Collaboration with other team
DUT_China is the very first team we collaborated with. We got to know each other through 2021 China iGEM Online Meetup on May 29th to 30th. We realized that there were other teams trying to use resolve the new pollutants environmental problems. That is where the initial idea of establishing a new pollutants alliance. Beside New Pollutants Alliance. Then we actively engaged in our very first collaboration: writing an article together to advocate for public awareness for the invasion of new pollutants. The article was published on our Wechat official accounts(social media) respectively, June, 5th, 2021 which is the date of the 50th World Environment Day. (Link:https://mp.weixin.qq.com/s/mzCshY8-hH6nMQMzvii0kg) The theme of this year’s World Environment Day is ecosystem restoration, which perfectly matched our theme. Because new pollutants has posed great threat on the ecosystem, and is badly in need of restoration. We wold like to introduce the concept of “new pollutants” to the public and appeal for immediate action.
This year, OUC-China are dovoted to create a whole cell biosensor which have higher sensitivity, lower leakage, better signal-noise ratio and larger dynamic range. Therefore, the selection of components is very important for us. The modeling group needs to accurately evaluate the system through parameter fitting and predict the system performance. We encountered problems in parameter fitting. We turned to XMU-China, which gave us great help in parameter fitting.
OUC-R is our sister team for both of our teams come from Ocean University of China. However, it it the first time for OUC-R to attend iGEM, while our team has been participating in iGEM since 2011. Thus, we generally have more experiences comparing to them. We had several offline meet ups to convey our experiences to them and discuss our projects. Our projects were quite different, but we found we still have a lot to learn from each other.
OUC-R tried to report their signals but finding the traditional reporting proteins are to big for them, because their design was based on short chain RNA. We introduced our part to them which include a RNA reporter 3WJdB which perfectly meet their need of short chian reporter to indicate the transcription of targeted gene. We chose 3WJdB in order to reduce the reaction time, but surprisingly found it can achieve other intentions!
Moreover, taking the advantage of the close geographical location of our two teams, we decided to work on local education together. We contacted a local NGO who bridged us with the local high school students. We initially planed to hold a lab tour and face to face lecture with them, but it changed into online courses instead due to epidemic situation. The online course turned out to be extremely successful, the feedback can prove it(See more detail on our Collaboration with NGO page).
LZU-CHINA’s Project this year is about gene therapy for virus infection. They reached us for OUC-China’s last year(2020) project is related with virus detection. We hold an online meeting gathering LZU-CHINA, NMU-China for these two teams’ project theme is gene therapy, 2020 OUC-China as well as 2021 OUC-China.
After the conversation about virus infection diagnoses and treatment between the gene therapy group and 2020 OUC-China. We surprisingly find that our team and LZU-CHINA both use CRISPR/Cas system to realize our complete different project objectives, one for gene therapy and another for antibiotic detection. We introduced our comic series to them and inviting them to create a thew chapter together with us, and the topic is the magical gene scissors CRISPR/Cas system. Our team created the script and LZU-CHINA drew the comic. On September 27th, 2021, we published the comic on both of our two teams’ We Chat official account. Link: https://mp.weixin.qq.com/s/VIIaPtx0_1o-LD_Rgjy3UQ
Our team and Tongji_Software got to know each other on the CRISPR Conference on September 12th, 2021. We two teams were paired by the sponsor to solve each other’s project problems about CRISPR. We hold an online meeting to introduce each other’s projects and discussed the problems we have met. It is very interesting and fruitful to stand on another team’s perspective to look for solutions. We may came up with several approaches targeting one problem, but found some of them unrealistic or had already been tried after discussing with our partner team. On the CRISPR Conference, we both presented our final solutions for each other after brainstorming, reading literature and discussing.
Since the plasmids have not arrived yet and some plasmid construction problems, we have not carried out the CRISPR module experiments, so we cannot ask very specific questions. But here are some of the questions that might be troubling in the future.
i.How fast does the Cas9 or dCas9 protein take effect? It has been pointed out in some studies that the time required for gRNA binding to Cas protein and guiding it to the target is not short. Therefore, will it affect the reaction time of the gene circuit, and is it suitable for the design of rapid detection? Is there any way to speed it up?
ii.As Cas9 protein is relatively large, how does it burden cells?
iii.What is the actual repression effect of dCas9 protein on the target in prokaryotic system?
iv.What are the advantages of using CRISPR to regulate gene circuits compared to traditional methods such as repressor or Riboswitch?
i.Cas9: RNA complex cutting DNA rate
The graph below shows the RNA complex cutting DNA rate
a.Changing the existing mode of transport is certainly not realistic, so one of the feasible ways is to change the concentration of the substrate, speed up, and, in general, more easily reach a certain amount of encounter and then reaction. Consideration may be given to enhancing the expression of the relevant genes. For example, increase the concentration of guided RNA or protein.
b.The rate of degradation of the original antonymic RNA may be more important than inhibiting the expression of antonymic RNA.
c.Specifically, tests are also needed to see the results.
ii.a. Cas protein 158KDa, green fluorescent protein 28KDa. The difference between the two protein molecular weights is not large. The OUC team wants to use Cas protein to regulate a specific line. Green fluorescence can detect the amount required should be relatively large. So don't worry too much about the burden of engineering cells.
b. The later design of manual detection devices, cell environment can be set.
iii.a. Compared to the characteristics of CRISPR in mammals, which requires the fusion of chromic glycoplasmic syringes with dcas9, the CRISPR system alone can significantly inhibit gene expression in bacteria.
b.If you want to describe specifically, you may be able to measure it with parameters such as dissocation constants.
c.If you want exact data, you may still have to experiment.
v.a. Editability can be achieved by regulating RNA sequences to regulate gene expression. Unlike riboswitch, which requires a variety of substrates to regulate, CRISPR simply changes the RNA sequence. But if you target a target, the advantage is not obvious.
b. The response speed is faster than the inhibitor protein, which is expressed in a fixed way. Synthesis guides RNA when needed, without having to resynthesis to deter proteins coming out.
i.Only using spacer comparison, the detection rate is low;
ii.It is difficult to find the validation set;
iii.At present, the most difficult thing is that some phages are lysogenic. We want to screen them, but there is no very effective tool and database in this regard.
i.Bacterial resistance to phages involves a variety of immune systems, and CRISPR / CAS system plays limited roles. Therefore, it is not comprehensive to predict the interaction between bacteria and phages only based on spacer sequence. In order to make the prediction more comprehensive and reliable, more parameters should be added. Among them, the first and second generation gene editing techniques, namely zinc finger proteins (ZFN) and talens are important immune mechanisms of bacteria.
Secondly, phage-bacteria recognition experiences nonspecific recognition and specific recognition in chronological order. The former is based on random collision, while the latter depends on the recognition mechanism between phage’ surface protein and bacterial cell receptor, and theses proteins correspond to a specific DNA sequences, which may become another comparison parameter.
Moreover, for lysogenic phage, its integration site on the bacterial genome is relatively conservative. As such, lysogenic integration site may be another candidate parameter.
A new question: the bacteria with spacer sequence represents that they can be infected by the corresponding phage, but it also proves that they have a certain resistance to such phage. Therefore, how to evaluate whether the phage is suitable as a candidate for phage therapy? We think a rational edition of the phage candidate might be needed to have better performance.
ii.Practical verification is needed to test the accuracy of database. One of the solutions is to cooperate with the teams with phage related experimental certificate and let the team assist the verification. Or it may work to cooperate with the laboratories exploring phage therapy. Ask them to use the database to select phage candidates to see whether the success rate is improved compared with the randomly selected candidates. However, if you want to verify the accuracy and universality of the database more comprehensively, the amount of data generated by cooperation with several laboratories is obviously not enough. Therefore, it is necessary to widely search the existing experimental data or the established database of phage infection of E. coli based on reality.
iii.Database collection is not our area of expertise, but we have also selected some databases or websites for reference.
a.Actinobacteriophage Database at PhagesDB（https://phagesdb.org/phages/）
The Actinobacteriophage Database at PhagesDB.org is a website that collects and shares data, pictures, protocols, and analysis tools associated with the discovery, sequencing, and characterization of mycobacteriophage—viruses that infect the Mycobacteria and also other bacterial hosts in the phylum Actinobacteria.
This is a database that can simultaneously query and identify CRISPR sequences and Cas genes online, providing valuable data resources and analytical tools for CRISPR-CAS system diversity research. It includes 16,990 complete prokaryotic genomes (16,650 bacteria from 2,973 species and 340 archaea from 300 species). The simultaneous identification of CRISPR sequences and Cas genes and the type and subtype of the system were realized using the CRISPRCasFinder program. And this website built in a variety of query screening and BLAST tools, very convenient.
It’s a knowledge base and web server to comprehensively collect and investigate the knowledge of CRISPR-Cas systems and generate instructive annotations, including CRISPR arrays and Cas protein annotation, CRISPR-Cas system classification, self-targeting events detection, microbe–phage interaction inference, and anti-CRISPR annotation.
d.International Nucleotide Sequence Database Collaboration (INSDC). ISDC is a long-standing foundational initiative that operates between DDBJ, EMBL-EBI and NCBI. INSDC covers the spectrum of data raw reads, through alignments and assemblies to functional annotation, enriched with contextual information relating to samples and experimental configurations. It may be possible to obtain genetic information beyond NCBI alone.
e.Streamlining CRISPR spacer-based bacterial host predictions to decipher the viral dark matter
In this study, the authors created a database of >11 million spacers and a program to execute host predictions on large viral datasets. Addtionally, they evaluated the performance using 9484 phages with known hosts and obtained a recall of 49% and a precision of 69%.
f.the Phage Receptor Database (PhReD)
This is a database of known and newly identified phage receptors. Specific receptors on bacterial surfaces are closely related to phage adsorption, and understanding the mechanisms and influencing factors is also crucial for studying host-phage interactions.
Note: the references mentioned above can be seen in the CRISPR APPLICATION GUIDANCES
ICII stands for “into China, into iGEM”, it was a combination of education and large-scale collaboration. There were totally 17 iGEM teams participated, including us. We walked into local science museums to spread synthetic biology knowledge, introducing ours as well as other teams’ projects. ICII was organized by NAU-CHINA and CPU_CHINA. Please visit their wiki to explore more detail. Here is the link:https://2021.igem.org/Team:CPU_CHINA/Map
The posters presents the brief introduction of the participated teams' project. We printed them out and brought it to the local science museum(you can find it one the photo of the scene below).
We think the organizer of ICII had an brilliant idea because local science museum is probably the best place to reach the children who live science. We immediately contacted the local science museum, namely Qingdao science museum and luckily got a chance providing an offline lecture for the local children(See more detail on our Education page).
Initiator of New Pollutants Alliance
New pollutants refer to chemicals that are not included in routine environmental monitoring, but may enter the environment and cause known or potential negative ecological or health effects, and may become the object of future regulations. At this stage, the main new pollutants of international concern include: environmental endocrine disruptors (EDCs), persistent organic pollutants such as perfluorinated compounds, antibiotics and micro plastics.
Through our previous questionnaire survey, some new pollutants have attracted public attention (such as antibiotics), while some (such as environmental endocrine disruptors) may have relatively low "popularity". Generally, the public does not understand the harm and pollution status of new pollutants. In fact, not only the public, but also the environmental monitoring departments and sewage treatment institutions (new pollutants may enter the environment from water bodies) do not have much involvement in the detection and treatment of new pollutants because new pollutants are not included in conventional environmental monitoring. At present, the detection, harm and degradation of new pollutants mostly stay in research institutions. There are many teams dedicated to promoting and solving environmental pollution in the iGEM team. Compared with the pollutants that have been included in the routine detection, there is still a greater vacuum in the detection and degradation of new pollutants, and it also creates opportunities for the application of synthetic biology. In 2021, we established the new pollutants Alliance (new pollutants alliance), hoping to gather forces to explore solutions to new pollutants from the multi-dimensional aspects of technology, system and culture, and make a better world together.
A.OUC_China：Due to the large scale use of antibiotic on human and animals, the remaining antibiotic in natural water has posed a threat on ecosystem and human health. Our project designed a whole cell biosensor with high performance of detecting antibiotic(taking tetracycline and macrolides as representatives). In order to overcome the restrictions of traditional restrictions of WCBs, we choose a fluorescent activated RNA aptamer(3WJdB) as the output signal to increase response speed.Moreover, we applied NIMPLY logic gate (comprising CRISPRi and strand replacement reaction) to improve signal-noise ratio and dynamic range.
DUT_China：Our work presents a simple strategy to improve PET plastic degradation in river, lake, soil and biosynthetic microbe factories. The first step is to degrade PET in biology method, then we can use the degradation chemical products to produce more useful things.
C.Nanjing_NFLS：The degradation of residual tetracycline antibiotics in the environment by antibiotics is a practical problem related to ecology and human health in recent years. There is an urgent need to develop efficient and convenient tetracycline (aureomycin) antibiotic degradation technology. We found that microbial fuel cells can degrade aureomycin through Fenton reaction by modifying the cathode. At the same time, in order to improve the degradation rate, we located the biofilm of battery anodethe for biofilm activity is closely related to the efficiency of microbial fuel cell. In the process of film formation, the process of bacterial communication through chemical molecules is called quorum sensing. Through over expression of quorum sensing system in electrogenic bacteria, we greatly improved the efficiency of fuel cell, so as to improve the degradation efficiency of aureomycin.
D.HiZJU：17- α- Ethyl estradiol, EE2 for short, is an artificial estrogen with strong estrogenic effect. It is also the main component of short acting contraceptives. The intake of EE2 will affect the endocrine system of fish and human body, and cause a series of diseases, such as feminization of male fish, hysteromyoma, precocious puberty of children, decline of sperm motility and so on. EE2 is mainly concentrated in domestic sewage and industrial sewage. Because there is no effective degradation method, the concentration of EE2 in water increases gradually. Hi ZJU team is committed to degrading and detecting environmental estrogen EE2 by synthetic biological means. We introduced amoA gene and Hao gene from European nitromonas into E. coli to make the latter obtain the ability to metabolize EE2. In addition, we developed a high sensitivity method for the detection of EE2 under natural conditions by yeast two hybrid technology and artificial intelligence assisted protein transformation. Green fluorescent protein was added to the plasmid expression sequence of yeast, so as to visualize the EE2 concentration in wastewater.
E.SJTU-BioX-Shanghai：In the traditional rapid detection technology based on monoclonal antibody, the sandwich method often requires the target to have more than two different antigen epitopes. The competitive method is often cumbersome and has limited sensitivity. Therefore, the types of targets that can be detected by rapid antibody detection have certain limitations. More importantly, if some molecules do not cause the immune response of model animals, the preparation of monoclonal antibodies cannot be completed. At the same time, although the antibody development technology has been relatively mature, the relatively complex experimental operation and the inherent cycle of the immune process make it difficult to greatly optimize the time, human and resource investment required for antibody development. The subsequent protein expression, purification, modification and storage also have many difficulties for the cost is high, and the production and preparation cycle is difficult to be further compressed.Aptamer based detection technology can avoid many of these limitations. Therefore, SJTU BIOX Shanghai team hopes to use aptazyme liquid phase automatic screening technology combined with colloidal gold flow measurement analysis technology to build a platform that can quickly and automatically develop test strips for various new targets. Compared with the traditional immunocolloidal gold test paper, the production cost and production speed of the test paper products developed by this platform are expected to be greatly optimized.SJTU BIOX Shanghai team expects that it will play a great role in emergency response, large-scale multi-target preliminary screening and investigation, etc.
New Pollutants Workshop
We hold a new pollutants alliance online meeting introducing each other and discussing the collaboration form. The first meeting had only 4 iGEM teams participated, for SJTU-BioX-Shanghai joined the New Pollutants Alliance later. We a consensus that besides cross collaboration, it would be a great achievement to hold a new pollutants workshop, inviting experts outside of iGEM, welcoming public to get involved. Since resolving new pollutants is a comprehensive problem, but all of our members were just trying to grope for solutions through synthetic biology. Thus, listening as well as having conversations with people of other fields must be beneficial.
We designed 4 modules for our workshop, which were synthetic biology, rapid test, ecotoxicology and law area. We hope the conversation can include both academic experts as well as experts from company. Thanks to SJTU-BioX-Shanghai, we invited Shanghai Ruixin high-tech private enterprise who produces rapid testing instruments, making it a key stakeholder of the issue the majority of our alliance members engaged in. As for the fields of academic experts, we expected it to include ecotoxicology to help us update the frontier progress of new pollutants and synthetic biology to match the approaches we chose. Professor Wang Baojun, invited by HiZJU, had just started his new career in Zhejinag University(he used to work at the University of Edinburgh), and one of his research directions perfectly fit our concept: develop biosensors by synthetic biology. Besides, he was an experts in gene components and circuit design, making him a qualified teacher providing suggestions for the designs of the alliance members. We felt really glad for we are one of the very first organizations inviting him to give academic report after he came back from University of Edinburgh, England. The professors we invited who studies ecotoxicology came from Ocean University of China and Nanjing University of Technology thanks to the invitation of our team and Nanjing_NFLS. One of the features of new pollutants is as “new”. They are the newcomers to the nature environment due to the development of industrialization. Most of them are not well studied in a lot of aspects, especially its toxicity. However, unless their toxicity is generally well studied can we promote related regulations and laws. Thus, we also invited Students in Ocean University of China law school to join the conversation.
We hope that not only our members, but also the public gain from the workshop. So we made the workshop open to public and recorded it to publish on social media after the permission of the participants. We also made mind maps of the main contents of the workshop, please check it in our Education page.
Agenda of New Pollutants Workshop
Honored guests of New Pollutants Workshop
A.Dr. Wang Baojun
Title: Professor of Zhejiang University, Director of Institute of synthetic biology, Hangzhou International Science and innovation center; Vice president of Institute of biological and molecular intelligent manufacturing
Research direction: Synthetic biology; Gene components and circuit design; Biosensor and biological manufacturing; Biological computing and intelligent diagnosis and treatment.
B.Shanghai Ruixin Co., Ltd.
Shanghai Ruixin Technology Instrument Co., Ltd. is a high-tech private enterprise committed to the development and production of food safety rapid detection instruments, water quality analysis instruments, food safety rapid detection reagents and related software. The products are sold to China's agricultural system, market supervision system, smart market, canteen, supermarket, environmental protection chemical industry and other units, and some products are exported to India, Taiwan, Malaysia and Thailand. Its products have passed ISO9000 quality system certification for 15 consecutive years. More than 500 products have been formed, including pesticide residue rapid tester, multifunctional food safety analyzer, veterinary drug residue detector, grain mycotoxin detector, water quality analyzer, food safety rapid detection box, food safety rapid detection reagent and agricultural products Internet of things traceability system. It has obtained more than 30 national patents and software copyrights.
C.Dr. Zhang Xiaona
Title: Associate professor of Ocean University of China
Research Direction: Endocrine disrupting effect of environmental pollutants on fish and its harm mechanism; Explore the environmental health effects and pathogenesis of new organic pollutants using zebrafish as a model organism; Study on hazard mechanism and health risk assessment of new organic pollutants on shellfish.
D.Dr. Yuan Qingbin
Title: Associate professor of Nanjing University of technology
Research Direction: Environmental behavior and control technology of resistant bacteria and resistance genes; Identification and control of biological macromolecules such as viruses and DNA in the environment; Environmental application of nanotechnology and environmental effects of nano materials; Pollution characteristics and eco-environmental effects of micro plastics.
E.Dr. Wang Jun
Title: Associate professor of Ocean University of China
Research Direction: Research on marine ecotoxicology and environmental remediation, including current situation investigation of marine pollutants, biological toxicity, ecological risk assessment, environmental remediation technology, etc.
F.Dr. He Yide
Title: Associate professor of Nanjing University of technology
Research Direction: Study on the fate and toxicology of new pollutants in the environment
G.Cai Shiyu, Guan Yuezhi, Tang Linrong
Shiyu Cai, Undergraduate in LLB of Law School, OUC & James E. Rogers College of Law, UA
Lezhi Guan, Undergraduate in LLB of Law School, OUC & James E. Rogers College of Law, UA
Linrong Tang, Undergraduate in LLB of Law School, OUC & James E. Rogers College of Law, UA
The feelings and rewards of joining New Pollutants Alliance
As the initiator of the New Pollutants Alliance and the organizer of New Pollutants Workshop, we feel that we are the team harvest the most. We initially focused on antibiotic only. But through the meet ups we attended, we found there are other teams who are also dedicated to new pollutants problem resolving by synthetic biology. We then came up with the idea forming a New Pollutants Alliance. All of our alliance member chose developing new pollutants detecting or degrading approaches, we then face some similar difficulties like visualizing signal reports, improving degradation rate, lowering detection rate and so on. A close alliance can culture cross collaboration, for example we had collaboration with DUT, and developed partnership with Nanjing_NFLS(See more detail on our collaboration and partnership page). Since we got to know each other through meet ups and workshops, we then came up with the idea holding a workshop together. But this time, inviting people outside of iGEM. Because scientific research may be at the forefront of solving social problems such as new pollutant pollution, but it must require the joint efforts of all sectors of society. From laws, enterprise, to public awareness.
The workshop turned out to be very successful. The conversations with synthetic biology inspired the members’ project design, and the conversation with the experts in ecotoxicology enriched every team’s background. There were even further collaborations between our members and the invited experts. We received the email of the participating experts who thanked us for holding this workshop and looked forward to attend more. This is definitely one of the most pleased moments as the organizer of new pollutants workshop. The alliance is temporary, but there must be “latecomers” in the future for there must be more iGEM teams in the future trying to use synthetic biology approaches resolving new pollutants related problems.
Honored to participate in the workshop of the New Pollutants Alliance composed of OUC-China, Nanjing_NFLS, HiZJU, DUT and SJTU-BioX-Shanghai and have a deep understanding of other iGEM team. Our team’s project is plastic degradation, which also includes the degradation of microplastics in new environmental pollutants. Our team’s ultimate goal is degrading environmental pollutants same as the one of OUC-China and other teams. In the meeting，we also learned about other pollutants besides microplastic——persistent organic pollutants, antibiotics, and the need for pollution control. New pollutants may enter the environment and cause known or potential negative ecological or health effects. These substances may become objects of regulatory management in the future. These compounds have attracted widespread attention from all walks of life, and they are also a hot spot in scientific research. We also learned about environmental related laws and testing standards at the meeting, which can help our projects go to the market better. Hope that our team can work together with other iGEM teams and contribute to the improvement of pollutant management and environmental problems.
We are pleased to be part of the New Pollutants Alliance. In previous Human Practices, our team has come to realize the same problems that new pollutants face in our current society: inadequate legislation, incomplete monitoring systems, and toxicology that needs to be studied. The New Pollutants Alliance is an opportunity for us to look at this issue from a holistic and common perspective. During the workshop, our team discussed with experts, iGEMers, and people interested in this issue from the perspectives of research, public policy, and detection systems. This has certainly inspired our own project and deepened our understanding of the issue of novel pollutants.
New pollutants refer to pollutants caused by human activities, which have clearly existed but no relevant laws, regulations and standards have been stipulated or are imperfectly regulated, and endanger life and the ecological environment. The environmental estrogen targeted by HiZJU-China is one of them. During this year’s iGEM trip, we met teams that targeted the degradation of the other new pollutants. We formed a new pollutant alliance, held online meetings to share ideas, and jointly organized a workshop to optimize our projects. The workshop also played a role in disseminating science, which made the relatively unfamiliar concept of new pollutants no longer the elephant in the room, and can enter people's hearts and arouse their attention. To act together for the same goal, I think this is the meaning of the iGEM competition.
Through the initiation of the OUC_China team, we participated in establishing the New Pollutant Alliance and conducted a series of discussions. Through the sharing lecture of other teams, we got a deeper understanding of new environmental pollutants, which greatly enriched our background and supported our idea of taking advantage of our platform to continue to develop new target detection kits.
In the process of participating in the workshop, we had detailed exchanges with teachers, company staff, and team members. Next, all of our projects are discussed, including the legislation issues on environmental pollutants. It is still a long way before the public realize the threatening of new environmental pollutants and regulating them formally, but it’s our duty to keep an eye on new substances. No matter detection or degradation, our goal is to come up with novel ideas and make the realization progress more quickly.
Finally, we jointly expressed our concern about the environment health and our expectation of making a difference to local environment.