Integrated Human Practices Overview

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This year, the NYCU-Taipei iGEM team aims to develop an advanced fundamental tool that provides real-time monitoring of bacterial status. Since the start of our journey, we have been continuously integrating opinions from stakeholders, experts and the public to add upon our initial project design. After gaining feedback from our human practice events, we reflected on potential challenges such as biosafety and the feasibility of real-life application. These reflections enabled us to reposition our core project value and construct a comprehensive end product.

Overall Evolution of Our Project

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The overall evolution of our project consists of five phases and six milestones. We adjusted our original project mission after receiving advice from Dr. Yu-Ling Shih, who specializes in basic microbial physiology. We further validated our project design and ensured our end product after meeting up with molecular genetic professionals Dr. Ting-Jen Cheng and Dr. Wei- Chieh Cheng. Consultation with Prof. Cheng-Yen Kao, whose expertise lies in bacterial pathogenesis and antibiotic resistance mechanisms, inspired us to think beyond boundaries to discover new possibilities for application. We were also encouraged to figure out how our project outperforms pre-existing technologies. After thorough consideration of safety issues, we reorganized our project mission and reached out to potential stakeholders, including fermentation company GeneFerm Biotechnology Co., Ltd. and members of the Taiwan Bacterial Conference to obtain a greater picture of how our product would be implemented in real world situations.

Throughout the process, parts of our wet lab and dry lab design have undergone significant changes. Addition of the CRISPR DNAi device prevents environmental leakage of engineered bacteria; The MCrg validation system was proposed; TEV and HIV-1 protease cleavage system enables observation on fluorescent signal change after addition of degradation tags. Adjustment of YOLOv5m increases model accuracy in object detection; Cloud platform- Heroku provides linkage between Python and our LineBot platform to enable transmission of bacterial information.

We understand that science and technology would never be complete without the integration of humanity. By gathering the advice and opinions from diverse groups of people, the NYCU-Taipei iGEM team seeks to make our project more accessible to both the community and the world.

Project Direction

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Our Original Project Mission

After recognizing the inconvenience users face while monitoring bacterial growth (see Description for more information), we came up with the idea of developing a convenient and effort-saving method for determining microbial growth status. While brainstorming our project, our mission was to strike a balance between “automaticity” and “preciseness” and provide an alternative method that could replace traditional techniques such as OD measurement and culture cell count. Moreover, we expected that our product could be applicable to any bacterial strain and culture condition.

Phase 1: Working Towards Our Project Design

We set off to design our construct after determining our project mission. Since our goal is to enable automatic and remote tracking of bacterial culture, we decided on using light signals emitted from reporter genes as apparent and rapid indicators of different bacterial status. Our dry members thought of installing a camera device inside the incubator to allow automatic detection. Thus, we nailed down the two main compartments of our preliminary design: “growth status reporters” and a “real-time detection device”.

Selection of Growth Status Promoters

While selecting suitable growth status promoters for the expression of reporter genes, we chose those that lead to high mRNA transcript levels to ensure that our reporter signal could be distinguishable. However, Dr. Yu-Ling Shih, an associate research fellow at the Institute of Biological Chemistry in Academia Sinica, mentioned that since our promoters will be transformed into the bacteria as a plasmid form, it is important to notice that promoter strength may not remain the same after it is cloned from genomic DNA. To deal with such uncertainty, we chose at least two promoter candidates for each growth status to enable future comparison. Dr. Shih also reminded us that high copy number plasmids may cause accumulation of toxic substances and thus affect the physiological status of the bacterial host.

TEV Protease Cleavage System for Precise Protein Degradation

Our growth status indicators involve those that point out cell division and specific phases within the bacterial growth curve. Expression of phase-dependent promoters were as expected, but our cell division promoter FtsI did not express specifically during bacterial division. Both Dr. Shih and team HKUST recommended us to apply the TEV protease cleavage system. By inserting a TEV protease cutting site within the fluorescent protein, and combining TEV protease-encoding gene with our cell division promoter, cell division could be timely tracked by loss of reporter signal. We also applied the TEV protease to our growth phase indicators in order to precisely manipulate protein degradation between consecutive phases. For more information, please visit our Design page.

Phase 2: Optimizing and Validating Our Design

Growth status indicators, or “Promoter-reporter pairs” are ligated to one another to form our final plasmid construct, which enables our engineered E. coli DH5α to display different colors according to its growth status. Experiments should be conducted to validate and optimize our design to ensure that our construct works correctly, which is vital for establishing a prototype for our end product.

Confirmation of Fluorescent Expression in “Promoter-Reporter Pairs”

Dr. Shih and Prof. Cheng-Yen Kao from the Institute of Microbiology and Immunology at NYCU gave us advice on the validation of our growth status indicators. Both experts recommended that we test the fluorescent expression of promoter-reporter pairs before combining fragments, and be aware of differences in expression patterns before and after ligation. Therefore, we did extra confirmation to check the fluorescent expression pattern of each promoter-reporter pair as well as the ligated pairs. For more information, please visit our Result page.

Inspiration for the Fluorescent Intensity Amplifier

Our growth status indicators turn out to be visible and could demonstrate color transition between consecutive phases. Although an apparent increase in fluorescent intensity is observed during phase-dependent promoter expression, colony colors show low saturation and require sufficient background light when observed by the naked eye. Dr. Ting-Jen Cheng and Dr. Wei-Chieh Cheng from the Genomic Research Center at Academic Sinica advised us to amplify the fluorescent signal by using an immersive objective under microscope or by applying fluorescent microplate readers for detection. Although we did not apply these techniques in our project, they provoked our interest and led us to the discovery of the fluorescent intensity amplifier developed by Cambridge 2007.

Phase 3: Evaluating Product Feasibility and Prepare for Application

Understanding the strengths, weaknesses and potential stakeholders of our product are vital for the implementation of our real-time growth status detection system in society. Most importantly, we have to evaluate product feasibility and its limitations. At first, we trained our detection device to recognize bacteria cultured on solid plates due to clear distinction between colonies. We further expanded our application to bacterial growth status detection in liquid medium after Prof. Kao mentioned that recombinant protein is more commonly expressed in liquid culture during industrial large-scale fermentation. For more information, please visit our Implementation page.

From Population to Single Cell Level

We reflected on the application range of our product after Prof. Kao provided us with the idea of narrowing the detection scope from a populational level to single cell level. We discovered the potential of our native color indicator to serve as a drug screening tool under single cell level, which enhances the preciseness of present methods* by providing quantitative measurement of live and dead bacteria after a certain drug treatment under a microscope.

Validation Biomarker as Proof of Concept

Both Dr. Jane, our PI, and Prof. Kao suggested that we find a validation method to verify that the expression of each growth phase indicator is credible. The E. coli MCrg strain consisting of fluorescent-labeled ribosomal proteins is used as our proof of concept, which reflects ribosome dynamics that show a phase-dependent pattern. By demonstrating that our growth phase indicators specifically express within the segmented timezones, we can prove that our product is viable for use.

Reorganization of Our Project Values

Prof. Kao also suggested that we think about our project core values. As an expert in microbiology who is experienced in collaborating with hospitals for the practical usage of their study in the clinical and medical fields, he emphasizes the importance of being able to bring “practical impact” to users. We re-examined whether detecting growth status on solid medium is meaningful from a users' perspective. After the consultation, we also abolished our thought of analyzing the percentage of different growth phases in a liquid culture**.

Dr. Ting-Jen Cheng and Dr. Wei-Chieh Cheng also informed us on how we should position our project while compared with other methods. We learned that we had to make a trade-off between “real-time convenience” and “preciseness”. E. color, with the advantage of time-saving and low technical threshold, may not provide results as accurate as that of flow cytometry or qPCR. However, we can view it as an auxiliary tool that assists in other traditional detection methods.

Phase 4: Dealing with Real-Life Challenges

Many challenges remain after the implementation of our project in real-life applications, which include the following:

1. Presence of pre-existing technology:

   Automated plate readers are capable of detecting bacterial growth every 15 minutes without taking the culture out of the incubator, which demonstrates a similar function as E. color. This may pose challenges and affect usage intention of consumers.***

2. Not as precise as international standards:

   One of the applications of our cell division indicator is to serve as an auxiliary tool for antibiotic susceptibility testing. Despite the fact that our indicators can provide a Yes/No answer on whether the antibiotic is effective in killing the bacteria, the CLSI guideline already established a standardized criteria for determining antibiotic susceptibility (S/I/R).

3. Safety issues regarding mixed culture of user and indicator strain:

   After consulting with our industrial partner, GeneFerm Biotechnology Co., Ltd., we acknowledged that the industrial fermentation and purification process is strictly standardized. Therefore, safety precautions must be made to ensure that no contamination occurs between user strain and our indicator strain, e.g. horizontal gene transfer, leak of fluorescent protein. For more information regarding biocontainment measures, please visit our Safety page.

Phase 5: Repositioning of Our Project Mission

After the progress of consultation and reflection, we reconsidered the feasibility and safety of our design, and realized the recomposition our project is required. The goals to developing a convenient and effort-saving method for determining microbial growth status has't changed. However, our initial goals of completely replace traditional techniques seems unable to achieve at the present stage. Our tool provide a faster and convenient way of monitoring the bacterial status, but its preciseness may not surpass current techniques. In addition, the initial thought of “applicable to any bacterial stain and condition” need to be adjusted. The application of our product have to be carefully considerated, for example, the implementation of our product in the current stages are not suitable in the food industry unless we think of a solution. Overall, our project aims to build an fundamental tool, which could assist the current methods, making the measurement more easy, convenient, and time-efficient.

Project Values

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Survey

Background & Proposal

After the overall concept of our project is complete, we would like to hear from potential stakeholders and collect public opinions to understand whether our product is responsible and good for the world. Therefore, we designed a project questionnaire, which was spread out on various platforms in both Mandarin and English versions. The designed survey aims to obtain background information on how bacteria is used in research, understand challenges microbiological researchers face and how they think our project could solve their problems. Valuable feedback from participants assisted us in developing products that are closer to user needs. By placing E. color under a bigger framework and through two-way interaction with potential users, we reassessed and solidified our project values.

Survey Results

Below presents the survey results of respondents who applied bacteria in their research. For more details regarding our survey, please refer to the document below.

1. How do you measure bacterial growth status?

The majority of the respondents measured bacterial growth status by determining OD₆₀₀ value using a spectrophotometer or by applying culture cell count to calculate colony forming units (CFU). We asked whether they have encountered any inconvenience while applying these methods. Almost all respondents that apply OD measurement mentioned potential problems of this method, which include the following:

Some respondents using OD reported that volume of the liquid broth decreases every time they measure the growth curve, which may introduce extra variables to the experiment. Those who apply culture cell count mentioned that this method requires them to wait long periods of time (hours~days) before the formation of visible colonies, and easily introduces contamination of other bacteria while plates are being spread out.

2. Are there any aspects that need improvement?

Next, we asked the respondents whether they think there is room for improvement for present methods used for measuring bacterial growth status. The following lists messages we received from some researchers.

Organizing the information obtained above, we understood that past methods for monitoring bacterial growth still pose disadvantages. Measurement may be imprecise due to presence of dead bacteria within the culture. Moreover, despite the fact that measuring OD₆₀₀ value is convenient because it provides quick measurement of bacteria concentration, it may not be suitable for strains that require special culture conditions.

3. How do you view our project?

Within the questionnaire, we introduced our project design and shared the four main core values with participants. Then, we asked all participants to rate our products on a scale of 1-10 based on the parameters below.

1) Capability of increasing accuracy

2) Capability of saving time

3) Capability of reducing manpower

4) Capability of lowering technical threshold

5) Capability of lowering cost

Conclusion and Reflection

From the survey results, we discovered that present methods for monitoring bacterial growth have their own advantages and downsides. OD measurement is the standard detection method, and is easy, fast and convenient when it comes to measuring the basic level of bacterial growth. Despite its major strengths, researchers mentioned that there are still aspects that require further improvement.

Survey respondents are generally supportive of our products in fulfilling our five project core values. Most of the respondents gave positive comments on E. color for its ability to save manpower and provide real-time visual monitoring, whereas some of the potential users express their concern about the ability of our device to lower cost and technical threshold. This valuable feedback enabled us to rethink which project values we should prioritize, and what adjustments our product should make in order to enhance its implementation success. In this situation, we integrated such advice with safety concerns Geneferm Biotech mentioned, and came up with an alternative approach by serving as an “internal control” instead of incorporating our indicator construct into user bacteria. This relieves the original cost and technical burden and empowers our new design to stick closer to our project values.

After the organization of our survey, we were recommended by the respondents to implement our project in many different fields, including education, basic research, biomedical science, nutrition, clinical medicine, and environment & conservation. These are the main application targets that we strive to work on. We appreciate the encouragement from all of the potential users, which motivated us to keep making our dream come true.

We also learned another lesson— the design of the survey problems is important. The questions of the survey were too hard to answer and lacked the attraction for people to answer. Next time, we should ask questions that are easier for the public to answer.

Human Practices Overview

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This year, the NYCU-Taipei iGEM team aims to develop an advanced fundamental tool for monitoring bacterial status. With the combination of fluorescent growth status indicators and a real-time detect-and-report hardware system, our product provides a convenient and effort-saving method for determining microbial growth status with low technical thresholds and high accessibility. Since tracking microbial status is crucial for many experiments and real-life applications, we hope our product could benefit microbiological researchers and widen our social impact in diverse aspects, including the basic scientific, industrial, and medical fields.

Here, we present the progress of our project, including background information that ignited our research motivation, project goals and solutions, proposed implementation, and efforts our team conducted to ensure that our product could bring practical impact to the community and the world.

Inspiration - Formation of Our Project

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Microbial organisms are commonly-used model organisms owing to their relatively simple and easily manipulable genome, fast replication rate, and strong adaptive capacity to environmental change. Besides its usage in basic scientific research, microorganisms also play a vital role in the development of novel therapeutics, possess high application value in the food and biomanufacturing industry, and provide alternative solutions to environmental issues. Microbial systems also work as one of the major cell factories for recombinant protein expression, producing up to nearly 70% of the 170 recombinant pharmaceuticals used worldwide.

Understanding bacterial growth status is extremely crucial in order to put these microorganisms to good use. Various approaches have been developed to monitor microbial growth, such as OD₆₀₀ measurement, culture cell count, and biochemical assays. Each method has its own advantages and shortcomings, shortages including but not limited to high cost, technical threshold, the requirement of culturing time, manpower, etc. Through conversations with microbiological researchers and the process of literature searching, we noticed a common need for the development of a more time-saving and convenient method.

With the core objective of improving the working environment of microbiological researchers in mind, we further nailed down our project approach to accomplish the following milestones: 1. Save time, manpower, and the possibility of parameter change to bacterial culture 2. Allow remote tracking of bacteria growth 3. Increase accessibility by reducing cost and technical threshold. Accompany by the intention of establishing inclusivity in the research field which enables users from different social, economic, and educational backgrounds to benefit from this foundational advance. We got the idea of designing a monitoring tool that does not require extra manipulation except visual distinguishment between colors. Our project goals include assisting laboratories that could not afford a spectrophotometer or PCR machine and hopes to reduce barriers between age, educational level, and country development status. Moreover, expand the application range of our product to enhance efficiency and productivity in several different fields. This auxiliary tool for traditional monitoring methods could increase the convenience and efficiency of microbial use in basic research and industrial settings, with the hope of bringing impact to the whole scientific community.

E. color - Solution to the Problem

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To meet our goal of “building an advanced fundamental tool that could monitor the bacterial growth status conveniently and automatically”, our project- E. color was born, which contains two main concepts: “Fluorescent growth status indicator” and “Real-time detect-and-report system”

Fluorescent Growth Status Indicator

The reasons we choose to use the fluorescent indicators in our project are based on their advantages of easy observation and obvious differences between colors. The characteristics of easy observation make our project more convenient to monitor the growth status compared to other parameters (e.g., concentration). Since our product is observable through the naked eye and easy to monitor, it lowers the requirement of measuring equipment and reduces the technical threshold. Moreover, with its characteristics of obvious differences between colors, we could indicate different statuses with different colors, making the distinguishment between statuses more obvious and noticeable.

Automatically Detect-and-Report System

The automatic system was designed due to its advantages of reducing time and manpower, better color distinguishment, and real-time tracking. Using this system, the color of the microbial organisms is automatically detected, real-time status is recorded, and relevant information is passed to the users using mobile devices. This way, time is saved and the manual process of measuring is no longer required, lessening the burden of the researchers. What's more, the machine distinguishes differences between colors better than human eyes, which could provide a more precise interpretation of the fluorescent indicator status, further obtaining more accurate information on the microbial status.

(Learn more information on the Design page)

Implementation- Impact on the World

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Since we aim to develop an advanced fundamental tool that could bring impact to the whole society, we wished our potential users are not limited to the researchers in the basic research field but extend to wider fields such as industrial and medical. With the expectation that our design could benefit and make improvements to the current technique.

(Learn more information on the Implementation page)

Human Practice - Works to be Better

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To make sure our project could bring a real impact to the world, we conducted several human practice events throughout the project. By the way of reaching out and communicating with other people, the experimental design and proposed implementation had been constantly modified, and novel ideas pop out in the middle of the discussions. In addition, a number of events were done to share the knowledge of synthetic biology with a broader community. Here, we roughly classify our work of human practice into three categories: Background Research, Collaboration, and Education & Communication.

Background Research

The Background Research category contains works that significantly influenced the formation of our project and proposed implementation. In this category, Experts consultation and Public survey were included. Here we acquired knowledge from the professionals, obtained a deeper understanding of the current situation in different fields, and received advice for our projects. Adjust and rethink our project, we hope to make it better and able to meet the requirements of society.

Collaboration

The Collaboration category contains activities that interact with other iGEMers. Events include the one-to-one discussion with HKUST and WEGO-Taipei iGEM team, and participation in the 2022 Asian Federation Of Biotechnology (AFOB) regional symposium and Taiwan Synbio Alliance, exchanging experience and learning from other excellent iGEMers over Asia and Taiwan.

(Learn more information on the Collaboration page)

Education & Communication

In the Education & Communication category, we have scheduled various activities to share the concept of synthetic biology with a broader community. Many different lectures, activities, and materials were designed in order to fit the audience from various backgrounds and ages. This category includes a five-week internship with Taipei American School (TAS), teaching them some of the wet and dry lab knowledge; Host a lecture about synthetic biology on our campus; Introduction to synthetic biology in our Instagram Posts, and making a video about synthetic biology in indigenous languages (Taiwanese Hokkien/Taiwanese dialect).

(Learn more information on the Education & Communication page)