Human Practices

1. Introduction

Our team believes in connecting science with the community, aiming to create a more caring society. Human Practice is a way of thinking important for our interaction with society. From brainstorming to now, Human Practice plays a vital role throughout our entire thinking and acting, ensuring our primary initiative and final design are responsible and secure.

This year, we conducted expert interviews, cooperation with other teams, field surveys, visits to biological enterprises, and in-depth collaborations with researchers. Throughout the series of actions, we followed the "problem - discussion - feedback" strategy.

Through these actions, we encountered many individuals and organizations that could provide valuable suggestions for our project. We believe that these cooperators will greatly benefit from our innovation, and we also hope to learn more about the needs of the groups affected by this method. For the good of the society, we are looking forward to apply this research of the black box system to the steps inside the industrial chain.

Our human practice page will take you through our journey of interacting and engaging with people from different backgrounds and professions, as well as how their perspectives, comments and needs have shaped our project into what it is today.

2. Expert interview: Thoughts turn into action

(1) Question

How can we solve many biological questions with a single strategy?
After studying various cases of problems in biology and even more questions derived from the problems in discussing with peers, we are desperate for a key to many different questions. Having researched a large number of synthetic biology topics, we realized that each specific problem has a variety of potential solutions and have indeed been intrigued to help with the progress in facing all of these issues. Therefore, we interviewed Dr. Wu.

(2) Communication

We had a conference online with Dr. Wu from Columbia University Medical School. Dr. Wu is an experienced scientist in the field of synthetic biology, and in response to our confusion, he provided us with the following advice:

1) There could be a universal solution to the problems we encountered.
2) Many of the biological problems stemmed from related genes.
3) By using mutagenesis, we could alter genes taht can address and tackle various conditions.

(3) Feedback

We realized that mutagenesis could be the key our dilemna, as proteins are in fact a universal contributor to so many biological systems. We asked Dr. Wu for papers on the subject of mutagenesis. In the several papers shown to us, we found some crucial points that could contribute to our solution. In the paper “Polymerase-guided base editing enables in vivo mutagenesis and rapid protein engineering,” we learned about the cytosine deaminase, an enzyme that could convert cytosine to thymine. This is the primary thought to induce mutagenesis by this enzyme.

(4) Action

The project is initiated, focusing on the effect of mutagenesis and how to induce mutations. We aimed to use the enzyme CBE to mutate cytosine to uracil and last to thymine.

3. Interview with Mr. Liu, Project Leader at a Biotechnology Company: Insights to the problem

(1) Question

Could the use of CBE (cytosine deaminase) induce controllable mutagenesis?
With the original plan of using CBE, we want to testify the thought with an expert in the area, so we arranged an interview with Mr. Zhenqing Liu, who currently works in a biotechnology company researching the efficient expression of human serum albumin in engineered yeast within the field of synthetic biology.

(2) Communication

We had a discussion with Mr. Liu and asked him to comment on our primary design. In his comment, He points out essential questions about our system:
1) How could we control the mutations we induce and not to mutate essential genes for survival in the organism?
2) How could the system be used in a variety of areas?

(3) Feedback

We researched about the effect of the Lac operon, an operon that synthesized repressor protein to bind to the operator. If Lactose is added, the repressor protein deactivates, initiating the transcription and possibly the mutation process. Apart from the controlled initiation of RNA polymerase, we used the concept of N terminal T7 RNA polymerase and C terminal T7 RNA polymerase from “Evolution of a split RNA polymerase as a versatile biosensor platform” and the concept of n-Mag and p-Mag from “Implementation of a Novel Optogenetic Tool in Mammalian Cells Based on a Split T7 RNA Polymerase” to create control over the time of mutagenesis inside the bacteria.

(4) Action

We fused the cytosine deaminase (CBE) with n-Mag and N-T7 RNA polymerase genes while p-Mag is fused to the C-terminal T7 RNA Polymerase, designing the product which can be controlled by the Lac operon, T7 promoters, as well as the presence of blue light. Adding up to our original plan, we proceed to satisfy as much demands and problems to formulate a universal solution.

4. Interview with BGI Genetics and VectorBuilder researchers: Feasibility study

(1) Question

Could we construct plasmids to help to demonstrate if our concept and action is feasible and could apply to real world end productions?
After Mr. Liu’s interview, we found a way of controllable mutagenesis by using the Lac operon as an initiator and using n-Mag and p-Mag as a time control of mutagenesis. Then, we asked the researchers at both facilities whether it is possible to produce such a plasmid and if they could help to conduct it.

(2) Communication

We interviewed online about controllable mutagenesis, and in the conference, we got valuable information that the production of the plasmid to possible, so we engaged with the firms to discuss about the role of the plasmids in a experiment.

(3) Feedback

Because we want to show the effectiveness for the plasmid, we created the first generation plasmid contained only the Lac operon and CBE, and the second generation plasmid containing both the contents of the first generation but with C terminal and N terminal RNAP with n-Mag and p-Mag as control. Then after the introduction of the plasmids in to a green florescent protein engineered bacteria, we will see the results of the two generation. Also, in order to prove the practicality of the plasmids, we will introduce two experiments containing two separate different plasmid, one for the degradation of nicotine and the other the metabollic process of indirectly transforming arginine to spermidine.

(4) Action

We asked VectorBuilder to produce the two generation plasmids for us. Apart from this, we asked BGI Genetics to produce the two different plasmids involving in the formation of spermidine and the degradation of nicotine to see the effects of controllable mutagenesis. The project proceeds to the next stage of experimentation and practical testing.

5. Project Collaboration with Zhou Lab

(1) Communication

During our project discussion, Professor Zhou highlighted the potential role of spermidine in treating neurodegenerative diseases and suggested that our design could be applicable to arginine metabolism. However, Professor Zhou emphasized two problems:
1) Is there the presence of an arginine-to-spermidine metabolic pathway in Escherichia coli? 2) How to address the containment of genetically modified organisms when using a "black box" system.

(2) Feedback

1) Arginine to Spermidine Metabolic Pathway in E. coli:
Through literature review, we confirmed the existence of an arginine-to-spermidine metabolic pathway in E. coli strain K-12. The pathway is as follows:

• Arginine → Agmatine: Catalyzed by Arginine Decarboxylase (AdiA or SpeA), which decarboxylates arginine to produce agmatine and CO₂. AdiA is active in acidic environments, while SpeA functions at neutral pH.
• Agmatine → Putrescine: Catalyzed by Agmatinase (SpeB), which hydrolyzes agmatine into ornithine and urea.
• Ornithine → Putrescine: Catalyzed by Ornithine Decarboxylase (SpeC or SpeF), converting ornithine to putrescine with the release of CO₂.
• Putrescine → Spermidine: Catalyzed by Spermidine Synthase (SpeE), which combines putrescine with a decarboxylated S-adenosylmethionine (dcSAM) to produce spermidine.

These findings support our strategy to utilize a "black box" system to engineer E. coli strains with higher spermidine yields through targeted genetic mutations.

2) Containment of Genetically Modified Organisms (GMOs):
To prevent the escape of GMOs, a biological containment strategy involving a self-killing system can be implemented. This strategy uses genetic engineering to introduce a "suicide gene" that triggers cell death under specific environmental conditions. In our experimental design, E. coli is engineered with an arabinose-inducible promoter controlling the expression of Cas9 nuclease.

• Absence of Arabinose: The arabinose promoter is repressed by AraC binding to the araI1 and araO2 sites, forming a DNA loop that blocks RNA polymerase binding, preventing Cas9 expression.
• Presence of Arabinose: The arabinose promoter is activated, allowing RNA polymerase to transcribe the Cas9 nuclease. Cas9, guided by specific single-guide RNAs (sgRNAs), targets and cleaves essential genes, resulting in indels that produce nonfunctional proteins. This leads to cell death, effectively preventing the escape of engineered E. coli from the laboratory environment.
By integrating this containment strategy, we aim to mitigate the risks associated with the use of genetically engineered E. coli in our experiments.

(3) Action

After the successful empirical testing of out two generations of plasmids, we proceed to improve our experimental precautions with our bio-engineered product. We added the plasmid containing L-arabinose operon to control the viability of the engineered bacteria to exterminate it if there is a potential contamination. The project, with satisfactory experimental results, is once again modified for universal needs, making the system to surpass on its self, constantly updating its function. This makes our program more adaptive for a myriad of problems.

6. Discussion with various other teams: Collaborate efforts aiming to exploit the potential of the system.

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(1) Communication

We arranged a united conference with various IGEM teams, aiming to use the project to the fullest of its potential for all of us. In the conference we will uniformly think about collaboration between different systems and how to unite them.

(2) Feedback

In the meeting, we introduced our system of controllable mutagenesis to the other teams, and after they introduce their design, we start to think unanimously about the possible utility of the other’s system. After some thinking, the conference as a whole began to share the possibility of the corporation between different systems, and to our controllable mutagenesis, we find two ways to utilize it:

1) Using the black box to mutate the given plasmid of a group aiming to enhance a bacteria’s tolerance to high lactic acid environment and binding to a tumor cell. In this discussion, we offered a chance to mutate the plasmid and select the most successful bacteria, and using the bacteria as a template, we will sequence it and send the results to the other team. this could greatly benefit the other team in the efficiency aspect of the bacterium.

2) Using the black box to mutate and select the most successful promoter for high DNA expression. In this discussion, the other team wants to increase DNA expression, as they want us to select the best promoter for this. We came up with the solution to use our T7 RNAP at the upstream of the plasmid and change the downstream to GFP sequence. Then after the random mutation with T7 RNAP, we could select the brightest bacteria and sequence it, finding the most appropriate promoter sequence.

(3) Action

We asked the first team to send the sequence of the plasmid by mail and negotiated to share the results as team collaboration effort. For the second team, we shared our thoughts with the team leader about the adding of T7 RNAP to the up stream of the promoter and educated them to formulate a plan. The system of controllable mutagenesis is shared and solves many problems inside the other teams’ projects. This could demonstrate our effectiveness of the system and how it changes to be a rather universal solution to many of our peers.

7. Enterprise Visit and Feedback from Shanghai BasalMedia Technologies Co., Ltd.

(1) Communication

During our visit and exchange with Shanghai BasalMedia Technologies Co., Ltd., a company specializing in cell culture medium development, reagent R&D and production, and fundamental research in biotechnology, the founder, Mr. Qiwei Sun, guided us through their culture medium R&D department. He introduced a variety of culture medium products and provided constructive feedback on our experimental designs:

1) Culture Medium for Screening E. coli Capable of Nicotine Metabolism Mr. Sun suggested that the medium used for screening E. coli capable of metabolizing nicotine could be formulated as a nutrient-depleted medium. The commonly used medium for E. coli cultivation is LB medium, composed of peptone, yeast extract, and salts. While nicotine itself, as a toxic agent, can be used as a selective condition to screen for E. coli strains with high nicotine metabolic capacity, this alone might not be sufficient. By using a nutrient-depleted medium—reducing the nutritional content of the medium—we could further screen for E. coli strains that are more adapted to the presence of nicotine. This is because the harsher survival conditions would require the bacteria to enhance their metabolic efficiency to form larger colonies, thereby leading to the selection of more efficient genetic combinations.

2) Culture Medium for Screening E. coli with High Spermidine Production Regarding the medium for screening E. coli strains with higher spermidine production, Mr. Sun pointed out that excessive spermidine production could inhibit the growth of E. coli colonies, which may make this approach unfeasible. Instead, he suggested we focus on establishing a spermidine reporter system rather than using external conditions such as the culture medium for indirect selection.

(2) Feedback

1) Culture Medium for Screening E. coli Capable of Nicotine Metabolism We have adopted Mr. Sun's valuable suggestion. In subsequent experiments, if we observe dense and large colonies under conditions where nicotine is the only selective factor, we will then prepare a nutrient-depleted medium by mixing the culture medium with 50% glycerol or water.

2) On the Infeasibility of the Culture Medium for Screening High Spermidine-Producing E. coli We first reviewed the literature to confirm the effects of spermidine production on E. coli. We found that excessively high concentrations of spermidine lead to cytotoxicity. In response, E. coli converts spermidine into its inactive form, N-acetylspermidine, through the action of spermidine acetyltransferase (SpeG), thereby reducing intracellular spermidine concentration. Another degradation pathway involves the enzyme glutathionylspermidine synthetase/amidase (Gss), which conjugates spermidine with glutathione to form glutathionylspermidine. This not only reduces spermidine concentration but also provides an antioxidant protection mechanism for the cells.

Furthermore, excessive spermidine concentration can inhibit biofilm formation. Biofilms are cell clusters formed by bacteria and other microorganisms on surfaces, encapsulated in a self-secreted sticky matrix composed of polysaccharides, proteins, and DNA, allowing them to grow extensively and form a community. In E. coli strains that produce high levels of spermidine, biofilm formation is inhibited, resulting in smaller colony sizes.

As for Mr. Sun's suggestion of developing a reporter system, we propose utilizing the QS (quorum sensing) system of E. coli to create such a reporter system to implement his recommendation. The QS system is a mechanism by which bacteria coordinate group behavior through the secretion and sensing of signaling molecules. When the bacterial density reaches a certain threshold, the QS system regulates gene expression, controlling behaviors like biofilm formation and toxin secretion. When E. coli intends to form a colony and rapidly proliferate, it activates all genes downstream of the luxS promoter, expressing various materials required for biofilm formation. We plan to introduce a plasmid carrying an mCherry red fluorescent signal downstream of the luxS promoter into E. coli. In this way, as soon as biofilm synthesis begins, the QS system will activate the expression of all genes downstream of the luxS promoter, including the exogenous plasmid's mCherry red fluorescent signal. When selecting colonies, we only need to pick those without the red fluorescent signal, indicating high spermidine production that suppresses biofilm formation and the associated gene expression.

(3) Action

In the final part of adapting the project to suit industrial firms, we adopted some aspect we can improve our project on. First, we uses the adaption of a nutrient poor environment to ensure the concentration of spermidine producing plasmid in a single cell bacterial colony: when we are selecting high spermidine-producing E. coli strains, we can target smaller colonies, as high level of spermidine is indicated by the inhibition of biofilm formation and, consequently, limited colony growth. Next, we modified the bacterial gene for rapid screening adaption by introducing the mCherry plasmid to signify the property of the cell. The adaption for situating the system in a chain inside the industry is the final target for the practicality of our system. We had modified the system for numerouse uses and it all corresponds tot he final target of producing an solution in biology.

8. Implementation of the system in a mushroom farm: a practical suggestion

(1) Communication

After communicating with Mr. Qiewei Sun and refining our physical implementation strategies, we are determined to solve pre-existing problems in the society with our system. To implement societal benefit, we cooperated with Fengyang mushroom farm for a chance to solve a essential issue with their mushroom production.

The following problem proposed by Ms. Qian is about the decrease in crop yield of Morchella due to Morchella white mold disease caused by pathogenic infections. With communication, we find the two essential component of the suggested solution:

1) The final solution must not effect Morchella yield its self.

2) The final solution needs to be a long term prevention of future diseases.

(2) Feedback

We formulated the solution on the wide range of pH that Morchella could thrive on: Morchella could produce significant yields in the pH range of 5.5 to 7.2. with additional information that the pathogen population could be greatly reduced due to the change of optimal pH of 7 to 5.5, we want to formulate the solution of introducing a genetically modified bacteria specie to reduce the environment pH.

(3) Action

We provided a proposal of introducing genetically engineered E. coli that contained antioxidant into the environment to lower the environment pH. With the use of high throughput screening controlled random mutagenesis method, we can determine the most successful gene for E. coli to regulate the pH value. When introducing the whole solution to the facility members at Fengyang mushroom farm, we received instant questioning, but upon discussing with facility members, they believed that with our further down-to-ground cooperation with the facility, the practical application of our system could be implemented.

9. Conclusion

Through the development of controllable mutagenesis, we have learned about various connection from the project to the society as a whole, ranging from medical treatment to bio screening. The question of solving various problems with a adaptive strategy seems to be solved naturally as we listen and alter our projects in to the right direction of controllable mutagenesis. By engaging in human practice, the whole black box system is supported substantially with comments from entrepreneurs to professors, from peers to doctors. The integration of human practice with science is the soul of the project.

We are here to solve the problems and we have solved them, one by one.