Team:UT-Tokyo/CTCD/Content

Please imagine that you are a police officer. You are searching for dangerous criminals who hide in crowd and it is difficult to find them. This situation is the same as finding cancer cells (dangerous criminals in our body) in our blood when they resemble to normal blood cells. In this way picking up cancer cells has been developed around the world, where every year a different method is developed. Don't you think that it is like a dream if there were drugs, which if someone take, they will confess their crime? What about drugs, which if the cancer cells take, they will shine brightly on the blood? Here our team focuses on microRNA (miRNA) and provides a special genetic circuit which exposes cancer cells in blood vessels.(Fig.1)

Circulating tumor cells (CTCs) tumor cells arising from epithelia, flowing through blood vessels and causing metastasis to other tissues. Focusing on these cells, there are ways of early detection of cancer, detecting CTCs in blood of patients with early-stage cancer. The effectiveness of these methods has been ensured through the results of researches especially in breast cancer and colon cancer (ref). These methods of detection are a technology developed in recent years, compared to the traditional methods of picking up the tissue of patients which has been used for a long time. However, these traditional methods produce a hard burden to the patients, because of the several and long procedures. On the other hand, the methods of detecting CTCs produce a non-invasive option for patients compared to traditional ones. From the utility points, CTCs have been used all around the world to develop several detection methods. Most of these methods emphasized utilizing anti-EpCAM antibody, which is an adhesion protein used as marker of cancers, and distinguish CTCs from blood cells by the difference of their cell size. However, because the concentration of CTCs in blood is very low, it is required to develop a new approach for the detection of CTCs.

Here we have developed a new detection method for CTCs focusing on two types of profiles; miRNA and EpCAM. It has given the more specificity for CTCs to utilize the two profiles. miRNA is a special short non-cording RNA and interferes the translation of the complementary mRNA, interacting with some proteins and forming the RNA-induced silencing complex (RISC) in wide-species from virus to human. Focusing on different expression patterns of miRNA in each cell type, we utilized miRNA profiles with a synthetic biological approach for detecting CTCs. The circuit that we have developed has EpCAM promoter, reporter and blood cells-specific miRNA (miR-142-3p/5p) binding sites. Two conditions, which are activation of EpCAM promoter and an absence of blood cells-specific miRNA, are required for translation of the reporter. If we inject this circuit into cells in blood, only in the case of CTCs the reporter is produced.(Fig.2)

[1] Friedlander, Terence W., Gayatri Premasekharan, and Pamela L. Paris. "Looking back, to the future of circulating tumor cells." Pharmacology & therapeutics 142.3 (2014): 271-280.

Fig.3 the construct of our circuit

EGP-2 promoter

EGP-2 promoter is the promoter of Epithelial cell adhesion molecule (EpCAM) protein.EpCAM is a trans membrane glycoprotein which is expressed in epithelial cells and shows high level expression in various type of human epithelial carcinomas[2].Using this promoter, reporter genes can be expressed only in cancer cells. EGP-2 promoter is originally about 3500bp and it is not convenient to make a DNA construct. But, in the previous research, it is known that EGP-2 promoter can work if a part of its sequence is deleted[3]. In this project ,We cloned a part of EGP-2 promoter, and made it useful as a biobrick parts.

miRNA-142

MiRNA is a class of non-coding RNA.MiRNA binds the miRNA-recognition element in the 3' untranslated region of the target gene. MiRNA shows the tissue-specific expression pattern. In our project, we use miRNA-142, which is expressed only in hematopoietic cells[4].After miRNA-142 is transcribed, it is cleaved to miRNA-142-5p and miRNA-142-3p. Adding the recognition element of miRNA-142 in the 3' untranslated region of the reporter genes, leaky expression in non-carcinoma hematopoietic cells can be reduced.

Fig.4 When this construct is transfected to the epithelial cells like cancer cell, pEGP-2 promote the transcription of EGFP, and the mRNA is not degraded by miRNA because miR 142 is specific to hematopoietic cells.

Fig.5 When this construct is transfected to the hematopoietic cells, pEGP-2 does not work as a promoter, and mRNA is not transcribed. Even if the leak of the promoter makes few mRNA ,this mRNA is degraded by RNAi caused by miR 142.

In our project, miRNA is used for degrading mRNA in cells that express miRNA. But if we transfect construct like [pCMV-LacI-miR A binding site] and [pCAG-LacO2-GFP][5], mRNA in cells that does not express miRNA is degraded. This system will makes many application of miRNA in iGEM possible.

We focused on a tissue-specific promoter and miRNA in order to detect CTCs.As well as hematopoietic cells, many other tissues shows specific miRNA expression patterns[3]. Therefore, if by making a circuit that returns an output to a certain miRNA expression pattern, we may identify where a CTC comes from.[5]

In addition to the detection of CTCs, combinations of tissue-specific promoter and miRNA can be applied to many treatments. For example, suicide genes can be expressed in cancer cells in a specific tissue.[6]

Characterization of BBa_K747096

During DNA assembly, we found that this promoter was activated in Escherichia coli. Then we decided to characterize this promoter activity by comparing with constitutive promoter (BBa_J23101) using GFP (BBa_E0040) (Figure 1).
As seen in Figure 1, this promoter shows weaker activity than BBa_J23101.

Figure 1

The activity of BBa_K747096GFP was activated by 501nm excitation laser.
a)Measured when OD600 is 0.9~1.1.
b)Measured after cultured O/N.

In order to calculate RPU (relative promoter unit [1]), we performed real-time measurement of GFP fluorescence (Figure 2). We measured activity of GFP and OD600, which are necessary for calculation of RPU.

Figure 2

Real-time measurement of BBa_K747096
a)Activity of GFP (activated by 488nm excitation laser).
b)OD600.

We calculated RPU(Relative Promoter Unit) of BBa_K747096 from the Fig. 2 using the equation (1).

This equation can be used only when Fluorescence per cell is in steady state. In this experiment, this condition was fulfilled as shown in Table1.

Table 1 : Fluorescence / OD

	K747096	J23101
t=4h	92.62	13.14
t=6h	99.37	19.86

From Table1, we can get RPU of BBa_K747096:

RPU = 5.8

[1]:https://2010.igem.org/Team:Kyoto/LearnMore#Relative_Promoter_Unit_.28RPU.29

Characterization of BBa_K747096

During DNA assembly, we found that this promoter was activated in Escherichia coli. Then we decided to characterize this promoter activity by comparing with constitutive promoter (BBa_J23101) using GFP (BBa_E0040) (Figure 1).
As seen in Figure 1, this promoter shows weaker activity than BBa_J23101.

Figure 1

The activity of BBa_K747096GFP was activated by 501nm excitation laser.
a)Measured when OD600 is 0.9~1.1.
b)Measured after cultured O/N.

In order to calculate RPU (relative promoter unit [1]), we performed real-time measurement of GFP fluorescence (Figure 2). We measured activity of GFP and OD600, which are necessary for calculation of RPU.

Figure 2

Real-time measurement of BBa_K747096
a)Activity of GFP (activated by 488nm excitation laser).
b)OD600.

We calculated RPU(Relative Promoter Unit) of BBa_K747096 from the Fig. 2 using the equation (1).

This equation can be used only when Fluorescence per cell is in steady state. In this experiment, this condition was fulfilled as shown in Table1.

Table 1 : Fluorescence / OD

	K747096	J23101
t=4h	92.62	13.14
t=6h	99.37	19.86

From Table1, we can get RPU of BBa_K747096:

RPU = 5.8

[1]:https://2010.igem.org/Team:Kyoto/LearnMore#Relative_Promoter_Unit_.28RPU.29

We will paste here some parts of the safety form of our team.

We also discussed safety of our team in human practice page.

Your Training

a) Have your team members received any safety training yet?

We have not had any safety training officially, but have been taught by learned people.

b) Please briefly describe the topics that you learned about (or will learn about) in your safety training.

We learned about techniques for preventing diffusion of Escherichia coli or other organisms including ogenetically modified organisms into environment and risks concerning DNA assembly experiments.

c) Please give a link to the laboratory safety training requirements of your institution (college, university, community lab, etc). Or, if you cannot give a link, briefly describe the requirements.

Division for Environment, Health and Safety (http://www.adm.u-tokyo.ac.jp/office/anzeneisei/index.html) in our university is responsible for training laboratory safety.
Taking lab safety training course is not mandatory, but strongly recommended for those who are involved in experiments. However, this course is for the graduate school students and senior stuffs, and not open to undergraduate students. We thus had a training directly from the PI.

The Organisms and Parts that You Use

Species name(including strain)	Risk Group	Risk Group Source	Disease risk to humans?	Part number/name	Natural function of part	How did you acquire it?	How will you use it?	Notes
Escherichia coli JM109	1	DSMZ	no			from our Lab	DNA asssembly
Escherichia coli MG1655	1	DSMZ	no			from our Lab	Assay
Pseudomonas fluorescens	2	http://www.absa.org/riskgroups/bacteriasearch.php?genus=Pseudomonas	yes	sigma factor	polymerize RNA with RNA polymerase	order the part DNA from a synthesis company	transcriptional control	The bacteria causes opportunistic infection and it affects with usually patients with compromised immune systems.
Pseudomonas protegens	1	http://www.dsmz.de/catalogues/details/culture/DSM-19095.html	no	anti sigma factor	prevent combination between RNA polymerase and specific sigma factor	order the part DNA from a synthesis company	transcriptional control
Pseudomonas syringae	1	DSMZ	no	sigma factor	polymerize RNA with RNA polymerase	order the part DNA from a synthesis company	transcriptional control
Pseudomonas syringae	1	DSMZ	no	anti sigma factor	prevent combination between RNA polymerase and specific sigma factor	order the part DNA from a synthesis company	transcriptional control
Chlorocebus aethiops COS-1	1	DSMZ	no			receive the cells from another lab	Assay
Chlorocebus aethiops COS-7	1	DSMZ	no			receive the cells from another lab	Assay
Homo sapiens HL-60	1	DSMZ	no			receive the cells from another lab	Assay
Homo sapiens oral epithelial cell	1	http://www.lifescience.mext.go.jp/bioethics/data/anzen/syourei_02.pdf	no	EGP2 promoter	the promoter of EpCAM	from the member of our team	transcriptional control

Risks of Your Project Now

Please describe risks of working with the biological materials (cells, organisms, DNA, etc.) that you are using in your project. If you are taking any safety precautions (even basic ones, like rubber gloves), that is because your work has some risks, however small. Therefore, please discuss possible risks and what you have done (or might do) to minimize them, instead of simply saying that there are no risks at all.

a) Risks to the safety and health of team members, or other people working in the lab

When we are exposed to E. coli cells, we have a chance to have irritation in our eyes, skin, and respiratory system. Furthermore, ethidium bromide which we use to detect DNA band is carcinogen. We also use mammalian cells in assay. These cells can be infected by viruses which can also infect us. We also use harmful reagent, such as membrane binding solution.

b) Risks to the safety and health of the general public (if any biological materials escaped from your lab):

As maintained above, our lab has harmful organisms and substances. If they diffused outside, there might be health hazard.

c) Risks to the environment (from waste disposal, or from materials escaping from your lab)

We might dispose chips or tubes which contain a bit amount of genetically modified organisms without sterilizing. It offenses the law of our country; they may effect on biodiversity in our country. The bacteria we used also have drug resistance against ampicillin or CP, and if escaped, they could not be killed by those drugs. Mammalian cells are so weak that they can hardly live in the natural world.

d) Risks to security through malicious mis-use by individuals, groups, or countries

There seems to be no risks with this subject. In today's cognition, parts that we use do not seem to lead the expression of harmful materials.

e) What measures are you taking to reduce these risks? (For example: safe lab practices, choices of which organisms to use.)

In order to avoid the risks about bacterial cells shown above, we autoclave all wastes, sterilize all equipments that contain the organisms with detergent or hypochlorous acid. When bacteria exposed outside the tubes or examiners, we immediately seterilize them with ehtanol. Furthermore, in order to keep ethidium bromide away from our skin, we use kimwipes to wrap the chips used to taking it up before throwing away, and we take care that our bare hands do not touch the gel polluted by the substance.

Risks of Your Project in the Future

What would happen if all your dreams came true, and your project grew from a small lab study into a commercial/industrial/medical product that was used by many people? We invite you to speculate broadly and discuss possibilities, rather than providing definite answers. Even if the product is "safe", please discuss possible risks and how they could be addressed, rather than simply saying that there are no risks at all.

a) What new risks might arise from your project's growth? (Consider the categories of risk listed in parts a-d of the previous question: lab workers, the general public, the environment, and malicious mis-uses.) Also, what risks might arise if the knowledge you generate or the methods you develop became widely available?

In sigma-Recounter project, the circuit we constructed has potential for the application for defeating pests or bacteria by means of the expression of toxic proteins. In this case, there is a risk that you mistakenly output the toxin.
In CTCD project, the role of the circuits is to detect CTCs by GFP, thus it is thought to be difficult to have a risk of doing humans or environment harm.

b) Does your project currently include any design features to reduce risks? Or, if you did all the future work to make your project grow into a popular product, would you plan to design any new features to minimize risks? (For example: auxotrophic chassis, physical containment, etc.) Such features are not required for an iGEM project, but many teams choose to explore them.

In the future study of sigma-Recounter project, in addition to the reset system, we intend to increase the number of nodes and to enable one state to move to any other states. Therefore, if our project is used to express a toxin to defeat pest or bacteria, you can prepare an anti-toxin node or a reset system for mistakenly expressing the toxin.

Our activities involved in iGEM were all conducted by undergaduates alone!!
Based on fund-raisings and public relations, all team members had a lot of brainstoming, investigations and discussions in order to select project carefully. And we conducted experiments and FINALLY saw results.
We also designed and composed all publish tools, and of course, polished all scripts and presentation by ourselves.

Project

All we had a lot of brainstorming, investigations and discussion when we decide our projects.
Yoichi Irie(Team Leader)
σ-ReCounter:
Shunsuke Sumi(idea)
So Nakashima(idea and comformation)
Takefumi Yoshikawa(comformation)

CTCD:
Masayuki Osawa(conformation)
Shigetaka Kobari(investigation and conformation)
Shunsuke Sumi(conformation)
Senkei Hyo(investigation)
Yoshihiko Tomofuji(conformation)
Yshiki Okesaku(investigation)

Experiment

Lab. Leader:
Takefumi Yoshikawa(construction, assay;σ-ReCounter)

Lab. Members:
Atsuki Ito(construction)
Hajime Takemura(construction)
Keisuke Tsukada(construction)
Kentaro Tara(construction)
Kento Nakamura(construction, assay;σ-ReCounter)
Naruki Yoshikawa(construction)
Nobuhiro Hiura(construction)
Shigetaka Kobari(assay;CTCD)
So Nakashima(construction, Assay;σ-ReCounter)
Yoshihiko Tomofuji(assay;CTCD)
Yuto Yamanaka(construction)

Modeling

Keisuke Tsukada, Kentaro Tara, Manabu Nishiura, Masaki Ono

Web

Almost all members wrote drafts of our team wiki.
Cristian David(check our English)
Hiroki Tsuboi(team website, implementation of our team wiki)

App

Naruki Yoshikawa

Design

Cristian David(parker design)
Yoshiki Okesaku(all design, all figure)

Presentation

Kento Nakamura, Manabu Nishiura, Masato Ishikawa, Yumeno Koga, Yuto Yamanaka

Poster

Public Relation

Ding Yuewen, Keisuke Tsukada

Adviser

Kota Tosimitsu

Promega KK.for chamical reagents

Teiyukai, Faculty of Engineering, The University of Tokyofor fund

Integrated DNA Technologies MBL

COSMO BIO Co., Ltd.for fund

Leave a Nest Co., Ltd.(Hiroyuki Takahashi)for advice for public relations & introduction of Promega KK.

This year, we set 2 goals in human practice and have made efforts to realize following goals.

1.Spreading iGEM in general public.

2.Activating iGEM in Japan.

We set these 2 goals because we want to remove prejudice against gene recombination and synthetic biology, and familiarize people with synthetic biology. By doing these, we think that people can understand synthetic biology better.

We also expect undergraduate students, who will lead next generation of biology, to improve their skills by activating iGEM in Japan.

"EcoLightsOut"

We have developed an Android app. to introduce our project. This is a puzzle game which uses the counting system we created. In the project, the state of E. coli is changed by stimulus of signal molecules. In the game, players can stimulate the E. coli by touching it. E. coli are displayed in 3x3 or 5x5 grid shape. When you touch an E. coli in the game, the colors of the E. coli and four adjacent ones are changed. This behavior is analogy of diffusion of signal molecules. The goal is to turn all E. coli green.

Synthetic biology is not well-known and thought to be unfamiliar to ordinary people. To introduce synthetic biology into public, an easy entrance such as playing game is effective. We hope that players will get interested in synthetic biology by enjoying this game.

You can download this game from Google Play.

Lectures to general public

School festivals

The university of Tokyo has two school festivals per year. The May festival is held in May and the Komaba festival was held in November.We explained iGEM and synthetic biology briefly and introduce our project to audience. We　invited other iGEM teams in Japan to May festival. We offered precious opportunities that Japanese iGEM teams meet through May festivals.

Techno-Edge

Techno-Edge is the event which the department of technology of university of Tokyo held. The purpose of this event was to appeal department of technology to junior high or high school students.Many laboratories and academic circles such as Robotech took part in this event. iGEM UT-Tokyo also participated in it to appeal synthetic biology.

Not only high school and junior high, but also primary school students came to our booth and were interested in our explanation.

Presentation

This year, iGEM Nagahama invited us to the genetics society of Japan, and we participated in it. We made an oral presentation workshop of synthetic biology and took part in poster session. There were many iGEM teams, and we could advertise iGEM in academic world. Moreover, specialists gave advice to us, and we were inspired by professors of synthetic biology.

We also joined in Japanese Society for Cell Synthesis Research.

A cram school

We held a seminar in which we explained synthetic biology and iGEM for high school students at a cram school in Komaba.

Collaborations

We collaborated on modeling with Nagahama.

Their project aimed cadmium collection using Escherichia coli which has positive chemotaxis for aspartic acid, then we constructed simplified model of chemotaxis and simulated behavior of E. coli by using probability and random function.

Their wiki explained the result of modeling.(→link) We constructed all equations in their simulation including the equation that determines E. coli to choose going straight or turning in probability, and explained equations to them.The figure describing the result of simulation is also made by UT-Tokyo. All code for modeling is here.(→link)

Ethics and regulation

We thought to confirm whether our project meets ethics, but ethics is very vague, so we decided to research about ethics.

iGEM Japan

iGEM Japan is an organization which was founded last year for iGEM teams in Japan to cooperate each other.

In March, iGEM Kyoto held iGEM-Japan West meeting. In this meeting, we shared each team's project and advised each other. This meeting was very useful because we could find our idea's weak point.

In August, iGEM TMU-Tokyo held iGEM-Japan East meeting, and we made presentations about our projects and criticized each other. Thanks to these meetings, we developed quality of our projects.