Team:OUC-China/Communication
Overview
Communication among OUC-China team members
Our team has always been wanting to create a biosensor. Our two candidate projects were biosensors detecting pathogenic bacteria and urine sugar respectively and both include fascinating ideas. For the pathogenic bacteria detector, we designed it to report the concentration gradient of targeted bacteria by expressing different chromoproteins, green, yellow and red more specifically, that is the reason why we named it “traffic light”. As for the urine sugar detector, we proposed it to be combined with the idea of intelligent furniture. We planed to make the biosensor integrated in the closestool. Every time the the user urinates, the biosensor would automatically measure his urine sugar concentration and report when its is abnormal, enabling a healthier and easier lifestyle. We found both these two concepts intriguing, so we had a team voting, and the urine sugar detection project won.
Communication with PI
Then we elaborated the design and presented it to our PI, professor Chenguang Liu. Our design was highly appreciated by professor Liu, while he provided us pertinent suggestions. He argued that urine blood had already became a routine test index , thus rapid detection methods must be well developed. He recommended us to reconsider whether our design can excel among all kinds of other mature detection methods. We seriously considered professor Liu’s advice, and started a new round of academic paper reading. Then we noticed that antibiotic has been proved to be able to be detected by biosensor, but the design is currently straightforward and conceptional which definitely needs redesign and modification for better performance and manipulation. The more backgrounds of antibiotic we explored, the more excited we became. One one hand, antibiotic contamination has been exaggerated quickly in this decades and surely needed to be monitored and controlled. One the other hand, as one of the most typical environmental new pollutants, it is still lacks of widely applicable detection methods, which calls for brand new approaches.
Despite feeling a little pity, we decided to abandon the idea of intelligent urine sugar detecting closestool. However, since a biosensor is generally composed by 3 segregated models, namely detect module, logic circuit and report module, we only changed the detect module from sugar detection to antibiotic detection but maintained the later two modules.
Communication with other iGEM teams
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.
This year, we built comprehensive collaboration with ZJUT which can be devided into 3 modules: Modle, hardware and wet lab.
Model:
We both built a deterministic model for gene expression and used simbiology for solving the ODEs. Discussions about simbiology were made to.
Since the tetR gene plays an important role in both of our projects, we have collected and shared the biochemical constants of tetR, such as the kinetic rate constants of its binding to tetO.
We shared many useful suggestions in data fitting with them. They also simulated the behavior of our gene circuits in a cell-free system, and the trend of simulation results matched to the literature well.
Hardware:
Both of our teams’ projects are biosensors. And the reporting module is believed to be the key part of a sensor. After discussion, we decided to try different fluorescence detection methods separately. We decided to measure fluorescence by analyzing the RGB(mainly G) value to report the intensity of fluorescence. Besides, we choose to integrate analysis program into a Wechat applet, enabling smartphone users to easily reach it. ZJUT-China decided to build a fluorometer using simple electronic components. After comparing the results, we found that the smartphone based method are pron to detect low density fluorescence, while electronic components have more accuracy on high fluorescence intensity.
Wet Lab:
Our first online communication was hold on July 17th. During the meeting, both parties presented their project principles and designs in detail. Both our team devoted on developing a biosensor. Our reporter was called 3WJdB which was a RNA fluorescent aptamer. This reporter was initially expressed in the Cell-Free system, but we applied it in the whole cell biosensor instead. ZJUT chose cell free system. We then came up with the idea that they can help us to prove the function of our genetic circuit in cell free system.
On the second meeting, which was hold on October 3rd, we discussed the difficulties we had met,. We both found that Cas9 related plasmids have very long constructing circle. This fact made us experimental process stacked. But luckily, the plasmid we chose, namely dCas9 was expected to be constructed earlier than spCas9 which was chosen by ZJUT. After comparing the plasmid map in SnapGene , we found these two plasmids were very similar (Figure 3). Meanwhile, we confirmed the sequence of gRNA, except for the 20nt targeting sequence the rest of the sequence is identical, which means ZJUT can directly replace their spCas9 plasmid with the dCas9 plasmid.
We mailed ZJUT our successfully constructed containing 3WJdB element.Eventually, they commissioned the construction of dcas9 plasmid and also validated the Cell-Free system with dCas9 plasmid. They found that the effect of dCas9 plasmid was higher than that of cas9 plasmid, which might be because dCas9 plasmid was induced by AHL to increase its expression.
What's more, they found that the cell free system was very sensitive to the concentration of antibiotics, which required only 0.5 ng/ml to activate the expression of fluorescence. Interestingly, however, as the antibiotic concentration in the system increased, the fluorescence expression showed a decreasing trend, which was exactly the opposite of our expected, intra-bacterial experiments conducted by our team
Here are the analysis and speculation of this phenomenon: There exists the phenomenon that the gradient dependent fluorescence trend may be different(usually opposite) within different concentration range. We can see from the graph below that the trend in the second testing point(1 ng/µl) has already opposite to our expectation. One possible speculation is that the cell free system is ultra-sensitive, and 1 ng/µl of antibiotic has already exceeded the operating range.We can also see that the fluorescence intensity corresponding to 0ng/µl antibiotic is also 0. This fact indicated that the basal leakage of our genetic circuit is pretty good in cell free system. In the future, we would also like to try cell free system to achieve our goal for it have better basal leakage rate and other adorable attributes such as controllability and safety. And to further prove the concept, we have to design more testing point with lower antibiotic concentration range(at least below 1 ng/µl according to the data ZJUT provided)
Our two teams have very similar topic that is solving the environmental problem of antibiotic contamination. Since iGEM is a competition problem oriented, thus we collaborate a lot on integrated human practice.
1.Interview stakeholders
Thanks to Nanjing_NFLS, we reached Mr.Jiao Yanlin, who is the director of Hengrui pharmaceutical biotechnology company, and had an online interview. The detail of the interview will be showed below.
2.New Pollutants Alliance
We along with other 3 teams who’s projects were also related with new pollutants, mostly detection and degradation, formed New Pollutants Alliance. We hold several online meetings and had cross collaboration. Most importantly, we organized an online workshop named New Pollutants Workshop. The workshop was hold on September 25th, and (See more detail on our Initiator of New Pollutants Alliance page)
3.Mutual proof of content
Our projects are logically connected just like upstream and downstream. The detection results consolidate the necessity of degradation and can also prove the effectiveness of degradation. Nanjing_NFLS mailed their samples to us, which contained a series of antibiotic level. These samples were extracted from their electrolytic cell in various time points, namely the later the water sample were extracted, the lower corresponding antibiotic concentration was expected. What we feel really grateful was that they tested the antibiotic concentration using liquid chromatography, thus the data was very trustworthy. That is to say, we could also test the accuracy of our whole cell biosensor by testing their sample, seeing their samples as standards.
4.Hardware
We also expected to have further collaboration on our hardware. For example, integrating our biosensor in the inlet of their electrolytic cell can report whether the contaminated water is worth antibiotic degradation. And if our biosensor is added on the outlet of their electrolytic cell, it can report whether the treated water is “clean” enough to discharge. However, since our hardware were designed respectively, the physically combination form(connection) still need more work to be down. We hope to have further collaboration in the future.
Entrepreneurship consultation
Both of our two teams see our products can be implemented in real world, having both necessity and possibility. We then wrote our own business plan respectively. Thanks to Nanjing_NFLS, we reached the vice president of Nanjing Wandes Environmental Protection Technology Co., Ltd, who’s name is Yuan Daoying. He read our business plans and provided us precious advice. His comments were also very encouraging!
The business plan below comes from Nanjing_FFLS. Please check ours on our Sustainable page
Communication with public
We designed a questionnaire and distributed on May 8th,2021. We totally received 362 responds, all the information have received the permission of respondents for publishing. The graphs below showed the origins of the respondents(divided by provinces) as well as their backgrounds(divided as whether have biology and medicine related educational or working background ).
Here are main results we can conclude from the questionnaire：
1.Chinese public generally have inadequate knowledge of antibiotic
2.People with biology or medical background know more about antibiotic related knowledge.
3.Public are generally unsatisfied with antibiotic education.
4.Public prefer new medias thantraditional medias for science popularization. School and community are also been expected to shoulder educational responsibility.
Communication with scholars
A.Professor of Ocean University of China: Ru Shaoguo
Professor Ru Shaoguo is a doctoral supervisor, registered environmental impact assessment engineer, editorial board member of Research of Environmental Sciences and Ansian Journal of ecotoxicology, executive director of Shandong ecological society and director of Qingdao ecological society.
Professor Ru Shaoguo’s research Fields: biological screening methods and biomarkers of environmental endocrine disruptors; Endocrine disrupting mechanism and ecological restoration of environmental secretory disruptors; Ecological risk assessment and ecological restoration of persistent organic pollutants.
After listening to a brief introduction of our project, professor Ru showed intense interests on it. He provided us abundant of information and suggestion, promoting our projects in the following aspects.
He firstly recommended us to read the paper of professor Zhu Yongguan, who is a academician famous for studying antibiotic in China. He did a thorough study of the distribution of antibiotic in China in 2013. Thus, reading his work may help us have a overall perception of the contamination status of antibiotic in China.
Professor Ru secondly told us a crucial feature of the usage of antibiotic in China, that is the proportion of antibiotics used for human is larger than that in developed countries. In China, the proportion of antibiotics used for human is almost as much as used for animals. This information helped us firm the idea of developing a biosensor detecting the antibiotics which is usually produced for human to adapt the circumstance in China.
Professor Ru had an abundant of experience contacting with aquaculture farms. After knowing that the antibiotics we planed to detect are erythromycin, chlortetracycline and tetracycline, Professor Ru told us that erythromycin and chlortetracycline are mainly for medical use of human, but they still exist in aquaculture industry due to misuse and illegal use. This information expanded our biosensor’ application scenarios that it not only can be applied on detecting the antibiotic in sewage of pharmaceutical factory, it can also be applied on farm sewage monitoring, serving both farmers and government departments responsible for supervising of water quality.
Professor Ru confirmed our guessing that the possibly being polluted water may exist in the sewage treatment plant, aquaculture pond, poultry and aquafarm wastewater. More specifically, sewage treatment plant may more likely to gather the human used antibiotic, while farm related regions may contain more animal used antibiotic. This information instructed our integrated human practice, for having more specific target people to interview and target places to conduct filed research and sample collecting(See more detail on our consultation with experts, filed research and sample collecting page)
Besides, professor Ru’s previous study showed that the nature water in estuary of Qingdao have considerable antibiotic concentration. This information inspired us to collect some nature water sample. We luckily took the scientific research ship owned by Ocean University of China, Dongfanghong No.2, sailed into Bohai, a continental sea of China, which had been influenced by human activity intensively and collected some water sample(See more detail in our sample collecting page).
Professor Ru kindly introduced us to a local sewage treatment plant for filed research and sample collecting. He also recommended us to collect the water sample in both water inlet and outlet, the comparison of the results may reflect whether the current sewage treating procedure can reduce the concentration of antibiotic. Even though this information may not very useful for our project design, but could be a crucial information for the Nanjing_NFLS, the iGEM team we later built partnership for their project is to develop a device degrading antibiotic(See more detail in our Partnership page).
Professor Ru showed great interests on our project design. He told us that the current method of antibiotic methods are mainly based on chemistry, and all of them need pre-treatment such as extraction. Our aim of developing a whole cell biosensor detecting antibiotic in the original water sample attracted him a lot. Professor Ru’s praise and expectation made us realize the importance of realizing this concept, which promoted us to design a series of experiments testing the activity of our designed whole cell biosensor in all kinds of possibly contaminated water sample. This definitely perfected our proof of concept section(See more detail on our proof of concept page).
Professor Ru also provided us a brain-fresh idea of biosensor. Comparing to chemical based sensors, biosensor can not only realize quantitative analysis just like chemical based sensor, but may also reflect the harm contaminants have on organism which traditional sensors can not achieve. Then we realized that we have met a dilemma: the fluorescence intensity which we designed to reflect the antibiotic concentration may also reflect the lethality antibiotic have on our whole cell biosensor. Because high concentration of antibiotic on one hand can initiate more cells(our projects’ chassis is E.coli) expressing fluorescence gene, on the other hand repressing the growth and replication of cells. Thus, in order to avoid the later circumstance, we have to make sure our whole cell biosensor is successfully inserted the antibiotic resistant gene(See more detail on our proof of concept page).
Professor Ru mentioned to us that different target users may have different requirements on sensor. For example, scientists usually seek for accuracy, while public prefers rapid and visualized reporting form. We didn’t seriously considered our target clients at the beginning stage of our project, but after this conversation with professor Ru, we tried to rethink of our strength and modify our design to meet the specific needs of our target clients(See more detail on our Entrepreneurship page ).
Professor Ru critically pointed out that the key difficulty of biosensor is to transform the chemical based input signal into electronic output signal. Before consulting professor Ru, our project design was ended with different tensity of fluorescence. But after professor Ru explained to us that in order to match the requirements of quantitative analysis, reporting the results by electronic signal is a must. And it is also a good way competing test paper analysis, whose results are usually evaluated by naked eyes, thus more of qualitative than quantitative. Being inspired by professors Ru’s recommendation, we designed our hardware which is a portable box containing the whole cell biosensors. We also developed a mini apps to cooperate with the hardware by analyzing the fluorescence intensity of the photo taken through a whole on the hardware, and reporting the corresponding antibiotic concentration(See more detail on our Hardware page).
Professor Ru told us that China is witnessing a series of policy reducing the usage of antibiotic for both animal and human use. This triggered the idea of having a thorough study of antibiotic related law, hopping to enrich our background knowledge on social level, and possibly provide some modification suggestions to resolve the antibiotic pollution issue beyond synthetic biology(See more detail on our Study and Refinement of Law page).
B.Experts in New Pollutants Workshop
Through New Pollutants Workshop we organized, we communicated with a lot of scholars in synthetic biology, ecotoxicology. Here are the brief introduction of them.
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.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.
c.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.
d.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.
e.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
Here are the mindmaps of New Pollutants Workshop. Hope people viewing our wiki can grasp the highlights of this activity.
Here are part of information and inspirations we got from the workshop:
1.Prof. Yuan Qingbin introduced another aspect of antibiotic resistance risk, that is extracellualr which our team didn’t used to concern. We were surprised at how abundant it is in the environment, and is definitely an inconvenient new pollutants.
2.After knowing one of the main resources of extracellular ARG is released after the death of bacteria, we realized an important fact the widely used safety control such as suicide switch may kill the bacteria and release the antibiotic resistance gene, thus not safe at all. We are then convinced that physical isolation is extremely important and probably is the only safe approach of preventing modified gene releasing into environment.
3.Prof. Yuan’s team have been actively developing smart material(Molecular imprinting technique plus photocatalytic material C3N4) which can bind and degrade specific DNA, making it possible to detect and degrade antibiotic resistant genes. We felt that the wisdom of his team is similar to that of new pollutants alliance members. The only difference was they choose material science while we choose synthetic biology. Especially for our team and Nanjing_NFLS, we all dedicated to resolving the antibiotic related environmental problem. We chose to detect and degrade different sources of the risk, antibiotic and antibiotic resistant gene respectively. Besides, we were so happy to know that prof. Yuan’ s team had already applied their product in sewage treatment plants and received very positive feedback(the removal rate was more than 99.9% after 30 min treatment). We look forward to learn more form the experience of prof. Yuan’s team!
4.Prof.Wang Jun told us that there are two main problems researchers studying microplastic face. One is the not unified methods and standards of isolation and identification of microplastic. There exist urgent need of establishing national wide or even international wide union to set the standards. We also value the the power of union, that is why we initiate New Pollutants Alliance. Unless we gained the participation and support of people in different fields can we reach a consensus and promote problem solving. Another problem is that the commonly used material of studying the ecotoxicity of microplastic is fluorescent microsphere, whose concentration is much larger than that of microplastic in environment, thus can’t reflect(often exaggerate) the real risk human and animals now facing. This fact illuminated us to focus more on the environmental concentration of new pollutants, this will enable us to design more practical products.
5.At the discussion section, Prof. Wang revealed that the degradation of new pollutants now can only be done in sewage treatment plant, because it is unrealistic to apply the technology in ocean. He then appealed that there should be more regulations on plastic using. We realized the limitation of our products, and also see the importance of law refinement.
Prof. He Yide mentioned a tricky problem that dose depended response curve is non-monotonic, making it difficult to determine the hazard threshold.
7.Prof. Wang Baojun introduced a lot of approaches(modules) to realize signal amplification, which was exactly what every team who designs biosensor dream to achieve. We learned several “tricks” and hope to optimize our design in the future. We also discussed the limitation of signal amplification. Prof. Wang reminded us that the output signal intensity is not simple addition signal-amplification-modules. For example, if the expression of house keeping genes are influenced, the signal might decline when more modules were added. Thus, there shall be more trying and testing in order to realize the idealistic design.
8.Prof. Wang’s team combined microfluidic system with biosensor, making it more controllable and safer. What is more, it can show some figures by the micro array wells. He provided us some new ideas of hardware, which we may apply in future.
9.We then discussed the bio safety of biosensor. Prof. Wang told us that besides the genetic safety, physical safety are also important. More importantly, it gave the user the feeling of safety. We then decided to add a filter membrane on our hardware to achieve better physical safety.
10.We discussed the consolidation of cells on paper, which was an approach both our team and prof.Wang’s team had applied. Prof.Wang’s team successfully achieved the concept but our team failed the experiments. We asked him why our cells have poor growth status. Prof.Wang told us that the living cells have higher request on nutrition, that is the reason why they chose cell free system. Prof. Wang suggested us to air dry in nutrient solution, and the addition of water can then revive the cells. This was an extremely good suggestion, we would definitely try in future!
Communication with stakeholders
We luckily interviewed the process Engineer of Haibohe Sewage Treatment Plant, who gave us detailed introduction of the plant. Haibohe Waste Water Treatment Plant The designed water quantity was 200000 tons per day, and actual water quantity was 170000 tons per day, serving 530000citizents who lives in 24km2. The plant follows the Standard for discharge of pollutants from urban sewage treatment plants. The standard includes strict discharge limits of COD(Chemical oxygen demand), ammonia nitrogen, ss(suspended solid), total phosphorus, total nitrogen and so on. But new pollutants such as antibiotic are not included. Engineer Liu believed that the responsibility of degrading antibiotic belongs to the units who produce it such as the drug factory. But if antibiotic contamination be become a common and serious problem one day, and letting the sewage treatment plant shoulder the degrading responsibility become the most effective way, then the government should set the discharge standards, provide the antibiotic detecting and degrading methods.
Mr.Xue firstly admitted that Haibohe Sewage Treatment plant aren’t required to treat antibiotic, thus having no demand of antibiotic detecting. We then asked his opinion on what scenarios he reckon our biosensor may possibly being applied to. Mr.Xue critically pointed out that there would be demands when there were standards. We realized that the sewage treatment plant was a vivid example. People there, from the leader to the engineer, didn’t reckon antibiotic detecting device match the needs of the plant because there was no requirements of degrading.
But sewage treatment plant did care about antibiotic, Mr. Xue told us that before entering anaerobic tank, which is a crucial part of sewage treatment, there might exist the necessity of detecting antibiotic. Because if the sewage with low biodegradability entered the anaerobic pond, the anaerobic bacteria’ activity would be depressed. The so called “low biodegradability sewage” is more commonly seen in pharmaceutical factory sewage than other kinds of sewage because it contains antibiotic that inclusively kill bacteria. If the biodegradability of sewage is too low, the plant might add an extra anaerobic pond to make sure there are enough anaerobic bacteria working. This could be a very suitable case for applying our ALLPASS whole cell biosensor, but Mr. Xue added that biodegradability is a general idea including far more other environmental indexes that influence the living conditions of bacteria, such as pH, salinity, not just antibiotic. This means that a single antibiotic sensor is not enough for deciding whether the sewage can enter the anaerobic pond or not, not to mention the concentration of antibiotic has already been attenuated when enter the municipal sewage treatment plant, which has little effect on bacteria basing on the plants’ former experience.
Mr.Xue told us that the up stream corporations and social organizations have to reach their own standards before discharging into municipal pipeline which leads to the local sewage treatment plants. For example, hospitals have to reach the Discharge standard of water pollutants for medical organization, and farms should meet the Discharge standard of pollutants for livestock and poultry breading. However, we found antibiotic was mentioned in neither of these two standard.
After the conversation with Mr.Xue and Ms.Liu, we deleted hospital and farm from our potential user list. We then focused on pharmaceutical factory. But we know that even though hospital and farm may not the unities who need our antibiotic sensor most at current time, but are still our potential users. Once the standards are published, the needs boom. Besides, if the organization who produces antibiotic have strong environmental protection awareness, a cheap, fast and user-friendly device would be a good choice contamination monitoring.
Talking of antibiotic detection, what firstly came to our mind if the detection of the antibiotic residuals in food. We then consulted the the local Food and Drug Administration officer for the procedure and standards of antibiotic detection. The officer told us that antibiotic concentration was an important index in food safety indeed. And different type of food would be tested of different kinds of antibiotic, for example beef and mutton would be tested for tetracycline, while chicken would be tested for chloramphenicol. We then asked about the detection procedure, and learned the important information that the government would entrust a third-party testing agency to test the concentration of antibiotics. This information instructed us to plan another interview with the third testing company for it is an important stakeholder(See more detail in our Third testing company page).
The officer also told us that antibiotics were not detected in domestic water (tap water), this information complement to what the pharmaceutical factory told us(See more detail on our Pharmaceutical factory page), and expanded our application scenario.
Thanks to Nanjing_NFLS, we reached Mr.Jiao and had an online interview for we live in different cities, Qingdao and Nanjing respectively.Mr. Jiao got his master degree of environmental engineering from Jilin University. From 2010 to 2021, he engaged in operation and maintenance management of sewage, waste gas and solid waste in the environmental protection station of Hengrui pharmaceutical branch company. At present, he is incharge of environmental protection management in Hengrui pharmaceutical biotechnology company.
The two main drugs the drug factory produces are Daptomycin and Caspofungin. The former is antibiotic based drug which targets gram positive bacteria while the later is non antibiotic drugs. That is to say, Hengrui drug factory do has the discourse power of discussing antibiotic contained waste water related issues, especially the antibiotics for human use.
Mr.Jiao told us that every drug factory has its own sewage water treatment system. Drug factories usually have to reach two criteria before emitting into municipal sewage pipeline. One is about some general index, such as chemical oxygen demand (COD), ammonia nitrogen (NH3-N) and so on. Another criteria is the indicators of acute toxicity, called Discharge standards of water pollutants for pharmaceutical industry Chemical synthesis products category, for Hengrui drug factory specifically. After reading the documents, we found that antibiotic was not included as one of the acute toxicity substances.
However, Mr.Jiao told us that the drug factories which mainly produce antibiotic drugs follow another discharge criteria, which is called Discharge standard of water pollutants for pharmaceutical industry Fermentation products category. In this category, the limited proportion of sewage containing antibiotic was regulated.
As required by the Environmental Protection Bureau, a third party is regularly entrusted to test the acute toxicity of sewage. Water Quality-Determination of the Acute Toxicity-Luminescent Bacteria Test and Zebrafish(Danio rerio) eggs method are the common used method. The mechanism is that the acute toxicity of water sample to be tested can influence the luminous intensity and survival rate of luminescent bacteria and zerbafish eggs respectively. But this two methods have to be done by third party testing companies. Thus, Mr.Jiao told us that sewage treatment plants of pharmaceutical factory are looking forward to rapid testing products to detect the toxicity of sewage, but not just antibiotics.
Mr.Jiao emphasized that there is no suitable means to judge the toxicity of sewage to microorganisms at present. Like what Mr.Xue in Haibohe sewage treatment plant had already told us, the sewage treatment process is mainly anaerobic process and aerobic process. The anaerobic microorganisms in the anaerobic process are mainly methanogens, which are particularly sensitive to toxic substances. If the antibiotic concentration is too high, the anaerobic process cannot be functional, and the operation efficiency is very poor. At present, there is no good rapid detection product in China.
Mr.Jiao told us a story that in the first half of the year, a detection instrument in Canada measured the content of active substance ATP in activated sludge(containing anaerobic bacteria) to indicate the toxic substances. More specifically, it is achieved by comparing the amount of ATP in 100ml activated sludge under normal conditions and the change after immersion in toxic sewage. Mr.Jiao was super excited about this products but didn’t buy it for the price is very expensive. As such, Mr.Jiao was very pleased to hear that we are developing a rapid testing products for antibiotic detection with generally low price. He praised our products very useful and meaningful. And he considered it to have great market potential. Because it is impossible for the sewage station of a private pharmaceutical factory to be equipped with a HPLC and hire technicians.
At the end of our interview with Mr.Jiao, he motivated our directions for improvement. He told us that Hengrui pharmaceutical factory used to collaborate with Zhejiang University who screened for high salt and high toxicity tolerant microorganisms , using the bacteria in sewage station of Hengrui pharmaceutical factory as the resource. Mr.Jiao recommended us to consider the attributes of the sewage we planed to test with our whole cell biosensor, such as high salinity and high acute toxicity. Combing with the intention of Zhejiang University research group, we may use a high salt and high toxicity tolerant bacteria as our chassis organism.
Another motivation is another possible application scenario: waterworks. Mr.Jiao told us that the duality of antibiotics: are the inhibition of activated sludge bacteria and the negative influence on the drinking water when flow to natural water. Drinking water containing antibiotics is a risk to human health, thus detecting the concentration of antibiotic in waterworks is meaningful.
SGS is an internationally recognized inspection, identification, testing and certification organization. It is a globally recognized benchmark of quality and integrity. More than 93000 employees operate more than 2600 branches and laboratories around the world. Its core services can be divided into the following four categories: inspection, testing, certification and identification.
SGS, formerly known as the French grain shipment inspection institute, was established in Rouen in 1878. In 1919, the company was registered in Geneva and named Société Générale de Surveillance.
SGS vision: Their goal is to become the most competitive and productive service organization in the world. They are constantly improving and perfecting our inspection, identification, testing and certification services, and always provide first-class services for local and global customers.
We were luckily welcomed by a head engineer of SGS, and visited the chemical laboratories in its Qingdao branch office. We got to know that there are strict standards even for different parts of animal, for example the skin and muscle. So the food to be tested have to be separated and crushed, and this procedure was called sample making. SGS had intellectualization control experiment system, their food weighting results would be sent to a database automatically, making us realize that the quantification and automation can make the testing more effective and trustworthy.
Most of the test items SGS conducted are basing on GB(national standard), thus the accuracy was important. There was a strict and a little bit complicated procedure of testing.
Firstly, sufficient extraction is required to avoid false negative and false positive. After adding extractant, oscillation, homogenizer and ultrasonic can all effectively extract the target. Secondly, the testing material have to be purified in order to remov non-specific extracted interferents. There were a lot of approaches such as extraction, adding purification powder and SPE column. The third step was concentration. The method include “nitrogen blowing” and rotary evaporation concentration. The last step of pre-treatment was determination, which means settling the volume, which was usually 1 ml. Then comes to the testing section. The principle of LC-MS is to separate the substance to be measured from the impurities first, and then use mass spectrometry. It is a very selective means according to the qualitative and quantitative of ion peaks. Test the standard, empty sample and sample in turn. The final concentration is obtained by multiplying the machine reading by the constant volume and dividing by the weight. The engineer in SGS told us, the better the specificity of the detection instrument, the less pre-treatment is required. We realized that one of our products’ advantage was there need no pre-treatment for it has high specificity. We also proved that our whole cell biosensor can work effectively in open sea water(See more detail on our proof of concept page).
There was an important procedure, that is to add interior label and external labels. The labels were usually made by changing the protium intodeuterium, which would not change the properties but distinguishable. The interior label was added at the very beginning link: the target extraction, while the external label was added in the very last link: testing. This way could detect the influence of interferents and correct sample loss. The sample loss could be really high in some cases, such as 80%. Thus, if there was no interior and external label, the real concentration could not be detected with accuracy. This method inspired us so much, that we deeply understand the importance of controlling.
The engineer in SGS told us that the current concentration of antibiotic in environment is on the level of µg/kg. Thus, if our product can’t reach this detection limit, our results would be all negative. We felt thankful for this advice.
We visited an aquaculture farm named Heshengyuan, which is located in Qingdao district. The boss of this private enterprise Sun Zhangde warmly welcomed us. Heshengyuan aquaculture farm mainly co-cultures sea cucumber seedlings and shrimp. The farm produced 178000 cubic meters of tail water a day. The antibiotic is used in the seedling pond only for it was uneconomic to apply antibiotic in culture pond with much more water. At present, the price of antibiotics was more about 100 to 200 yuan per kilogram. A Small and medium-sized farms like Heshengyuan would spent at least 100000 yuan a year on buying antibiotics. The antibiotic the farm used include neomycin sulfate, florfenicol and so on. The choice of antibiotic were made according to the regulations on the use of antibiotics in aquaculture. It is a common knowledge that the antibiotic legal using are the ones can be degraded quickly(usually 24 or 48h). Mr.Sun told us the list of antibiotic in the regulation has the trend of shrinking. The antibiotics which this farm used before such as Penicillin, oxytetracycline and furans are all banned now.
The local government made some other actions. From this year on, the Environmental Protection Bureau began to set the aquaculture wastewater discharge standard, namely 3ppm for legally used antibiotic. The Fisheries Bureau and Ocean Bureau had held a meeting to issue standards to the private aquaculture farms. The farms were informed to reform sewage treating methods, for example installing protein filter, and get ready for the spot check.
We asked the usage of antibiotic in detail. Mr.Sun told us that the injured sea cucumbers were treated with antibiotics when changing pools. The use of antibiotics on the farm has decreased by 50% in the past two years. These years, China as well as the world has witnessed a decreasing trend of antibiotics usage, and the national requirements are becoming more and more strict. Nowadays, there are many antibiotic substitutes, for example applying probiotic spores, making it dominant bacteria to inhibit pathogenic bacteria, antimicrobial peptides and chitosan can to improve immunity. However, even the cost of probiotics is lower than that of antibiotics, the effect was slow to see. Besides, probiotics mainly focus on prevention of diseases, while antibiotics mainly focus on treatment. Especially when there were burst of large scale disease, antibiotics were required immediately. In conclusion, antibiotic are now still irreplaceable in some circumstances. The vaccine against shrimp disease has made rapid progress in China in the past two years.
We then asked whether the farm are interested in biosensor for antibiotic detection. Mr.Sun expressed his interests, but added that he would only prefer it when the cost is not very high. After knowing more detail of our products’ design, he was surprised at that our product can detect specific antibiotic. Even though our biosensor can only detect 3 antibiotic currently, he suggested us to extend it into a platform wich can detect a series of antibiotic, exploring a bigger market. We realized that the request of farm is different from that of pharmaceutical factory. Pharmaceutical factory wants something has broad spectrum detection ability which can indicate the general toxicity of sewage water, while farm wants the product to be sensitive to a specific antibiotic only.
At the end of the interview, we mentioned that antibiotic was probably a sensitive topic for farm, for the customers concerned a lot. But surprisingly, Mr.Sun believed that antibiotic related problem was unavoidable facing. Strictly following the regulations giving his confidence facing the customers and government honestly.
Communication with children and young teenagers
This year, we actively conducted a series of educational activities. People of different ages and regions have been reached.
Lab tour
Taking the advantage of the summer camp Ocean University of China organized for the high school students from all over China, we invited them to have an offline lab tour in our iGEM lab. We showed them around the lab environment we daily work in at great length to the students who mostly have intense interests on biology and planing to choose biology as their future major in college.
We are glad that we introduce them the basic molecular biology and microorganism experiments and the corresponding equipments. We demonstrated to them how to correctly use the clean bench doing bacteria related experiments, explained to them the significance of the invention PCR, introduced to them how the electrophoresis apparatus works. The students showed great interests on every corner of the lab because everything is so fresh to them. They also asked some questions about iGEM and our current projects. We then introduced some principle ideas of synthetic biology, and found them generally had solid basic knowledge of biology which surprised us a lot.
We believe that an offline lab tour is the most straight forward way of arousing interests of students. But unluckily, the later education activity designed for high school students are online due to pandemic(See more detail on our Collaboration with NGO page).
For Children in Underdeveloped Area
Lvchun county, Yunnan province, used to be one of the national poor counties in Yunnan Province in China. In January 2014, Chinese government launched the "targeted poverty alleviation" project. A lot of universities in China was paired with specific poor counties in order to backup the local industry and local education. Ocean University of China, the university we OUC-China belongs to, was paired with Lvchun county. One of the programs conducted successfully was a long term supportive education program. Every year, a teaching team composed by some graduate students with various knowledge background went to lvvhun county for a one-year teaching journey. Once their job completed, there would be another teaching team arrive to replace them making it a long-term activity. OUC-China 2020 was the first iGEM team reached the teaching team for collaboration. Due to the epidemic condition on 2020, they chose to conduct online course which turned out to be nice. We also believe that one of the key point of education is its continuity. So this year, we reached out the 2021 teaching team in Lvchun and received warm welcome. The leader of the teaching team Mingyu Wang recommended that we may build a long-term collaboration. Since Qingdao where Ocean University of China locates and Lvchun have thousands miles’ distance, the best way of collaboration is that we provide lectures on line and they help us organize the students in the real classroom. After participating in the activity in person, we are amazed at how dramatically internet access have grown in China. The lecture slides and video went smooth without any lagging or blur, which allowed we to have great interaction with the local children almost like face to face.
On May 16th,2020, the People's Government of Yunnan Province announced that Lvchun county had finally be lifted out of poverty after more than 6 years’ multiple efforts. The name Lvchun in Chinese means greenish spring. We all hope that Lvchun county had already welcomed its new era just its Chinese name indicates. And even though it had already got rid of poverty, its education is still been lag behind the urban area of China. For this reason, we hope our iGEM team along with the supportive teaching group would continuously work together to promote the local education of Lvchun, making our effort to bring them a brighter future!
The online course was well organized thanks to the supportive teaching group. They had projection of the live video on a big screen so that every children can see the slides we prepared. When we raised a question, the co-teacher would unmute the microphone and bring the computer to the children who would like to answer. At first, the local teacher reckoned synthetic biology must be too difficult for children in this age, but we insisted this topic and promised to make it understandable. We made an analogy that the intention synthetic biology is to create a “little goblin” who is extremely hardworking and loyal, just like the scenario in Harry Potter. He may help us degrade toxic pollutants, produce fuel, diagnose diseases and so on. But nowadays, they are bacteria like. The results turned out to be very good. When we discussed the boundary of gene editing, we led them to think whether can they modify their babies’ gene to gain some talents if one day they become parents. We assumed that children might just think on their own perspective, thus support this technology. But surprisingly, almost all of the pupils who voluntarily answered this question were against it. What impressed us the most was one little boy said that parents should love their child who they are, instead of turning him into what they like. Through this answer, we felt the warmth and sparkles of humanity
When we occasionally mentioned that we had created a comic series to popularize synthetic biology, Wang Mingyu, the leader of 2020 OUC supportive teaching teams in Lvchun encouraged us to print the comics out and send them some. We happily accepted this wonderful idea and printed our first edition of OUC-China made comic series: E.coli Spaceship. The comic series are now put in the library of Lvchun first middle school, students can borrow them freely!
Communication with social organizations
1.Collaboration with NGO
"Zhongyi Society"(it means public welfare in Chinese) is a newly established private non-profit voluntary service organization that provides students and community residents with social practice, public welfare services and professional experience. It aims to gather the strength of social loving units and people, organize and carry out voluntary services and social practice activities for children, adolescents, community residents and other groups.
Its activity center locates only hundreds meters away from the campus of Ocean University of China. We then contacted them and organized an online lecture for local high school students. Our first option is to conduct an offline activity so that we can make good use of the resources of university for visiting a university campus and laboratories can be really beneficial for high school students. However, our university has strict restriction for outsiders visiting university due to pandemic. We then decided to hold a online lecture and have an “online lab tour” for the local high school students. The photo below shows the registration interface of our activity. There are 43 students signed up and attended the activity.
Our course was divided into three sections. We firstly shared an inspiring news lately that a Chinese scientific group successfully achieved transforming carbon dioxide into starch through synthetic biology, possibly solving green house effect and food crisis at the same time! After the lead in, we introduced the basic and classic ideas of synthetic biology, and discussed the possible implementations. The second section was an online lab tour, OUC-R team pre-recorded a video, and introduced the details of every instruments. The third section was more academic, our team elaborated some deeper knowledge of synthetic biology such as logic gate and switch, enabling the students to have a brief idea of how our team projects were designed.
2.Local science museum
Science museum is the science hub for local people, especially the children who love science. How well a science museum established and activities arranged represents the science atmosphere of science in a city. Thus, we believe that the best place of spreading science knowledge to children is the local science museum. We actively reached the Qingdao Science Museum and expressed our willingness of providing some lectures to local children and luckily received active responds. We felt honored to be invited to attend the 10th Qingdao Science Popularization Lecture of 2021 which is a public welfare activity organized by Qingdao science and Technology Museum regularly. Experts in various fields were invited to provide high-quality popular science activity resources to the public, especially young people hopping to promote the scientific quality of the whole people in our city.
Fan Zhenxiang, the head of Exhibition and Education Department of Qingdao science Museum provided us a lot of support by giving us some suggestions after previewing our prepared syllabus. He firstly arranged our lecture on August 7th, for it is the birthday of Alexander Fleming who is one of the scientist we would introduce to the children. But unfortunately, China experienced a mild and regional epidemic on August, 2021, so we then changed the date to October 3th, 2021.
October 3th was among the national holiday of China, and the activity attracted more than 100 children as well as their parents. In the WeChat official account(social media) of Qingdao Science Museum, this activity was described as follows:
The teacher first taught the knowledge of bacteria and antibiotics, using multimedia and interactive activity to deepen the audience's understanding of the relationship between bacteria and antibiotics, and consolidated the knowledge of health and epidemic prevention. Then, through the analogy of "building blocks", the audience understood the knowledge of "synthetic biology". After then, the contribution and challenge of gene editing technology to society are further discussed. The last part of the activity is the observation and experience section. The teacher prepared bacteria containing fluorescent genes placed in the safety equipment. Through close observing, the audience had a more intuitive understanding of gene editing technology. The audience were very satisfied with this popular science lecture. After class, the children expressed that they should study hard, grow up and engage in relevant scientific research.
We really enjoy having face to face interactions with the local children. We were so surprised to see that the local pupils in Qingdao have a really wide range of knowledge. For example, when introducing the concept intestinal flora, we raised the question why human can’t digest grass but some other mammal such as rabbits and cattle can do so. We received the answer “they have longer intestine” for surprise, because we expected the answer that different animals have different composition of intestinal flora. We praised the boy who offered the answer, and extensively asked them how many stomachs do cattle has. The pupils chorused “F-O-U-R” together. Another thing we were very pleased to see is every child attended the activity was accompanied by at least one parents. It is so precious that parents would like to spare their time, accompanying their child to do something children may gain and grow from. Actually, the parents not just came to the science museum to be with the children only, they listened to the course as well and even participated in the interaction section. When we asked for the attitude they hold toward gene therapy on disease treating. One of the parents questioned how many diseases are caused by gene mutation. It was a really good question, for the common diseases public know are mostly not gene based. Most of the monogenic genetic diseases are rare to see. We are glad that a parent raised this question for children generally know little about diseases. Taking this chance, we explained that even though a single monogenic genetic disease may be rare to see, but the accumulation of total population monogenic with genetic disease is huge. Besides, some mutation may not directly cause specific disease, but may increase the susceptibility of having a specific disease. Thus, gene therapy do has its potential, but still need serious consideration.
We brought some ferrules and badges having “iGEM” images as for the gifts encouraging the children to answer the questions actively. But actually, they are super into the class and actively answered whatever questions we raised with or without encouragement. After the lecture ended, a little girl came to us, wanting to take a photo with us for she wanted to study in Ocean University of China in the future. We express our best wishes to her at there.
We also designed some activities for the local children, that is we brought a medium containing fluorescent bacteria in picachu pattern. The picture below shows children gathering around the blue light meter to see the bacteria excited to emit green fluorescence. The medium was sealed up tightly to ensure safety.
Communication with people in social science
We contacted the law students in Ocean University of China.They provided us another perspective for solving environmental problem. Here is their legal proposal
