microbiology*, *73*(19), 6125–6133.
[37].Hui, N. , Min, B. W. , Mei, G. K. , Jun, L. W. , & Chun, H. D. . (2009). The comparative research on intestinal flora population of patients with colorectal cancer and normal people. *Journal of Kunming Medical University*
[38].Xu, M., Chang, B., Mathews, S., & Gao, B. (2014). New drug targets for alcoholic liver disease. Hepatology international, 8 Suppl 2(Suppl 2), 475–480.
[39].Xing, W. W., Zou, M. J., Liu, S., Xu, T., Wang, J. X., & Xu, D. G. (2011). Interleukin-22 protects against acute alcohol-induced hepatotoxicity in mice. Bioscience, biotechnology, and biochemistry, 75(7), 1290–1294.
[40].Gao, B., & Shah, V. H. (2015). Combination therapy: New hope for alcoholic hepatitis?. Clinics and research in hepatology and gastroenterology, 39 Suppl 1(Suppl 1), S7–S11.
[41].Gao, B. (2012). Hepatoprotective and anti‐inflammatory cytokines in alcoholic liver disease. Journal of gastroenterology and hepatology, 27, 89-93.
[42].Na, D., Yoo, S. M., Chung, H., Park, H., Park, J. H., & Lee, S. Y. (2013). Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nat Biotechnol, 31(2), 170-174. doi:10.1038/nbt.2461
[43].Keane, J., Gershon, S., & Wise, R. (2001). TUBERCULOSIS ASSOCIATED WITH IMFLIXIMAB, A TUMOR NECROSIS FACTOR ALPHA-NEUTRALIZING AGENT. Infectious Diseases In Clinical Practice, 10(9), 498. doi: 10.1097/00019048-200112000-00012
[44].Nowotarski, S. L., & Shantz, L. M. (2010). Cytoplasmic accumulation of the RNA-binding protein HuR stabilizes the ornithine decarboxylase transcript in a murine nonmelanoma skin cancer model. The Journal of biological chemistry, 285(41), 31885–31894.
[45].Emerald, B. S., Chen, Y., Zhu, T., Zhu, Z., Lee, K. O., Gluckman, P. D., & Lobie, P. E. (2007). AlphaCP1 mediates stabilization of hTERT mRNA by autocrine human growth hormone. The Journal of biological chemistry, 282(1), 680–690.
[46].Geva-Zatorsky, N., Issaeva, I., Mayo, A. et al. Using bleach-chase to measure protein half-lives in living cells. Nat Protoc 7, 801–811 (2012).