We have observed that one unavoidable aspect of promoting synthetic biology today is the issue of biosafety. Currently,
the primary methods for controlling biosafety are suicide genes and hardware-based solutions. However, neither of these
methods can guarantee a 100% prevention of gene leakage. Furthermore, developing new genes related to biosafety is a
challenging endeavor, such as rare codons and auxotrophic chassis microorganisms. These limitations significantly hinder
the advancement and application of genetic engineering.



In order to rapidly reduce the risk and probability of gene leakage in synthetic biology projects, we propose and
advocate for the use of multiple biosafety measures instead of focusing solely on improving the effectiveness of
individual measures.



We conducted a random survey of 50 teams from previous years that used biosafety mechanisms: 86% of the teams showcased
their considerations for biosafety on their wikis, 64% had specific biosafety measures, but the majority had only a
single biosafety measure. Only 12% of the teams opted for two or more safety measures, and just 4% chose three or more
multiple biosafety measures.



For example, achieving a risk reduction of around 0.01% using a single measure would be challenging. However, it is
relatively easy to find measures that reduce the risk of leakage to around 5%. Yet, a 5% risk of leakage remains too
high for real-world applications. If we identify three measures, each with a 5% risk, working together in a project, the
overall project's risk of leakage would be reduced to 0.0125%, which is an acceptable level of risk.

Based on this discovery, we have actively promoted this approach. We shared these results in the iGEM China regional
exchange group, encouraging more teams to use multiple biosafety methods, and received responses from several teams.

Furthermore, in our own project, we have implemented multiple biosafety measures to reduce the overall risk of gene
leakage.through various efforts, Thinker-China aims to incorporate "safety" into every aspect of this project. We have
divided safety into four categories to elaborate on: project design safety, hardware safety, biosecurity, and laboratory
safety.

Our ultimate goal is to prevent and treat colorectal cancer by providing a novel approach. In
this scheme, we generate glucoraphasatinase to convert glucoraphasatin into sulforaphane, and incorporate ferulic acid
to produce curcumin. To ensure the safety of the entire process, we have added a suicide gene system that can express a
suicide gene at 42 degrees Celsius. In the following image, we clearly dissect our project into three distinct parts:

Part1:Why

During the process of our project, we discovered that the commercially available sulforaphane
is mostly synthesized in factories. The industrial synthesis and purification of sulforaphane involve the use of
substances such as ethyl acetate and dichloromethane. Additionally, preservatives need to be added during packaging. All
of these substances are harmful to the human body. As a result, we have decided to explore an alternative method for
producing sulforaphane.

Part2: Choosing a Safer Chassis Microorganism

Before designing the specific project, we need to decide on the chassis microorganism. After
reviewing the literature [1], we have chosen two chassis microorganisms: Escherichia coli and cyanobacteria. Both of
them possess the characteristics of fast replication, simple structure, and convenient genetic manipulation. However,
considering the potential presence of neurotoxins, hepatotoxins, cytotoxins, and highly toxic secondary metabolites such
as microcystins in cyanobacteria, we have opted for Escherichia coli as the chassis microorganism.

Part3：Choosing a Safer Production Method

While searching for relevant information, we discovered that most of the proposed methods
for synthesizing sulforaphane involve its production within the gastrointestinal tract. However, using genetically
modified microorganisms directly in the human body poses certain safety concerns, and it is nearly impossible to
completely prevent the leakage of bacterial colonies. Additionally, many consumers have reservations about ingesting
genetically modified Escherichia coli directly. Therefore, we aim to find a better way to prevent biological leakage
while generating sulforaphane. In exploring other strategies to prevent biological leakage, we came across the concept
of generating sulforaphane ex vivo, which involves designing a device for its production. This approach minimizes the
risk of genetic leakage as much as possible.

Part4： Designing a Suicide Gene System

To further ensure the safety of daily production of sulforaphane and prevent gene leakage,
we have designed a suicide gene system.

a.Screening for Safer Promoters

During the process of screening promoters, we prepared several options, including
tetracycline-inducible promoters, lactose-inducible promoters, and temperature-controlled promoters. In subsequent
studies, we discovered that tetracycline is an antibiotic that can cause harm to the human body when added in large
quantities. Using lactose induction would require the use of IPTG, which may also have potential adverse effects on the
human body. Therefore, we ultimately decided to utilize a temperature-controlled promoter.



We have selected three suitable temperature-controlled promoters: Tclwt, TlpA39, and Tcl42. Our objective was to choose
a promoter that exhibits minimal expression at an appropriate temperature, as the accumulation of the suicide gene could
have adverse effects on the human body. During our literature search, we discovered that the Tcl42 promoter achieves
minimal expression at 37 degrees Celsius while significantly increasing expression at 42 degrees Celsius. Compared to
the other two promoters, Tcl42 demonstrated a more pronounced advantage. Therefore, we ultimately chose Tcl42 as our
promoter of choice.

Note: Quoted from paper [2]



In our experiment, to validate the functionality of the Tcl42 temperature-sensitive promoter, we first fused it upstream
of the mRFP reporter gene and cloned it into the pSB1A3 plasmid. Subsequently, we transformed the recombinant plasmid
into E. coli DH5a. The transformed strains were cultured for 12 hours at both 37 degrees Celsius and 42 degrees Celsius.
Using a spectrophotometer, we measured the initial fluorescence intensity of mRFP at the two temperatures and normalized
it to the OD600 to evaluate the activity of the Tcl42 promoter.

Additionally, in our experiment, we further tested the time dependency of the Tcl42 promoter. At the initial stage, all
samples showed no fluorescence. As time progressed, the samples at 42 degrees Celsius exhibited the fastest growth in
fluorescence intensity due to the activity of the Tcl42 promoter. On the other hand, the samples at 37 degrees Celsius
and 25 degrees Celsius only showed minimal leakage of fluorescence.

b.Screening for Safer Cytotoxic Genes

In the selection of suicide genes, we have prepared two options: the SRRz gene and the mazF
gene. However, during the subsequent investigation, we discovered that the mazF gene is a ribonuclease, raising concerns
about its potential harm to human cells. On the other hand, the SRRz gene enhances cell membrane permeability, while R
and RZ facilitate penetration of the cell wall. Since human cells lack a cell wall, SRRz is virtually harmless to the
human body. Therefore, we have chosen the SRRz gene as the suicide gene to effectively eliminate the bacterial strain
and prevent biohazardous leaks. Additionally, the SRRz gene has laid the foundation for testing the
temperature-sensitive promoter Tcl42 in our experiments.

To evaluate the effectiveness of the Tcl42 promoter in driving the SRRz gene at 42°C, we cultured the samples in a 42°C
shaking incubator for 12 hours. Every 2 hours, we took out 500µL samples and measured their OD600 values to assess
bacterial growth. Under the same conditions, we also cultured DH5α strains carrying only the pTcl42 plasmid (pSB1A3) and
wild-type DH5α as controls. All experiments were performed in triplicate to ensure the reliability of the results.

Part5: Alternatives to Animal Experiments

To avoid animal experiments in project design, we have chosen to conduct cell-based experiments to validate the
effectiveness of our theories. The bacterial strains we have selected are listed in the iGEM Biosafety White List,
minimizing safety risks as much as possible.

Part1:Constructing Safer Devices

Our device is based on enzymatic degradation, requiring the addition of broccoli and ferulic acid each night to obtain
completely harmless beverages containing sulforaphane and curcumin the next day. Our hardware is assembled using
stainless steel, and during the synthesis of sulforaphane and curcumin, only broccoli and engineered bacterial strains
are added to ensure the safety of the entire synthesis process. Additionally, we have designed a suicide system (see
Part 5) and incorporated a biological filter membrane (see Part 6) to ensure the safety of the final product. We were
inspired by the Wego-Taipei team in 2022 (https://2022.igem.wiki/wego-taipei/safety), and believe that by limiting the
bacteria within the hardware container, we can naturally suppress bacterial growth, ensuring both biosafety and
maintaining the efficiency of bacterial functions.'

Detailed description can be found in hardware part: https://2023.igem.wiki/thinker-china/hardware

Part2: Second Layer of Safety

We believe that relying solely on the suicide gene system is not enough to guarantee the
safety of the entire device. To enhance safety, we have added a detachable filtration device at the bottom, consisting
of two layers of membranes. The first layer performs coarse filtration to remove larger molecules like cellulose. The
second layer is a replaceable bacterial filter membrane that filters out substances larger than 0.2 micrometers,
effectively removing any bacteria that did not activate the suicide gene. This ensures that the beverage is free from
live bacteria and can be safely absorbed by the human body.



While we generally believe that the residues pose no danger, we include a biologically safe collection box as an
additional safety measure. After filtering out the residues, consumers can detach the filtration device and transfer the
residues into the provided disposable collection box. Once a certain quantity is accumulated, consumers can contact us,
and we will arrange for professional personnel to properly dispose of the residues.

Part1:Safety seminar

To reduce public fear of synthetic biology and genetically modified products, we have
decided to organize an online lecture and engage more people by distributing flyers.

Part2: Legal Research

After completing the hardware design, we decided to commercialize our device. Therefore, we conducted research on the
legality of selling our hardware to households. In China, genetically engineered strains of Escherichia coli (E. coli)
cannot be directly sold to households. As a result, we decided to sell the metabolic products of the engineered strains
to ensure that the product can be legally and safely sold to households.

 All laboratory members always wear gloves, lab coats, and safety goggles. The laboratory is
equipped with emergency showers, eye wash stations, fire blankets, and first aid kits. If there are hazardous
experiments, they are conducted under supervision. The laboratory is classified as P1, ensuring a relatively safe
environment for experimenters. Our team follows the safety protocols provided by iGEM and has established laboratory
safety management regulations. All personnel involved in the experiments undergo rigorous training prior to conducting
any experiments.

Part1：Safety Committee

a. Purpose：

Considering the need for laboratory management and to prevent experimental accidents, as
well as to ensure that team members can verify the correctness of their own experimental operations, we have decided to
establish a safety committee.

b.Composition and Responsibilities:

The safety committee consists of 2 teachers and 3 team members. The teachers are responsible
for providing training and testing before entering the laboratory, as well as supervising the team members' operations.
The students are responsible for checking laboratory hygiene and ensuring that instruments are returned to their
original positions after use.

Part2: Training

a.Why

Through a questionnaire survey, we assessed the understanding of laboratory safety among our
team members and those from other teams we interacted with. Out of the 50 members we surveyed, only 5 members had a
complete understanding of how to safely operate laboratory equipment before entering the lab. There were 40 members who
had a partial understanding of the correct operating procedures, indicating a significant safety risk.

b.Implementation

We have decided to provide training to all team members through short videos before entering
the laboratory. These videos will help them understand and follow the laboratory safety management regulations. After
the training, a test will be conducted to ensure that everyone can correctly operate the laboratory equipment.

c. Exam Standards

The exam will be conducted on a platform like Google Forms, with a total score of 100. Team
members will only be allowed to enter the laboratory when they achieve a perfect score. To prevent members from
memorizing the questions, the safety committee will prepare four sets of questions, and each time a random set that has
not been used before will be selected. If a team member fails to achieve a perfect score in all four attempts, they will
need to undergo retraining by the teachers from the safety committee, who will create new questions for a retest. The
member can only enter the laboratory after passing the exam.

d．Physical biosecurity

Before entering the laboratory, we require each member to record their name and entry time.
When using instruments, each member should record their name, time of operation, and the nature of the operation in a
logbook nearby. This allows the safety committee members to verify and review the operations and check if any member has
failed to return the instruments to their proper places. When leaving the laboratory, each member should also record
their name and the time of departure. For detailed instructions on operating specific instruments, please refer to the
Instrument Operating Procedures document.

Part3: Safety Management Regulations

Before entering the laboratory, the teachers and students in the safety committee have
collaborated to create a set of safety management regulations. These regulations cover common experimental operations
that can lead to errors, as well as measures to protect the laboratory and operators. Please refer to the uploaded PDF
document for detailed information on the safety management regulations.



Note: Considering the use of ultrasonic disruption of bacteria during experiments, we have prepared headphone-style
sound attenuators in the laboratory to prevent hearing damage to the operators.

Part4: Risk Assessment and Mitigation

Before conducting experiments, we thoroughly assess and evaluate all potential biosafety
risks that may occur during the offline laboratory procedures. We have identified that despite undergoing extensive
safety training, it is possible for team members to forget the correct operational steps during the experiment.

Through communication with other teams, we learned that the RDFZ-China team faced the same problem as us. As a result,
our two teams decided to collaborate on this matter. We started with an online meeting where we exchanged management
advice regarding laboratory safety. After extensive discussions, we formulated a solution.

To address this issue, we have planned the following steps. Firstly, we will affix detailed operating procedures and
slogans on the instruments. Secondly, we have decided to place QR codes on the laboratory instruments. Scanning these QR
codes will allow users to access comics illustrating the operational steps and associated risks, enabling a more
intuitive understanding of the procedures. During this process, we will share our slogans and QR code resources to
simultaneously address the challenges faced by both teams.

Detailed operating procedures

slogans



[1]:ZENG Xiao-Mei, SU Li, LIU Ya-Feng, XIE Shang-Xian. The practice of application and biosafety of classis microbial
cells in the training of innovative undergraduate students[J]. Microbiology China, 2020, 47(4): 1224-1229.

[2]:Abedi M H, Yao M S, Mittelstein D R, et al. Ultrasound-controllable engineered bacteria for cancer immunotherapy[J].
Nature Communications, 2022, 13(1): 1585.