Integrated Human Practice runs throughout the IGEM project. It is essential to developing the lab's internal and external parts. As such, the HP component consists of two main elements - the receipt of professional feedback and the iterative enhancement of the project. To advance this year's IGEM adequately, we went through meticulous planning and thorough interviews with palaeoarchaeological restoration personnel/stakeholders/professional genetic engineering practitioners and ancient architecture experts. To further refine and improve our project, we have invested a great deal of time, energy, and resources in visiting renowned ancient city sites/architectural restoration studios/clay brick factories in China, as well as two of China's top universities, deepening our knowledge of genetic engineering while engaging with industry experts to iterate and improve our project through their feedback.We also understand that our projects need to keep pace with the times, so we have done a series of activities to advance our impact on the world and accumulate resources to promote the future development of our projects.

Idea Source:

While visiting the ancient town of Zhujiajiao in Shanghai with a group, one of our classmates noticed that the roof tiles on these old buildings had small cracks visible to the naked eye after years of wind and rain damage. The deterioration of these culturally rich historical buildings saddened us, as rainwater could seep through the tiny cracks and accelerate the erosion.
After observing the damage to the rooftops in Zhujiajiao, we wondered if this phenomenon occurred in almost all ancient towns. So, our team interviewed people about damage to old buildings in other rain-abundant areas like the gardens of Suzhou and Hangzhou's West Lake. We found damaged buildings there, too. We then went to some inland ancient architecture areas like the Hui-style buildings in Anhui. After touring various old facilities of different sizes, we discovered the deterioration was broadly similar along the coast or deep inland. Upon realizing this phenomenon was quite prevalent, we formally implemented our project.

First, we decided to contact and consult the person in charge of Zhujiajiao Ancient City in Shanghai.

After the interveiw:
Our team learned that most of the roof restoration work in Zhujiajiao Ancient City was in the form of outsourced studios. So we looked for information on the Internet and inquired whether we could find the studio responsible for the restoration of Zhujiajiao Ancient City in the online database. Fortunately, we see in the introduction of Zhujiajiao Ancient City that its restoration work is in charge of the Eastern Ancient Architecture restoration room. So we went to visit the restoration room of Oriental Ancient Buildings and interviewed the staff engaged in roof restoration to understand the pros and cons/effects and costs of the current restoration plan.
Interview with the person in charge of Zhujiajiao Ancient City, Shanghai
Format: Offline, face-to-face consultation
Key points: We asked what methods (programs) are currently being used for the restoration of the roofs of this ancient city.
Response: The person in charge of the restoration program needed to learn. Still, he informed me that an external studio specializing in restoring ancient buildings handled the main restoration work. Still, he needed to find out the full name of the studio.

Oriental Ancient Architecture Restoration Studio

Format: online, videoconference/telephone contact
Key points: Presentation by restoration professionals of the restoration tools they currently use, e.g., clay, knowledge of the existing means of restoration of ancient buildings, thus assessing the feasibility of biologically based restoration
The Details:
-Ms. Chan, a restoration professional, explained in detail the existing methods of repairing old buildings, such as replacing broken, decayed, or missing parts with new clay masonry.
In the interview, they mentioned the limitations of this repair method, such as
-Early techniques may have needed to be recovered.
-Failure to find available materials to make tiles that look similar to the original building.
-Replacing the entire tile for small cracks is not cost-effective
So, we thought of repairing small gaps rather than replacing the whole tile to reduce the impact on the aesthetics of the old building and, at the same time, to enhance the solidity of the structure and follow the guidelines of "repairing the old as the old," and asked the feasibility of the idea of the Oriental Ancient Architecture Restoration Office, The answer was that the concept has the possibility of implementation.

Online Project Seminar

Format: web/telephone chat
Key points: Conceptualisation of possible branches of the project and their feasibility, definition of initial directions.
What we got in response: Initial advancement of human practices while identifying targets for improvement by examining the pros and cons of current roof restoration solutions, brainstorming with teammates, and initially identifying experts to contact to brainstorm ideas.

End of July Offline Meeting

Team members arrived in Shanghai on 1st August. At the follow-up meeting, we met with Mr. Yin Xianqi, a Ph.D. student from the Department of Bioengineering of Shanghai Jiaotong University. We exchanged the pros and cons of remediation technologies and our initial project idea, which was ultimately rejected due to the cost control and biosafety loopholes. Still, the MICP microbial precipitation technology was mentioned during the exchange and was discussed and confirmed to be feasible. Determined the feasibility of its implementation, the team began further research

Format: Personal visit.
Key Points: MICP Biomineralisation Technology
Biotechnology can be used to create an alkaline environment
Core gene: Urease, which breaks down urea into NH and CO2 ➕, combines with carbonate to form calcium carbonate precipitate and creates an alkaline environment
Our response: Using urease to break down urea to produce ammonia and carbonate gas can create an alkaline environment without causing excessive damage to the roofs of ancient buildings. Moreover, CO2 can provide sufficient carbonate ions for the reaction, which will combine with divalent calcium ions in an alkaline environment to form calcium carbonate precipitates and fill in the cracks of the roofs
Details: Dr. Liu pointed out that urease (Urease) is an enzyme that catalyzes the hydrolysis of urea into ammonia and carbon dioxide. It has a high specificity, which makes it widely exist in microorganisms and plant and animal tissues; our project needs NaOH KOH. For creating an alkaline environment, but considering that this kind of solid alkali will cause damage to the cultural relics, the color of the surface of the cultural relics is caused by damage. Urease can be used to create an alkaline environment, using urease to break down urea and produce ammonia and carbonic acid gas, which creates an alkaline environment. Urea will not cause corrosion of the building, while other substances will not cause excessive damage to the artifacts, and carbon dioxide can provide sufficient carbonate ions for this reaction. It can form calcium carbonate precipitation with divalent calcium ions in an alkaline environment and accumulate on cultural relics, which can repair the cracks or cavities in the dinosaurs. At the same time, it can form a protective film on the cultural relics to avoid the relics' erosion by the environment's unfavorable factors, which plays a dual role of repair and protection.

Stakeholder Interviews

Considering the unique tourist nature of ancient towns, we visited Zhujiajiao Ancient Town in Shanghai. We conducted questionnaires and interviews to understand the general public's attitude towards the restoration of ancient buildings. According to the statistics, most people prefer the "old flavor of the old town," i.e., to keep the main body of the old structures as much as possible without seriously affecting the living conditions, and not to replace the facilities if they can be repaired. The people living in the old city also believe that the old roof is the essence of the ancient city, and modern concrete high-rises have less trace of human flavor.

At the same time, we found that there are still some people living in the old buildings; we also conducted interviews with them, asking them whether the damage to the old buildings had affected their lives, and found that most of the people living in the old buildings mentioned the damage to the old roofs, which led to the leakage of rain and the impact on their lives. So, we briefly described our project to these interviewees and asked them whether this type of biological glue to repair the roof would cause them any inconvenience. Statistically, 90% of them answered that it was not an inconvenience. Moreover, this bio-glue dramatically improves the efficiency of restoring old buildings and saves them from leaky roofs
In addition to stakeholders with direct links to old roofs, we also interviewed workers restoring old buildings, who said that most of the ways of repairing old roofs nowadays involve direct replacement of tiles and that they often have to climb up to the tops with heavy tiles when repairing old roofs to replace the tiles. We asked if the restoration could be changed to utilize a bio-based glue, which would need to be applied to the broken area. They responded that this would significantly reduce their workload and the probability of injury. However, it might also result in lower wages and a lower barrier to entry for the job, which would also significantly increase employment and allow more people to have reliable jobs, reducing the poverty rate in the region
We also asked local governments if our technology would damage the ancient buildings and negatively impact cultural heritage preservation or tourism and employment in the region. The answer was that it would not negatively affect any industry because this technology replaced the cumbersome process of replacing roof tiles, significantly improving efficiency while ensuring the buildings' aesthetic appeal and even creating some jobs. Moreover, it effectively preserved the original look of the ancient structures, contributing to the promotion and inheritance of ancient culture. It also injected fresh blood into cultural heritage protection, drawing more attention to the old buildings.

Exclusive interview with Shanghai Brick Factory

Format: Personal visit
Results:
1. Learned the making process of clay bricks
2. Understand the characteristics of clay bricks that are prone to cracking after drying.
3. Got several clay brick samples

Boss Chen, the owner of Shanghai Big Brick Factory, is very knowledgeable in the firing of clay products such as bricks and tiles. Through an interview with he, we learned that the strength grade of clay bricks is divided into MU30, MU25, MU20, MU15, and MU10, with MU30 being the best with an average compressive strength of 30.0 and MU10 being the most common with an average compressive strength of 10.0. The roof tiles of ancient buildings are even weaker due to years of wind and sun exposure. Also, due to the immature firing techniques of the past, their products were rough-surfaced primarily and porous internally, prone to cracking, with high water absorbency that impacted their strength when wet.

The first offline laboratory with biological researchers from Shanghai Jiao Tong University

Form: Offline lab 
Key points: Discussed with researchers and determined the method of using microbial-induced calcite precipitation (MICP) via biological engineering to repair roof tiles of ancient buildings
Details:
- The researchers and we used the decomposition of urea by urease to form calcium carbonate to fill cracks
- Specific methods:
- Commercial synthesis companies synthesize the target genes that control urease synthesis. 
- Urease decomposed the provided urea into NH3 and CO2
- CO2 combined with the provided calcium ions to form calcium carbonate in the alkaline environment created by NH3
- Repaired the cracks between bricks and tiles through calcium carbonate precipitation

Interview with Dr. Xiao Zhihong

In previous urease experiments, we found that the natural outdoor environment could not guarantee the same standard conditions as in the lab (37°C). In actual conditions, the low-temperature setting affected enzyme activity, resulting in inefficient expression. So, we consulted Professor Xiaozhihong on maintaining sufficiently high enzyme activity below 37°C. To solve this issue, Professor Xiao suggested using a cold-inducible promoter, so we redesigned the experiment.
Details: Extracted the cspA promoter from Pcold E.coli and inserted it upstream of the plasmid containing the target gene. Under cold stimulation, more cspA protein can be expressed.
Also, under low-temperature conditions:

The CspA cold shock protein is activated and binds to the cold-inducible promoter to form a complex. This promoter complex recruits RNA polymerase II to the target gene's promoter region. Once RNA polymerase binds to the promoter, it moves along the DNA template strand to synthesize new mRNA molecules. So, by activating the cold-inducible promoter, transcription factors increase RNA polymerase binding to the gene's promoter region, enhancing the transcription process of the target gene.

CspA cold shock protein prevents the formation of stable secondary structures in mRNA resistant to ribonuclease, maintaining the stability of the cellular RNA environment and indirectly ensuring the regular expression of the target gene.

Thus, the mRNA yield of the target gene controlling urease synthesis increases, ultimately leading to more synthesis of the target protein and achieving better expression of the target gene at relatively lower temperatures.

Second interview with Dr. Xiao Zhihong

We resolved the temperature issue through previous discussions and improvements to the experiment. But soon, a new problem arose before us: cracking the clay bricks themselves. Carrying this issue, we knocked on Professor Xiao's door once again. After some discussion, one of our team members proposed an exciting idea - compare the clay bricks to lotus root slices and make them have some stickiness to achieve a connected effect like lotus roots. Very quickly, Professor Qin gave professional opinions on the idea of using biological polysaccharides to increase stickiness. 
Details: The galU gene encoding UDP-glucose pyrophosphorylase is transcribed and translated to form GaiU. Initially, UDP-glucose would become UDP-glucose phosphate, but the yield and production are too low. So we use GaiU to catalyze this reaction, providing energy, and then, through a series of intracellular responses, form UDP-glucose phosphate. The bacteria's endogenous enzymes can convert UDP-glucose phosphate into polysaccharides (EPS). Finally, EPS forms chemical bonds to wrap the objects (clay tiles and calcium carbonate) - this is biological crusting. In summary, the formation of biological crusts involves initial microbial attachment, EPS production, and fixation, as well as interactions and ecological functions of the microbial community. EPS exopolysaccharides are essential in promoting microbial adhesion, stabilization, and symbiosis, forming complex and diverse ecosystems.

Introduction of "exopolysaccharide part"

Form: Offline discussion
Key points: Through discussion with Dr. Xiao, we returned to the Shanghai Jiao Tong University lab to discuss how to increase stickiness to maximize the reaction with biology experts. (Biological polysaccharides increasing stickiness)
Details:
-We told Professor Xiao about the need to increase stickiness and discussed it, eventually introducing extracellular polysaccharides to improve microbial adhesion to soil particles. 
-However, as the experiments and discussions progressed, we found the exopolysaccharides produced by bacteria needed to be improved. So, we improved this by enhancing the metabolic enzymes involved in precursor nucleotide sugar production and cloning the galU gene.

Online interview with Tongji University architecture professor

Key points: Discussed with Professor Liu to determine the final output form of our team's product for ancient building roofs, and asked the professor to point out problems with our current product (paste form/spray) and whether our product has other potential applications as a branch direction for us.
Details:
-We asked the professor what form would adhere to buildings without falling off quickly, and he suggested a paste form applied to buildings.
-He said we could try using this bio-cement to enhance the durability of earthen buildings. Still, we found safety issues needed discussion, so we had a meeting and asked about safety.
-We had another discussion with the professor, and based on his advice, we decided to extend our technology to improve earthen building materials' durability. First, we assessed the feasibility of this project through an output report. Considering the real-world use of earthen building materials and project safety, we devised a solution - mix our bacterial strains with the soil in an enclosed space, then, after the reaction, mix in a high-concentration sodium hypochlorite disinfecting solution to sterilize and prevent strain leakage.

Lunch with PhD students from the Shanghai Jiao Tong University laboratory

I met several doctoral students in the same laboratory by chance in the student cafeteria. I communicated with them about our topic and learned about their experience in participating in the IGEM competition. When we parted, we gave them souvenirs from our team as a souvenir.

Ask the person in charge of Zhujiajiao again:

Form: Offline, face-to-face consultation
Key points: Now that our bio-cement can initiate at room temperature and we've increased toughness to prevent easy cracking, can it be therapeutic for ancient architectural relics in real-world applications?
The response we received: In real situations, they feel our bio-cement repair solution has gone through three rounds of improvement, and they would like to experiment with the final product in real-world tests. Suppose the experimental results are ideal and the cost of using this method is low. In that case, they may arrange for their studio to use our form to restore ancient buildings in Zhujiajiao, achieving cost reduction while promoting our product and solution so more ancient building roofs can be preserved.

Ancient Architecture Restoration Studio:

-Existing methods are limited by technology and materials, with low cost-effectiveness. They believe using our product could significantly improve efficiency and reduce costs.
-Replacing damaged parts with new bricks often causes secondary damage to ancient buildings, increasing restoration difficulty. They feel our product may reduce problems and minimize secondary damage risks.

Tourists:

-Current severe ancient building deterioration and modern material replacement compromise aesthetic appeal, impacting the viewing experience. They believe our product can best preserve aesthetics and the "old flavor of the ancient town."

Brick Factory:

-Different buildings use bricks with varying strength, porosity, and other metrics, making imitation difficult. They think using our product for restoration, rather than imitation, significantly reduces difficulty.

During our research, we discovered a new issue: ancient building rooftops have varied colors, so extensive use of MICP could easily damage aesthetics, preventing our product from achieving the desired effect. Our team will research whether biotechnology solutions can fundamentally address this issue. If bio-solutions are not feasible, we will consider using dyes to resolve aesthetic problems.

Once our product is successfully developed and widely applied, it will not only innovatively improve roof restoration methods for ancient buildings, maximizing efficiency and aesthetics, but the technology can also be expanded to more areas:

-Repairing tiny cracks in other structures like dams

-The biological polysaccharide technology could be applied to greening deserts by increasing sand stickiness to meet plant growth needs

In summary, our product has a wide range of expected future applications and excellent effects, delivering immense social and economic benefits.