Team:Tongji China/Poster

SHARK: Seek Heme And Reflect cracK

Siyuan Li¹, Jiacheng Jiang¹, Wenting Lu¹, Haocun Wang¹, Ziliang Chen¹, Zongyu Li¹, Tianzeng Sun¹, Min Ma¹, Zhenglong Xiang¹, Xianglei Wang¹, Yiren Shao², Xiangqi Huang⁴, Yingying Yue⁴, Qile He⁵, Weisi Wang⁵
Instructor: Yunzhe Kang¹
Advisors: Huan Zhang¹, Yuxiao Fan¹, Weijie Xu³
Primary PI: Jing Zhang¹
Secondary PI: Ye Leng¹

¹School of Life Science and Technology, Tongji University
²School of Mathematical Sciences, Tongji University
³School of Physics Science and Engineering, Tongji University
⁴College of Design and Innovation, Tongji University
⁵College of Arts and Media, Tongji University

Abstract

SHARK (Seek Heme And Reflect cracK) put forward a new solution for bleeding site detection of chronic lower gastrointestinal bleeding. Through engineered bacteria Hetoul (HEme TO ULtrasonic imaging), SHARK can realize rapid and accurate localization of bleeding sites in lower gastrointestinal tract. Compared to traditional endoscopy examinations, SHARK would not depend on doctors' technique and would bring a more comfortable experience to patients. Hetoul carries two main functional modules, HBS (Heme Biosensor System) for bleeding detection, and BAS (Bilateral ARG System) for location report. We have verified the HBS for its linear expression with hemin, and have constructed bridge from HBS to BAS successfully. For implementation, we designed DDS (Drug Delivery System) for uniform distribution of Hetoul in lower gastrointestinal tract, and established mathematic model to understand the object movements in gastrointestinal tract.

Introduction

Background

Chronic lower gastrointestinal bleeding can be caused by inflammation, vascular diseases, tumors and so on. It is hard to diagnose and cure, due to the unobvious bleeding manifestation and difficulties in the localization of the bleeding sites. The current detections usually use endoscopes, which highly depend on doctors' technique and put great burdens on patients.

Hetoul

To solve these problems, Tongji_China 2020 designed Hetoul (HEme TO ULtrasonic imaging). Hetoul can sense the blood and express acoustic reporters. The aim of us is to provide a more convenient diagnosis tool to realize rapid localization of bleeding sites in lower gastrointestinal tract.

Inspiration

We were initially inspired by a classmate's experience of acute stomach bleeding, her treatment faced difficulties in detection of the bleeding site.

After that, we communicated with doctors, and learned that the bleeding site detection can provide great help to the diagnosis of chronic lower gastrointestinal bleeding.

Problem

The current diagnostic process of chronic lower gastrointestinal bleeding is complicated. Patients would have medical consultations and routine examinations at first. Then the fecal occult blood test (FOBT) would be implemented. If FOBT showed positive results, colonoscopy or gastroscopy examinations would be conducted to localize the bleeding site. When colonoscopy or gastroscopy examinations failed, capsule endoscopy or enteroscopy examinations with higher accuracy (but also more expensive) would be conducted.

However, the accuracy of FOBT is not high enough. The success rates of endoscope examinations, especially for chronic lower gastrointestinal bleeding, highly depend on doctors' technique. The whole process is expensive and time-consuming, and puts great discomfort to patients.

Idea

Sensor: Detect bleeding

The Heme Biosensor System (HBS), which can respond to the heme and active the expression of downstream reporters, can enable Hetoul to detect the bleeding sites.

The system was first put forward by Mimee M. et al. in 2018^[1]. We found it during literature research.

Reporter: View in ultrasonic imaging

The Bilateral ARG System (BAS), which can form gas vesicles visible in ultrasonic imaging, can enable Hetoul to report the location of bleeding sites.

The Acoustic Reporter Genes (ARGs) we use in BAS was designed by Bourdeau R. W. et al in 2018^[2], and also used by SJTU-BioX-Shanghai in 2018. We found it when viewing their team wiki.

Carrier: Help distribution

The Drug Delivery System (DDS) of Hetoul, in form of lyophilized powder packaged in MAHBs (Multilayer Alginate Hydrogel Beads), can help Hetoul to distribute uniformly in the lower gastrointestinal tract.

The DDS was designed by Li Y et.al in 2017^[3]. We found it during literature research.

Reference
[1] Mimee M, Nadeau P, Hayward A, et al. An ingestible bacterial-electronic system to monitor gastrointestinal health. Science. 2018;360(6391):915-918. doi:10.1126/science.aas9315
[2] Bourdeau RW, Lee-Gosselin A, Lakshmanan A, et al. Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature. 2018;553(7686):86-90. doi:10.1038/nature25021
[3] Li Y, Feng C, Li J, et al. Construction of multilayer alginate hydrogel beads for oral delivery of probiotics cells. Int J Biol Macromol. 2017;105(Pt 1):924-930.

Design

Heme Biosensor System (HBS)

HBS was based on a synthetic promoter HrtO regulated by the heme-responsive transcriptional repressor, HrtR, and ChuA, an outer-membrane transporter of extracellular heme^[1].

Lambda repressor cI was expressed after HrtO, thus bridging Heme Biosensor System (HBS) and Bilateral ARG System (BAS).

👆The characterization result of HBS by GFP. The GFP expression showed a linear relationship between hemin concentration, in range of 0 to 4 μM.

Bilateral ARG System (BAS)

BAS was based on two Acoustic Reporter Genes (ARGs) for two kinds of gas vesicles, ARG1 and ARG2^[2], with different critical collapse pressures. They can be distinguished from each other in ultrasonic imaging by exerting acoustic pressure in the interval of twice ultrasonic imaging and comparing the imaging results.

We used promoter R0051 for ARG2, and I12007 for ARG1. Through the regulation of cI, BAS can express ARG2 when heme does not exist and express ARG1 when heme exists.

👆The characterization result of R0051 by RFP, and I12007 by GFP, simulating a all-or-none status in conditions of with or without HrtR.

FliC knock-out for colonization

We planned to knock out FliC gene, which is essential for bacteria flagellum synthesis ^[3], to reduce the autonomic movements of Hetoul.

Killing Switch

Internal killing switch: designed by SJTU-BioX-Shanghai 2018 Arabinose-induced E protein expression

External killing switch: designed by BNU-China 2019 Temperature-sensitive RBS and RelE-RelB system

Reference
[1] Mimee M, Nadeau P, Hayward A, et al. An ingestible bacterial-electronic system to monitor gastrointestinal health. Science. 2018;360(6391):915-918. doi:10.1126/science.aas9315
[2] Bourdeau RW, Lee-Gosselin A, Lakshmanan A, et al. Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature. 2018;553(7686):86-90. doi:10.1038/nature25021
[3] Mays ZJS, Chappell TC, Nair NU. Quantifying and Engineering Mucus Adhesion of Probiotics. ACS Synth Biol. 2020;9(2):356-367. doi:10.1021/acssynbio.9b00356

Model

The location information provided by Hetoul would have errors in the practical application, mainly caused by the time lag of gene expression and its movement along the gastrointestinal tract. To erase the errors of the location information in the practical application, and to determine how long Hetoul needs to distribute in gastrointestinal tract, we constructed models on the movements of objects in the gastrointestinal tract.

Before modeling, we put forward several assumptions of the geometry and movements of the stomach, small intestine and large intestine, and the properties of dietary residue.

After mathematical calculations, we obtained the formulas as follows:

Finally, ignore the infinitesimal and we get:

Then, we can know the velocity of particles, such as peels, and small molecules, such as heme, in different parts of gastrointestinal tract.

Conclusion

1. In the middle of the inner mucus layer, the average axial velocity of ﬂow ﬁeld is 2.7761x10^-7 m/s.

2. From the results above, the ultrasonic imaging should be implemented more than 16.5 hours after Hetoul is taken.

3. In the large intestine, at about 6 mm upstream to the bleeding site, the reporter of Hetoul could be detected.

4. In the small intestine, the Hetoul could be triggered at least at, or even downstream to the bleeding site.

Attribution

Poster Writing: Wenting Lu
Poster Art Design: Yingying Yue, Xiangqi Huang, Jiacheng Jiang
Poster coding: Siyuan Li, Weisi Wang

Team members

Siyuan Li
Team leader, Academic Group leader.
In charge of experiments;
Overall project design;
Design and experiment conduction of Heme Biosensor System characterization;
Design and experiment conduction of Bilateral ARG System characterization;
Experimental data analysis;
Plasmid construction and primer design;
Project wiki writing.

Jiacheng Jiang
Team leader, HP Group Leader.
In charge of human practices;
Content provision of the promotion video, the presentation video and the poster;
Project wiki writing.
Contact with the instructor and PI.

Wenting Lu
Academic group member.
Overall project design;
Parts design;
Design of Heme Biosensor System;
Design of Bilateral ARG System;
Experiment design and conduction;
Plasmid construction and primer design;
In charge of project wiki writing and translation.

Haocun Wang
Team Leader. Project manager.
Overall progress control of the project;
Participation in the video content;
Project wiki writing;
Contact with the instructor and PI.

Min Ma
Academic group member.
Plasmid construction of Bilateral ARG System;
Design and experiment conduction of K731250 part characterization;
Project wiki writing.

Ziliang Chen
Academic group member.
Design of Drug Delivery System;
Plasmid construction of Heme Biosensor System;
Project wiki writing.

Tianzeng Sun
Academic group member.
Literature research on Chronic lower gastrointestinal bleeding related diseases;
Plasmid construction of Heme Biosensor System.
Project wiki writing.

Zongyu Li
Academic group member.
Literature research on Chronic lower gastrointestinal bleeding related diseases;
Plasmid construction of Bilateral ARG System.
Project wiki writing.

Yiren Shao
Academic group member.
In charge of Modeling Work;
Project wiki writing.

Zhenglong Xiang
HP group member.
Participating human practices;
Video content writing;
Project wiki writing.

Xianglei Wang
HP group member.
Participating human practices;
Video content writing;
Project wiki writing.

Qile He
Video director.
Scriptwriting of promotion video and presentation video;
Video recording and production.

Yingying Yue
Art designer.
Visual design of videos and wiki pages.
Animation production.

Xiangqi Huang
Art Designer.
Visual design of poster, videos and wiki pages.
Animation production.

Weisi Wang
Coder.
Wiki page coding.
Visual design of wiki pages.

Instructor and Advisors

Yunzhe Kang
Instructor. Team leader of Tongji_China 2019.
Help in building the team Tongji_China 2020;
Guidance of project and experiment design.

Yuxiao Fan
Advisor. Wet Lab Manager of Tongji_China 2019.
Guidance of modeling.

Weijie Xu
Advisor. Student of School of physical science and Engineering, Tongji University.
Guidance of modeling.

Huan Zhang
Advisor. Member of Tongji_China 2019.
Guidance of experimental operation.

General Support

Dr. Jing Zhang: Professor and Deputy Dean of School of Life Science and Technology, Tongji University. Our primary PI, helped us in various aspects.

Dr. Ye Leng: Associate professor of School of Life Science and Technology, Tongji University. Our secondary PI, kept following our work and project.

Dr. Ping Li: Professor of School of Life Science and Technology, Tongji University. She shared lab resources with us.

Dr. Shaorong Gao: Professor and Dean of School of Life Science and Technology, Tongji University. provided us with lots of experimental materials.

Dr. Chong Li: Associate professor of School of Life Science and Technology, Tongji University. Helped us get in touch with companies that could support us.

Dr. Jiayu Chen: Associate professor of School of Life Science and Technology, Tongji University. Helped us get in touch with companies that could support us.

Dr. Aiping Lv: Teacher of School of Life Science and Technology. Helped us a lot in education process.

Dr. Chao Zhang: Professor and Deputy Dean of School of Life Science and Technology, Tongji University. Kept following the process.

Dr. Changsheng Du: Professor of School of Life Science and Technology, Tongji University.

Mrs. Libo Xing: Teacher in Tongji University, supports us in experimental materials.

Mrs. Xin Gui: Teacher in Tongji University, supports us in experimental materials and lab facilities.

Financial Support

Vazyme: Provided us with lots of experimental reagent.

Olympus Corporation: Provided us with financial support.

Becton Dickinson and Company: Provided us with financial support.

Snapgene: Provided us with a free license to use the software.

Geneious: Provided us with a free license to use the software.

Acknowledgement

Dr. Zhigang Huang
Deputy director of Gastroenterology Department of Shanghai Eastern Hospital.
In the early project selection stage, his suggestion drove us to adjust the aim from acute gastrointestinal bleeding to chronic lower gastrointestinal bleeding.

Dr. Yaozong Yuan
Director of Gastroenterology Department and Deputy director of Department of Clinical Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine.
From Dr. Yuan, we have confirmed that tumors and other related diseases are main causes for gastrointestinal bleeding.
Therefore, we think it valuable to transform Hetoul into a universal detection tool for gastrointestinal bleeding in our future work, which greatly expands the significance and application range of our project.

Dr. Qian Cheng
Professor, School of Physical Science and Engineering, Tongji University.
She provided many suggestions and supported experimental devices for us in terms of ultrasonic imaging equipment.

Dr. Bing Wang
Associate researcher, Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
She proposed many improvements for our DDS 3.0, reminding many aspects neglected by us in the DDS design and greatly helping to complete our DDS design.

Dr. Hongbing Wang
Professor and Deputy dean, School of Life Science and Technology, Tongji University.
He proposed many improvements for our DDS 2.0 and helped us a lot in DDS design.

Dr. Tintao Chen
Ganjiang Distinguished Professor, Director of Department of Microbiology, Nanchang University Institute of Translational Medicine.
From Dr.Chen, we learned about the current supportive policies of engineering probiotics in Shanghai, the technology and process of drug preparation in microbial drug enterprises and the current market situation, which greatly expanded the connotation and application value of our project.

Dr.Bing Lu
Associate chief physician of the Department of Anorectal Surgery, Shanghai Eastern Hospital.
In the early topic selection stage, he provided us with a lot of advice and helped us timely adjust the direction of the project.

Special Acknowledgement

Tongji_Software 2020 iGEM team give us a lot of advice on our modeling work and provided much guidance on the details.

SJTU-BioX-Shanghai 2020 iGEM team provided us with particles of gas vesicle protein and exchanged relevant information about the in vitro ultrasonic testing experiment, which provided us with a lot of guidance in the experimental work.

Tongji University

School of Life Science and Technology of Tongji University

School of Physical Science and Engineering of Tongji University

Shanghai Ruijin Hospital

Shanghai Eastern Hospital

Shanghai Institute of Materia Medica, Chinese Academy of Sciences

Nanchang University Institute of Translational Medicine

Specification

What's Hetoul？
Chassis Organism: E.coli Nissle 1917.
Genetically Modification: Heme Biosenser System, Bilateral ARG system, FliC-KO, Internal and External Killing Switch.
Drug Delivery System: Lyophilized powder of Heotul in MAHBs (Multilayer alginate hydrogel beads) , packaged by multi-phase emulsion technique.
Who needs Hetoul？

Hetoul was intended to people with chronic lower gastrointestinal bleeding. It can detect the bleeding sites and provide location information to doctors.

How to use Hetoul？

Preparation before imaging

Hetoul should be taken one day before the ultrasonic imaging, on an empty stomach. 8.5 to 10.5 hours after dining is best.

Hetoul would be released in users' lower gastrointestinal tract and work. Food in the intestine would affect proper distribution of Hetoul.

Ultrasonic imaging

The ultrasonic imaging should be implemented more than 16.5 hours after Hetoul is taken. Ultrasonic imaging would be implemented two times: the first imaging will use 1.5 MPa acoustic pressure to display the distribution of Hetoul in the gastrointestinal tract, and the second imaging will use 3.5 MPa acoustic pressure to collapse gas vesicle protein ARG2, leaving ARG1 to display the bleeding point detected by Hetoul. The site of bleeding could be confirmed after image processing program based on modeling.

Elimination of Hetoul

Take arabinose to eliminate remaining Hetoul in the gastrointestinal tract.

Hetoul released to the environment will autonomically die due to low temperature.