iZJU-China

Reflection tool

Throughout the project, we use “The Integrated Reflective Cycle” (1) as an essential reflection tool to reflect on our communication with different stakeholders and to integrate all the feedback from them (Figure 1). The voices of various stakeholders help us prioritize the requirements in designing the product and shape our project. The integrated reflective cycle is a simplified version of Gibb’s reflective cycle, containing fewer steps to make sense of and learn from an experience (2). We adapted the integrated reflective cycle in a way that helped us achieve the best realizations and learnings from the experience throughout the project. The use of such kind of reflection tool was suggested by the iGEM Team TU-Eindhoven 2022, which proved to be extremely helpful in reflecting on the experience and shaping the project. We, therefore, encouraged the future iGEM teams to adopt such kind of tool to better integrate human needs into the biological product design.

Figure 1 The Integrated Reflective Cycle that we used to reflect on our communications with stakeholders and integrate their needs into our project.

The experience

To solve the problem of turpentine's odor, we made investigations from the perspective of the turpentine’s odor, market condition, user group, business, product form, and so on through our stakeholders. According to our demand of investigation, we divided the stakeholders into 3 main types and made subtypes or detailed classifications in each type, which helped us design more targeted and specific consultation to contribute to further understanding of the problem and practical advice. Besides promoting the process of each perspective, using this standard method in each interview provide us with a more efficient analysis of feedback and most importantly integrate all the stakeholders in distinct perspective together to deepen understanding and advancement of the project.

Stakeholders

Effective stakeholder management can provide us with a clear and specific direction of our project. To clarify our stakeholders, we first merged and divided all the stakeholders into three main categories. Then, Mendelow's Matrix was used to identify the gold, silver, and bronze stakeholders.

Communication

Stage 1

Stage2

Stage3

Survey

Part 1: Knowledge contest: Understand the public awareness of synthetic biology and iGEM

1) For pupils: Cooperation with volunteer teaching teams

In cooperation with the volunteer teaching team, our team has brought many courses about the basic knowledge of biology, the fundamentals of synthetic biology and the introduction of iGEM competition to the primary school students in the countryside. In this questionnaire survey, we mainly focus on students' mastery and interest in this new knowledge, hoping to feedback the effect of our teaching through the questionnaire.

The outcome of the questionnaire

Reflection:

In conclusion, through this knowledge competition in cooperation with the volunteer teaching team, we learned that most of the primary school students in rural areas also have enthusiasm and interest in biology, but they may not have much knowledge of this aspect in their usual courses, and they do not have the conditions for doing experiments or making cell models. Through the courses of the volunteer teaching team, they have improved their understanding of biology and synthetic biology and planted the seeds of love for biology in the hearts of children.

2) For students in biomedicine related majors: offline questionnaires

We surveyed undergraduate, graduate, and doctoral students in biomedical related majors about their knowledge of synthetic biology and iGEM competitions through offline questionnaires. In order to achieve a certain educational significance, after completing the questionnaire, we will provide reference answers and ask the participants about their satisfaction with the questionnaire.

The outcome of the questionnaire

Reflection:

In conclusion, we can see from this survey that most biomedical major students have solid professional knowledge. Although interested in iGEM competitions, they don't really know much about them. Through this questionnaire, we conducted science popularization of synthetic biology and iGEM competition in colleges and universities and obtained good feedback.

In combination of these two surveys, we realized that there is still a big gap in public awareness of bacteria and synthetic biology. Thus, we produced a serious of popular science videos, hoping to raise the public awareness in basic biology knowledge and synthetic biology.

Part 2: User survey: Understand what they need and what they want

The target user group of our products is oil painting creators, including oil painting artists, students learning oil painting and oil painting enthusiasts. Therefore, it is important to understand their needs and their suggestions for our products. Based on this, we designed a questionnaire specifically for the potential user group, hoping to understand their demand for removing irritating odors from oil paintings and what factors are important to them in the process of removing irritating odors from oil paintings.

The outcome of the questionnaire

Reflection:

Through this questionnaire, we can see that our potential user group uses turpentine oil more frequently, and most of them think that turpentine oil has a very strong punting odor, indicating that our products are very necessary for them. In addition, when it comes to products, most people rank effectiveness as the most important value, easy to use and safety issue are also important things they value, indicating that when we design products, we need to focus on efficiency and safety.

Part 3: Online survey: Understand the public awareness of bacteria safety and product design

Since our products involve engineered bacteria, and considering that the public may have concerns about the use of bacteria, we have decided to publish a questionnaire online to survey people's awareness of the safety of bacteria and whether they accept products involving bacteria. Our questionnaire aims to understand the relationship between the level of education and the level of awareness of bacterial safety, and to understand the level of awareness of bacterial safety and the acceptance of engineered bacteria in nearby products.

The content of the questionnaire:

We collected a total of 701 questionnaires from 17 provinces in China.

The outcome of the questionnaire

Reflection:

Through this questionnaire, we learned that the higher the education level, the more profound the understanding of bacteria. In China, people generally have a relatively comprehensive understanding of bacteria, but there are also some misunderstandings. People are more hesitant to use equipment containing engineered bacteria in the surrounding environment, which may be related to the lack of awareness of engineered bacteria. Based on the above investigation, our team independently wrote biosafety handbook and published it on the social platform, hoping to contribute to improving the public's awareness of bacterial safety.

Safety concern

Safety is always the thing that needs to be considered.

Why?

Safety concerns hold significant meaning to the public in the context of our project, where we are utilizing engineered E. coli to absorb the pungent odor of turpentine. Beyond our interests, it is essential to emphasize the broader societal implications of prioritizing safety in such endeavors. Public safety is a fundamental ethical responsibility, and safety concerns underscore our commitment to transparency and accountability. In an age of rapid scientific advancements, public trust in scientific research is paramount. Our project's commitment to safety is not only a legal and ethical obligation but also helps promote public awareness of bacteria-related products.

Through volunteer teaching and questionnaires, we have learnt that the public has a strong concern with our project, as we use bacteria in our product. And perhaps in their minds, bacteria are something dangerous and something that needs to be kept away from. Thus, we published our biosafety handbook, telling the public what bacteria are and the function of engineering bacteria, hoping to provide more knowledge for everyone.

What do we do?

Except for our efforts on raising public recognition of bacteria, we also devoted ourselves to creating a better and safer product. After consulting with oil painting artists (eg. Ouyang Donghua), we learned that the first version of our project was unreasonable and safety was not considered, for if we spray the engineering bacteria on canvas it may influence the color of the paintings and the exposure of bacteria also seems dangerous. Hu Jue also provided us with another idea that we can make it more like an air purifier which can absorb the smell in the air. It seems safer for users to avoid direct touch with bacteria. In the whole group discussion, we talked about the feasibility of this new idea and then contacted our PI to discuss specific implementation plans. Finally, we designed and made our final product.

Safety about our project

1. Will the users come into direct contact with E. coli? Will it affect human health?

No, there is no direct contact or touch and it will not affect human health.

In the process of scientific research and industrial production, relevant staff need to wear laboratory coats, disposable gloves and masks, and operate in fume hoods. After using the fume hood, wipe the countertop with alcohol and turn on the UV lamp to kill bacteria. In this way, cross contamination of the experiment and harm to the staff can be avoided to the greatest extent. In the process of user use, according to our product design, the user will not directly contact the filter membrane where the bacteria are located, and the bacteria will not enter the atmosphere, so as to avoid the generation of biosafety problems such as microbial invasion.

The two engineered bacteria we used were both BL21 Star (DE3), which are commonly used in scientific research. Escherichia coli (E. coli), a Gram-negative bacterium, was first named by Dr. Theodor Escherich in 1885. E. coli is mainly found in the intestinal tract of animals, but there are some naturally occurring E. coli strains that are lethal to humans. Escherichia coli has the advantages of whole genome sequencing and a mature gene cloning and expression system and has been approved by the US FDA as a safe genetically engineered recipient organism. Most commonly used commercial E. coli strains are now derived from two isolates, K-12 and strain B. These two strains and their derivatives have been widely used and have had a tremendous impact on the development of basic biology, medicine, and biotechnology. BL21 and its derivatives are the most common examples of strain B and have been widely used for the mass production of recombinant proteins, biofuels, and biorefining. The BL21 Star (DE3) used in this project is one of the BL21 derivatives.

2. Is it safe to replace the filter in the product?

Yes, it is safe.

Changing the membrane does not require contact with bacteria. There is a cover outside the filter membrane, and when replacing the filter membrane and the cover, the whole sealing device is taken out.

3. How do bacteria die? Do the dead bacteria produce substances that are harmful to humans?

It will die naturally, and no harmful substances would exist.

α-Pinene is toxic to E. coli and will cause the death of E. coli after a certain period. In the previous research using BL21 (P450BM-3 QM/GlcDH/GLF) for α-pinene biotransformation, α-pinene was used as the organic phase in the aqueous-organic two-phase system. Experiments found that the production formation rate decreased after 30 min and no product was detected after 1.5 h, suggesting the molecular and phase toxicity effects of α-pinene to E. coli (3). To further improve process productivity, following research explored the effects of different organic phase to reduce adverse effects. In the experiment using DINP as organic phase to dissolve α-pinene, the reaction stopped after 4-5 h (4). In our project, coin oil was used as organic solvent since it has a relatively low NADPH consumption rate. Related experiments are yet to be conducted to test the survival time of the bacteria.

Product design

Aim

Establish a device that can utilize biological methods (engineering bacteria) to degrade α-pinene emitted from turpentine oil in the air.

To solve

Where to put the engineering bacteria?

Overview

Versions of our designs: Turpentine bottle → Color palette → Canvas → Air purifier

Initial thinking: Turpentine bottle

Ideas:

Directly add the engineering bacteria to turpentine and degrade α-pinene in the turpentine bottle.

→ α-pinene does not evaporate which solves the pungent odor problem at its source.

Draft 1: The engineering bacteria were directly added to the turpentine bottle.

Concerns:

1. Through a literature search, we found that Pseudomonas Verona cannot survive in a turpentine environment. The growth curve of Pseudomonas verona at different concentrations of α-pinene showed that Pseudomonas Verona hardly survived when the concentration of α-pinene was higher than 1000mg·L-1, while the concentration of α-pinene in the turpentine bottle was very high, far exceeding 1000mg·L-1. In addition, with the increase of α-pinene concentration, the survival rate of ZW strain and α-pinene degradation rate decreased significantly (5).

In high concentrations of α-pinene, the bacteria will die and may not be able to degrade normally.

2. During the wet lab discussion, it was found that the bacterial growth environment was not suitable for bacteria. The growth of bacteria requires a water-based environment and may not grow normally in oily turpentine.

3. By investigating the background information of turpentine, we found that this product form may affect the role of turpentine (5). α-pinene accounts for up to 80% of turpentine, and degradation before use may affect the effect of turpentine as a thinner for oil painting and affect painting.

Color palette

Ideas:

Idea 1:

Pre-add the engineered bacteria to the palette and start painting. The engineered bacteria will work on the palette to degrade α-pinene, which will degrade when a brush dipped in turpentine touches the palette.

To a certain extent, turpentine can ensure the normal dilution of pigments, and can degrade α-pinene as early as possible.

Draft 2.1: The engineering bacteria were directly added to the color palette.

Idea 2:

When a small device is added to the color palette to place the engineered bacteria in advance, the volatilized α-pinene will float upward and react with the engineered bacteria to degrade.

It can ensure the normal dilution of turpentine to pigment and degrade α-pinene as soon as possible.

Draft 2.2: The engineering bacteria were added in a small device above the color palette.

Concerns:

1. Through communication with the teacher, we realized that the open palette space may lead to the problem of low degradation efficiency. The gas volatilizes and diffuses rapidly in all directions, and the local placement may be exposed to less α-pinene, and the degradation efficiency is low.

2. The engineering teacher pointed out that the mechanical structure of this design is too complicated and difficult to implement. The idea of Idea2 is complicated, and the design of the mechanical part of the product is complicated and difficult to realize.

Canvas

Ideas:

The engineering bacteria are added to the culture solution to make a spray or coating, which is sprayed/coated on the canvas before painting to form a bacterial membrane and degrade the volatilized α-pinene when the brush is in contact with the canvas.

Ensure the normal dilution of turpentine to the paint, increase the contact area of engineering bacteria and αpinene, and improve the degradation efficiency.

Draft 3.1 The engineered bacteria were applied to the canvas.

Draft 3.2 The engineered bacteria were sprayed onto the canvas.

Concerns:

1. During the interview with artists, we found that bacteria grow on the canvas and form plaque which will affect the painting

2. During the interview with artists, we learned that spray/paint bacterial liquid, that is, water phase contact canvas. However, oil painting needs an oily environment, the direct contact between the water phase and canvas will affect the painting. (Also overturns the idea1 palette, where brushes contact with water-based bacteria)

3. From communications with an artist and the surveys we had done, we noticed that the safety issue of bacteria should be considered since we had not verified whether the bacteria may continue to grow, and whether the bacteria itself is harmful to the human body and the environment.

4. During product design group meeting, we thought that oil painting painters need to pretreat the canvas, which is troublesome to operate.

Air purifier

Ideas:

The engineering bacteria were added to the culture medium and placed in an air purifier. Before painting, the device was turned on to absorb the volatilized alpha pinene and release the scented gas.

Ensure the normal dilution of turpentine to the pigment, avoid bacteria and water-based liquid direct contact with the canvas, does not affect the painting, engineering bacteria placed in the container, safety is better guaranteed. The addition of air pump is conducive to the adsorption of α-pinene and the efficiency of degradation is improved.

Please see the Implementation part for detailed information.

Draft 4: The engineering bacteria were put into the air purifier.

Close the loop

With the intention of closing the loop between what we designed and what is desired, we iterated over the design of our product and conducted several interviews with the same stakeholders again to ensure that we have correctly integrated their needs into our product. Through the reflection on the views of different stakeholders, the product iteration, and attempts to solve safety concerns, we finally closed the big loop of our project, from the defined “gap” or problems to the final product which provides the optimal solution.

Throughout the communication with different stakeholders, we also closed several smaller loops in which we found new problems, reflected on our actions to solve them, summarized our solution, and made action plans to implement the solution.

Human Practices are embedded into the entire process of our project, informing our technical, safety, and communication decisions. The reflection tool helps structure feedback loops which shape our project, promote product iteration, and help us find the optimal solution.