Team:UT-Tokyo/Counter/Project/Humanpractice

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<img src="Sub_human_practice.png" class = "contTitle" />

Contents

Introduction

In Japan, few people know about synthetic biology, so we decided to offer an opportunity to get closer to it . Following activities were not only for general public but also for high school students, who will lead the next generation of synthetic biology.

We also succeeded in introducing iGEM and synthetic biology to expert researchers as well as to young generation.We also made effort for iGEM Japan. What we did is written in "Activity for iGEM".

To spread iGEM in general public

"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 <a href="https://play.google.com/store/apps/details?id=com.ironkayak.ecolightsout1"target="_blank">Google Play</a>.

<img src="Screenshot1.png" class = "figure" style="width:380px;float:left;" /> <img src="Screenshot2.png" class = "figure" style="width:380px;float:left;" />

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 all iGEM teams in Japan to May festival, and 6 teams participated in poster session. We offered precious opportunities that Japanese iGEM teams meet through May festivals.

<img src="Festival1.png" class = "figure" style="width:380px;float:left;" /> <img src="Festival2.png" class = "figure" style="width:380px;float:left;" />

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.

<img src="Technoedge.png" class = "figure" />

Presentation

We participated in the annual meeting of the genetics society of Japan at the invitation of iGEM Nagahama team. We made an oral presentation in the workshop of synthetic biology and took part in poster session. There were many iGEM teams, and we could introduce iGEM in Japanese academic society.

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.

<img src="Presentation1.png" class = "figure" style="width:380px;float:left;" /> <img src="Presentation2.png" class = "figure" style="width:380px;float:left;" />

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.

Activity for iGEM

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.(→<a href="https://2014.igem.org/Team:Nagahama_project#Modeling" >link</a>) We constructed 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 made by UT-Tokyo. All code for modeling is here.(→<a href="https://2014.igem.org/Team:UT-Tokyo/Counter/Project/Humanpractice/nagahama.c?action=raw">link</a>)

Ethics and regulations

We thought to confirm whether our project meets ethics, but ethics is very vague, so we decided to research about <a href="https://2014.igem.org/Team:UT-Tokyo/Counter/Project?page=Humanpractice-block&contents=Humanpractice-2">ethics</a>.

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.

<img src="Igemjapan1.png" class = "figure" style="width:380px;float:left;" /> <img src="Igemjapan2.png" class = "figure" style="width:380px;float:left;" />

<img src="Sub_ethics.png" class = "contTitle" />

Overview

From ethical codes, we examined whether our projects and their application can be sanctioned. However, “ETHICS” is not steadfast rather mutable. Each SOCIETY, RELIGION, and CULTURE, which one belongs to, ingrains us what we call “ETHICS.” Even in the same society, religion, and culture, a slight difference among educational levels and living situations lets us have nonidentical moral values. Being in such dissimilarity, researchers must take the diverse values into consideration in order to justify their researches and applications.
The regulation including the law, which is standardized from prevailed beliefs and ethical values among the society, permits individual’s act unless not violating others’ rights. We deduced that the regulation is involved with attitudes toward the research along with various ethical values. It is essential for each iGEM member to understand and comply with the law, regulations, and guidelines which are determined by international organizations and domestic government agencies.
However, it is difficult for undergraduate students such as iGEM members to fully understand these laws, regulations, and guidelines and apply them to their projects. Here, we edited a brief version of the complicated matters for other members. We also assessed our current projects in terms of their safety and risks, which can be caused by their application, and then we clarified that our anticipated risks would not surpass our risk management ability. Be conscious of what we talked about here, we hope that all iGEM members will develop judicious research ethics and achieve eloquent projects.

Laboratory Biosafety Guideline

In this section, we will talk about the fundamental guideline which WHO provides.This guideline shows researchers unique set of safety equipment, facility, and practices to conduct safe and sure experiments.

Biohazard Containment and Safety

The relative hazards of infective microorganisms are grouped by risk group (WHO Risk Groups 1, 2, 3 and 4).

Laboratory biosafety manual Third edition says as follows:

Risk Group 1 (no or low individual and community risk)
A microorganism that is unlikely to cause human or animal disease.

Risk Group 2 (moderate individual risk, low community risk)
A pathogen that can cause human or animal disease but is unlikely to be a serious hazard to laboratory workers, the community, livestock or the environment. Laboratory exposures may cause serious infection, but effective treatment and preventive measures are available and the risk of spread of infection is limited.

Risk Group 3 (high individual risk, low community risk)
A pathogen that usually causes serious human or animal disease but does not ordinarily spread from one infected individual to another. Effective treatment and preventive measures are available.

Risk Group 4 (high individual and community risk)
A pathogen that usually causes serious human or animal disease and that can be readily transmitted from one individual to another, directly or indirectly. Effective treatment and preventive measures are not usually available.

Laboratory facilities are designated as basic ? Biosafety Level 1, basic ? Biosafety Level 2, containment ? Biosafety Level 3, and maximum containment ? Biosafety Level 4. Biosafety level designations are based on a composite of the design features, construction, containment facilities, equipment, practices and operational procedures required for working with agents from the various risk groups.

Risk groups relate biosafety levels but do not “equate” to the biosafety level of laboratories designed to work with organisms in each risk group.
High risk groups require more rarefied composite of the design features, construction, containment facilities, equipment, practices and operational procedures. We mainly talk about BSL1 and BSL2 because it is uncommon for undergraduate students to handle the facilities above BSL 3.

Laboratory biosafety manual Third edition says as follows:

Guidelines for the surveillance of laboratory workers handling microorganisms at Biosafety Level 1
Historical evidence indicates that the microorganisms handled at this level are unlikely to cause human disease or animal disease of veterinary importance. Ideally,however, all laboratory workers should undergo a pre-employment health check at which their medical history is recorded. Prompt reporting of illnesses or laboratory accidents is desirable and all staff members should be made aware of the importance of masintaining GMT.

Guidelines for the surveillance of laboratory workers handling microorganisms at Biosafety Level 2
1. A pre-employment or preplacement health check is necessary. The person’s medical history should be recorded and a targeted occupational health assessment performed.
2. Records of illness and absence should be kept by the laboratory management.
3. Women of childbearing age should be made aware of the risk to an unborn child of occupational exposure to certain microorganisms, e.g. rubella virus. The precise steps taken to protect the fetus will vary, depending on the microorganisms to which the women may be exposed.

Biological Safety Cabinets

Laboratory biosafety manual Third edition says as follows:</p> <p>
Biological safety cabinets (BSCs) are designed to protect the operator, the laboratory environment and work materials from exposure to infectious aerosols and splashes that may be generated when manipulating materials containing infectious agents, such as primary cultures, stocks and diagnostic specimens. Aerosol particles are created by any activity that imparts energy into a liquid or semiliquid material, such as shaking, pouring, stirring or dropping liquid onto a surface or into another liquid. Other laboratory activities, such as streaking agar plates, inoculating cell culture flasks with a pipette, using a multichannel pipette to dispense liquid suspensions of infectious agents into microculture plates, homogenizing and vortexing infectious materials, and centrifugation of infectious liquids, or working with animals, can generate infectious aerosols. Aerosol particles of less than 5 μm in diameter and small droplets of 5?100 μm in diameter are not visible to the naked eye. The laboratory worker is generally not aware that such particles are being generated and may be inhaled or may crosscontaminate work surface materials. BSCs, when properly used, have been shown to be highly effective in reducing laboratory-acquired infections and cross-contaminations of cultures due to aerosol exposures. BSCs also protect the environment.

Training

Laboratory biosafety manual Third edition says as follows:

Human error and poor technique can compromise the best of safeguards to protect the laboratory worker. Thus, a safety-conscious staff, well informed about the recognition and control of laboratory hazards, is key to the prevention of laboratory-acquired infections, incidents and accidents.

Waste Handling

Laboratory biosafety manual Third edition says as follows:

The overriding principle is that all infectious materials should be decontaminated, autoclaved or incinerated within the laboratory.</p> <p>The principal questions to be asked before discharge of any objects or materials from laboratories that deal with potentially infectious microorganisms or animal tissues are:
1. Have the objects or materials been effectively decontaminated or disinfected by an approved procedure?
2. If not, have they been packaged in an approved manner for immediate on-site incineration or transfer to another facility with incineration capacity?
3. Does the disposal of the decontaminated objects or materials involve any additional potential hazards, biological or otherwise, to those who carry out the immediate disposal procedures or who might come into contact with discarded items outside the facility?

Risk Assessment

GMO means genetically modified organism. Assessing following criteria, BSL is decided. <p>Laboratory biosafety manual Third edition says as follows:

Hazards arising directly from the inserted gene (donor organism)

1. Toxins
2. Cytokines
3. Hormones
4. Gene expression regulators
5. Virulence factors or enhancers
6. Oncogenic gene sequences
7. Antibiotic resistance
8. Allergens.

Hazards associated with the recipient/host
1. Susceptibility of the host
2. Pathogenicity of the host strain, including virulence, infectivity and toxin production
3. Modification of the host range
4. Recipient immune status
5. Consequences of exposure.

Hazards arising from the alteration of existing pathogenic traits
1. Is there an increase in infectivity or pathogenicity?
2. Could any disabling mutation within the recipient be overcome as a result of the insertion of the foreign gene?
3. Does the foreign gene encode a pathogenicity determinant from another organism?
4. If the foreign DNA does include a pathogenicity determinant, is it foreseeable that this gene could contribute to the pathogenicity of the GMO?
5. Is treatment available?
6. Will the susceptibility of the GMO to antibiotics or other forms of therapy be affected as a consequence of the genetic modification?
7. Is eradication of the GMO achievable?

Acquiring knowledge of what criteria are based on for each BSL is helpful and instructive for us.

Laboratory Biosecurity

Laboratory biosafety manual Third edition says as follows:


It has now become necessary to expand this traditional approach to biosafety through the introduction of laboratory biosecurity measures. Global events in the recent past have highlighted the need to protect laboratories and the materials they contain from being intentionally compromised in ways that may harm people, livestock, agriculture or the environment. Before the laboratory biosecurity needs of a facility can be defined, however, it is important to understand the distinction between “laboratory biosafety” and “laboratory biosecurity”.
“Laboratory biosafety” is the term used to describe the containment principles, technologies and practices that are implemented to prevent unintentional exposure to pathogens and toxins, or their accidental release. “Laboratory biosecurity” refers to institutional and personal security measures designed to prevent the loss, theft, misuse, diversion or intentional release of pathogens and toxins.

We must understand these concepts and cooperate to gather appropriate information.

Declaration of Helsinki

Medical applications are often seen in iGEMs projects. The effect of the application of GMOs on human beings is still unknown. Moreover, we must also consider patients’ health and privacy. Concerning this sensitive issue, World Medical Association established Declaration of Helsinki.

Declaration of Helsinki says as follows:


2. Consistent with the mandate of the WMA, the Declaration is addressed primarily to physicians. The WMA encourages others who are involved in medical research involving human subjects to adopt these principles.
9. It is the duty of physicians who are involved in medical research to protect the life, health, dignity, integrity, right to self-determination, privacy, and confidentiality of personal information of research subjects. The responsibility for the protection of research subjects
12. Medical research involving human subjects must be conducted only by individuals with the appropriate ethics and scientific education, training and qualifications. Research on patients or healthy volunteers requires the supervision of a competent and appropriately qualified physician or other health care professional.
16. In medical practice and in medical research, most interventions involve risks and burdens. Medical research involving human subjects may only be conducted if the importance of the objective outweighs the risks and burdens to the research subjects.
23. The research protocol must be submitted for consideration, comment, guidance and approval to the concerned research ethics committee before the study begins. This committee must be transparent in its functioning, must be independent of the researcher, the sponsor and any other undue influence and must be duly qualified. It must take into consideration the laws and regulations of the country or countries in which the research is to be performed as well as applicable international norms and standards but these must not be allowed to reduce or eliminate any of the protections for research subjects set forth in this Declaration. The committee must have the right to monitor ongoing studies. The researcher must provide monitoring information to the committee, especially information about any serious adverse events. No amendment to the protocol may be made without consideration and approval by the committee. After the end of the study, the researchers must submit a final report to the committee containing a summary of the study’s findings and conclusions.
24. Every precaution must be taken to protect the privacy of research subjects and the confidentiality of their personal information

Human Genome and its Domestic Ethical Guideline

We may assay whole human genome in iGEM projects. It needs to be justified ethically. At least in Japan, a chief of the research facility which conducts gene recombination is obligated to establish an ethical committee and the member of concerned research facility must follow the regulations. Principal Investor (PI) must submit a research proposal taking risks into consideration to the chief. Therefore, it is important for members to conduct risk appraisal and to draw up a plan in advance. It is also significant for us to recognize the concept and importance of informed consent.

Domestic Law Related to Cartagena Protocol on Biosafety


Living Modified Organisms must be restricted to the laboratory and under control. We consider their potential adverse effects on biological diversity, taking also into account risks not only to human health but also to the environment.
In Japan, after the results of Cartagena Protocol on Biosafety, the Diet enacted “Act on the Conservation and sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms.” This domestic law aims to implement Cartagena Protocol on Biosafety properly.
In this Act, “use” means use for provision as food, feed, and other purposes, and use for other acts including cultivation, other growing, processing, storage, transportation and disposal. There are two types of “use” in the Act. “Type 2 Use” means use which is conducted with the intention of preventing the dispersal of living modified organisms into the environment. “Type 1 Use” means use which is not subject to taking measures similar to “Type 2 Use.” When we use E. coli, which collect objective biomarkers or toxic substances and release profitable substances, under natural environment, our experiments are categorized “Type 1 Use.”

When we make, import, and/or create Type 1 Use of Living Modified Organisms, we must stipulate regulations concerning usage which is based on the evaluation criteria about potential adverse effects on biological diversity provided by the competent minister. We also must obtain the approval of the competent minister. The evaluation criteria are close to the risk appraisal. The criteria also consider host’s properties including distribution, usage history, and competitiveness. In the case that iGEM members create Living Modified Organisms and plan to conduct field tests, we need consider these criteria. After the approval, usage in conformity with the regulations can be restricted only in order to prevent potential adverse effects on biological diversity. If new findings after the approval suggest adverse effects on biological diversity are suggested, experimenters need not take the responsibility. However, if they do not obey the restrictions, they will be imposed a penalty including imprisonment.

In Type 2 Use, if containment measures are prescribed, experimenters must obey them during the period of use. If containment measures are not prescribed, we need the competent minister confirming them. The containment measures are related to the biosafety guidelines. When we use BioBrick, we follow the BSL according to its sequence and host.

There are various penalties and seems a unique Act concerning Living Modified Organisms. The Act restricts a wide range of act and is grounds to punish for not following the biosafety guidelines. However, the object of penalty is involved with misconduct including false application. Penalties are difficult to happen as far as we naturally act as a scientist. It is much more instructive for us to understand what regulations justify Living Modified Organisms than what penalties are.<P>

UT-Tokyo 2014 Projects

<p>In our experiments, we used E. coli K12 JM109. K-12 strain is genetically knocked-out in order to prevent colonization human and animal intestine. Plasmid backbones were pSB1A2, BBa J61002, and pSB1C3 from pUC19. These plasmids lack genes which are necessary for living things other than E. coli to express.

counter

A sigma factor does not have special resistance against breakdown. When the sequence of a sigma factor is inserted into Pseudomonas syringae pv. tomato str. DC3000(ecf11) and Pseudomonas fluorescens Pf-5(ecf20), a sigma factor might be expressed, but not in plasmid form.

CTC

We handled cell line which is no chance for multiplication outside laboratory because of its frail character. No dangerous gene was expressed when we assayed because we checked only expression of promoter.

Both counter and CTC projects are confined to BSL1 and do not contain high risks parts compared with present BioBrick parts.

Advisors

On doing experiments, we handed in our plan of experiments to Professor Wakamoto, Ikeuchi and Ishiura, the bosses of the laboratories in which we conducted our experiments. These professors confirmed that our project observed the regulations. Professor Wakamoto provided us with almost all the materials for our experiments, Professor Ikeuchi provided us with a fluorescence microscope, and Professor Ishiura provided us with experimental facilities for the CTC detector project.

Our all experimental operations were under Professor's control and permission.