- Wiki
- Project Promotion Video
- Team Presentation
- Judging Form
On this page, we introduce in detail the work of our team members during their participation in the iGEM competition. At the same time, we thank Li Dingchen and Xu Xueying for providing our video with dubbing and text polishing, so that we can better exhibit our achievements. We also thank our PI and the laboratory of Professor Bingjian Zhang for their guidance in the design and experiments of our project.
The protection of cultural relics has always been a social concern, 2022 ZJU-China is dedicated to developing technologies for the integrated conservation of stone artifacts based on engineered Bacillus subtilis induced biomineralization. On the Project Description page, we briefly introduce our inspiration origin: the Asian games and the Linying Temple. We also introduce our conservation method: extracellular calcium carbonate precipitates are brought in for filling the tiny cracks of stone artifacts, with an extracellular scaffold system to further improve the physicochemical properties of the materials. We also add a quorum sensing module and a biosafety module to make our materials environmentally friendly and efficient. Therefore，we believe our project cast light on the synthetic biology application in relics restoration.
For troubleshooting, we validated a method for the chemical transformation of Bacillus subtilis WB600. We
also designed a pair of universal primers for selecting and sequencing exogenous fragments on plasmid pHY300PLK.
For measurement, we measured the activity of firefly luciferase and LRE from BBa_K325210 in vitro experiments. We also built hardware to
reliably detect a concentration of fluorescent protein as well as the oxidation process of D-Luciferin.
For existing part BBa_K4043004, we provide the application
experience on constructing the kill switch. For BBa_K2232000, we provided practical experience in its
application.
We completed a total of 14 iterations in the process of project promotion. In the biosafety module, we conducted three iterations, and finally transformed PsacB into Pveg nx leader RNA spoVG RBS with better induction activity and expression activity. In addition, six iterations were carried out in the carbonic anhydrase module, which finally enabled Bacillus subtilis to obtain the ability to precipitate calcium carbonate with a fixed structure outside the cell. In the hardware module, we have carried out five iterations, and obtained optimum measurement parameters for characterizing luciferase in the process. Finally, we successfully built a multi-channel fluorescence detection device. In all steps, our iteration follows the "design-build-test-learn" process recommended by the iGEM official website.
For Dry Lab, we organized an online modelling workshop with NAU-China and learnt new modelling needs and new ideas from each other. For wet lab, we did inter-lab work with ZJUT-China and GXU-China, validated the function of a few promoters. We also held and participated in several online and onsite meetups including CCiC. This year we have connected to more than 50 teams. We are also invited to mentor high school teams such as LZU-HS-Pro-A, Thinker- Shenzhen in safety and biosecurity.
Our human practice continues from the very beginning of the project. We have fully considered the scientific and academic value, environmental friendly value and ethical value of the project, as well as the public's expectations for the protection of cultural relics. To this end, we conducted an extensive questionnaire survey, interviewed synthetic biologists, environmental scientists, architectural engineers, historians and anthropologists, and determined the feasibility of “Story Light” through field visits to temples and heritage conservation sites. Through continuous summarization and reflections, we constantly update the design and promotion of “Story Light”. In addition, we also interviewed experts from different countries and regions to learn about the status of world cultural heritage protection, and fed back the results to more than 8,000 audiences through the Internet, conveying the concept of synthetic biology restoration of cultural relics.
We focus on the application of our new solution“Story Light” to promote cultural relics restoration and provide a reliable solution for heritage restoration practitioners. Before use, the engineering strains, containing three basic function modules individually, need to be cultured in a liquid medium with 10% sucrose. Together with the thickening agent, we infuse the mix into a syringe and inject it into the target crack, covering a wet dressing to keep it moist and clean. After a few days, the crack would be filled with the newborn mineral. Apart from the basic function modules, we design a toxin-antitoxin system for safety concerns and a quorum sensing module for precise restoration. Besides, “case by case” and customization are of great advantages for our project. Despite some challenges, like high cost and difficulties in colored heritage application, we firmly believe that our project will be able to meet the needs and become the first-line solution for cultural relics restoration in the future.
At the beginning of our project, our communication with synthetic biologists and environmental scientists established the design of Story Light's two core biomineralization modules. Our exchanges with museologists provided ideas for the proposed implementation. Based on the feedback from the questionnaires carried out in our silver medal work, we improved the design of the biosafety module.Through interviews with historians and anthropologists, team members formed a consensus that the project needs to be both scientific and humanistic. In the process of project design, we always adhere to the "authenticity" and "integrity" of the cultural relics, and conduct experimental design on the basis of avoiding secondary damage to the cultural relics. At the beginning of the project, we conducted offline research on the public’s expectations for cultural relics protection.Based on our public survey, expert interview and field research, we carried out our experiment.Finally,through laboratory results, lectures feedback, offline visits and exchanges between teams, we evaluate and further improve our project in scientific, artistic and educational dimension.
For improvement, we completed the job of improving existing parts and creating new parts. For existing part BBa_K4043004, we characterized it and constructed a set of
new parts by replacing its constitutive promoter and multiplied the regulatory sequence.
For BBa_K2232000, we characterized it and created a new part
through carrying out codon optimization for Bacillus subtilis.
We combine microscopic molecular dynamics with macroscopic flow-curing behaviour to simulate and predict the entire process by which the restoration fluid completes its work.
We discretise the hydrodynamic behaviour and incorporate stochastic flow processes, using a cellular automaton to illustrate the hypoxia condition that can be achieved with our restoration fluid; we simulate the workflow of the quorum sensing module using a hybrid model; molecular dynamics simulations based on ODE illustrate the generation of cures; the solidification process is validated by the implementation of a cellular automaton to verify the feasibility of our idea. We made homogeneity assumptions for the restoration fluid in our liquid flow modelling and concluded that density and viscosity were the most important factors influencing the flow of the fluid; most of the parameters in our ODE were obtained from the literature and those not queried illustrate the reliability of our chosen values through sensitivity analysis. The final results of our model illustrate the feasibility of our solution.
In this part, we have developed a proof of concept for our project by fusing the four parts of Lab work, modeling part, implementation and the experts' opinions and suggestions, in order to make it reflect our project as a whole. For the lab work, we do a co-culture experiment to confirm the cooperation of calcium carbonate production module and biological scaffold module as a whole. For the modeling part, we combine microscopic molecular dynamics with macroscopic flow-curing behaviour to simulate and predict the entire process by which the restoration fluid completes its work. For implementation, we try to give our guidelines for how to use it and mention the great advantages of our project are the “case by case” and customization. For experts' opinions and suggestions, they stated that natural mineralization products do exist on some cultural relics, which provides empirical evidence for our project design. At the same time, they also pointed out that there are many problems to be solved in both restoration technology and restoration materials for cultural relics restoration.
This year, ZJU-China built a strong partnership with CAU_China throughout the year. We collaborated from May to October. Our project focused on restoring tiny cracks of stone artifacts, while CAU_China focused on weathering conservation of stone artifacts. Both teams intended to use biomineralization in our projects. We held online meetups and did a lot of inter-lab works to help each other. Since the inspiration of CAU_China is Lingyin Temple located in our city, we helped them contact with a team whose work was the basic of CAU_China’s project. We also led members from CAU_China to do field trips investigating stone artifacts in the wild. On the other hand, CAU_China helped us gather images and analysis of the problems faced by stone artifacts around the world. Moreover, for education, we and CAU_China conducted an online lecture about synthetic biology to more than 200 young students in Beijing.
Before carrying out education and communication activities, our team often conducts brainstorming, preliminary research, planning and other processes, and integrates the feedback from the previous activity into the next brainstorming process. We mainly adopt PBL(Problem based learning) as our education method. We hope that this energetic education method can benefit as much as possible the teenagers and college students who are also energetic. Our team initially determined to use multiple media to transmit knowledge content related to synthetic biology and cultural relics protection, We released many science articles and videos on the social platform. Also, we held a debate contest, propaganda meetings, with souvenirs and promotional leaflets distributed. What’s more, we carried out theoretical knowledge education related to synthetic biology and laboratory education in high school, which obtained a highly degree of certainty.
Our team is aware that young people are the main body of creating the future, but we are also aware that most of our local young students are stuck in the rigid knowledge in textbooks and have little opportunity to contact the forefront of synthetic biology progress. Without understanding, there is no thinking. Therefore, our team contacted local high schools and designed theoretical knowledge learning of synthetic biology for high school students, which was introduced in the form of cases. At the same time, we also carried out laboratory education in local high schools, which made high school students experience the fun of molecular biology experiment PCR. Our educational activities have not only won the praise of teenagers, but also attracted teachers from local high schools to make changes. The teachers decided to improve the conditions of the high school biology laboratory and carry out laboratory experience education on a regular basis. We also promised to carry out science lectures for local high schools on a regular basis.
At the same time, this year is also the first year for our team to establish iGEM community. We started with the people around us, invited the seniors of Zhejiang University who had participated in iGEM competitions to join our community, and then gradually expanded the influence of the community. Up to now, the community has organized one meeting and many online communication activities. We believe that a group of people with the same experience and hobbies can produce some wonderful sparks.
We often use reporter genes (eg. luciferase and fluorescent proteins) in part characterization. Our new hardware (LviSense) is low-cost, multichannel and can sensitively detect concentration of fluorescent proteins as well as the activity of luciferase.
We invited Jerrie (pseudonym), a student majoring in biological sciences, and Prof. Yang to test our device. Based on their feedback, a temperature control module was added to the device, and we used a laser module with better uniformity to replace LED lamp as the excitation light source.
We successfully used LviSense to draw the concentration standard curve of mRFP1 and determine the detection limit. Moreover, we characterized the function of LRE and explored the thermal stability of luciferase.
We open source Ardunio code, blueprints of the circuit board and schematics of LviSense, which makes it easier for other teams to reproduce.
This year, our team is dedicated to breaking the barriers between science, art and ordinary people, and making more people contact and feel the beauty of science and art. We designed simple and easily comprehensible brochures with the theme of cultural relic, arranged regular content output through online channels, and went to community, schools and other places to share our promotional materials. At the same time, in view of the phenomenon that ordinary people are afraid of the scientific field, we decided to take laboratory life as the theme. We recorded vlogs, took interesting theme photos, created poems, and published them on our team's social media, hoping to convey to the public that science is not boring and unreachable as in the stereotype. At the same time, we have considered the needs of gender equality, visual impairment, hearing impairment and other aspects in the presentation form, so that more people can receive the information we want to convey. Finally, we also pay attention to inclusivity in all aspects of the team, whether it is recruitment of members or brainstorming in the later stage.
The feasibility, practicability, ethics and social value of an excellent project all need to be evaluated, and the pursuit of these values is the main concern of our iHP work. We clarify the public's core concerns about the restoration of cultural relics through public surveys and interviews, and build Story Light's biomineralization module and biosafety module through communications with synthetic biologists, cultural relics restoration workers, historians and anthropologists. Through questionnaire and interviews, we were surprised that the public, experts and practitioners all expressed their expectations for Story Light. Professor Li Qiang engaged in the restoration of cultural relics, also expressed his interest in carrying out follow-up laboratory cooperation. The iGEM competition is an outlet for our team to communicate with the outside world. We use "cultural relics restoration" and "Bacillus subtilis" as the key words for inter-team communication and public presentations. Through the Internet, more than 8,000 listeners and more than ten scholars from all over the world appreciated Story Light. Story Light is beyond laboratory work, bridging the gap between science and art, making cultural relics vivid and palpable.
Firefly luciferase is currently being applied as analytical reagents and reporter genes for bioimaging and biosensors. Luciferin-regenerating enzyme (LRE) plays an important role in the recycling of oxyluciferin into luciferin, improving the luminescent signal of firefly luciferase. BBa_K325210 contains the coding sequence for a mutant light-emitting enzyme (luciferase) and luciferin regenerating enzyme (LRE) from the Japanese firefly. To provide more laboratory data for these enzymatic reactions, we measured the activity of these two enzymes in vitro experiments. We also completed kinetics measurements and KM determination for luciferase. We also built hardware to reliably detect concentration of fluorescent protein as well as the oxidation process of D-Luciferin.
We modelled and predicted the entire workflow of the restoration fluid, which was divided into five main parts, namely the flow of the restoration fluid, quorum sensing module, carbonate anhydrase production, biological scaffold part and the solidification process, with each part being related to each other by certain conditions or substances. We have analysed and modelled the problem using fluid dynamics equations, cellular automation, hybrid model, and ODE. We constrain the viscosity and density of the restoration fluid in terms of our engineering objectives and to illustrate the feasibility of our designed genetic pathway through quantitative analysis，which includes consideration of speed limiting steps and prediction of material generation. The model system considers both physical and mathematical models and performs sensitivity analysis on some parameters. Most of the parameters used are taken from references and the final work can illustrate the feasibility of our project.
To ensure the safety and security of our project, we have identified the potential risks from three
perspectives: 1) the risk of leakage of our engineered organisms; 2) the risk during our lab activities; 3)
the risk during implementation.
To avoid the leakage of our engineered bacteria, we have designed a kill switch utilizing a pair of proteins,
mazE and mazF. A sucrose-induced promoter has been employed to activate the kill process. We have also
modified the promoter to improve efficiency.
Lab security is also an important aspect that can’t be neglected. We had to pass a knowledge test about lab
safety and security before we gained access to our lab. Our instructor gave us a lesson to inform us the dos
and don’ts extensively in our lab.
Last but not least, we have also considered the damage our bacteria may bring to stone relics and tried to
reduce the damage as much as possible through special injection methods.
①We use 3C1P model and some basic analysis tools in our Business Plan Part, and we did a good job in market analysis beyond others. ②We have considered ESG, digital foundations, long-term goals, etc. ③Our product focuses on innovative industry, which is different from traditional consumer goods, and we also do well. ④Our solution towards achieving a star business case is impressive. ⑤We combine sustainable resources with other modules, and make it useful for future teams.
BBa_K4202015
BBa_K4202045
BBa_K4202002
BBa_K4202003
BBa_K4202006
BBa_K4202009
BBa_K4202015
BBa_K2232000
This part collection was used for biomineralization experiments with Bacillus subtilis chassis. Among them, BBa_K2232000, BBa_K4202009 are used to accelerate calcium carbonate production and precipitation; BBa_K4202006, BBa_K4202015 are used to construct self-assembled extracellular bio-scaffolds; BBa_K4202002 and BBa_ K4202003 are a pair of fluorescent probes for characterizing the formation of bio-scaffolds. Each of these parts has been well characterized and documented at Part Registry.
