Bronze
Competition Deliverables
We have created a wiki page, and shown it online.
We have uploaded our presentation video in time.
We have submitted the judging form and safety forms before the deadline.
Attributions
We have received generous help from teachers, professors, doctors, experts, companies, other iGEM teams, and nearby communities. All of them gave us precious advice on different parts of our project, which inspires us to complete our project. We really appreciate their help, and have built an Attributions page for the all the people that have helped us along this iGEM journey. Visit our
Attributions page to check it out.
Project Description
Deep vein thrombosis (DVT) is developed from blood clots in the veins of the lower limbs. It hinders the bloodstream, which could cause tissue necrosis, putting patients at risk of amputation. There is a high chance for DVT patients to develop fatal complications with high mortality rate, especially pulmonary embolism (PE). Thus, this disease couldn’t be ignored.
We developed a strain of
E. coli Nissle 1917 that could secrete Nattokinase, a serine protease that has high thrombolytic activity. The genetically modified bacteria could constantly release Nattokinase into one’s small intestine, and therefore prevent thrombosis. Additionally, we developed a home-aid testing kit, which detects the D-Dimer amount in a person’s saliva sample. The D-Dimer testing result and the person’s thrombosis risk would be calculated and displayed on our app. Finally, with our optogenetics design, the production of Nattokinase by the
E. coli could also be enhanced through a tap on the app. For more information, please visit the
Description page.
Contribution
Two main contributions are provided by Natto It Out to pass on our experiences to the next generation. Firstly, we've developed a cost-effective software that predicts the cleavage sites targeted by S8 protease family through the hidden features in their amino acid sequences. Secondly, we've built up a preliminary 3D printed device for thrombus level detection, and developed a user-friendly application on the phone, in which the users control Nattokinase release through Bluetooth signal transduction.
Silver
Engineering Success
We’ve designed a kill switch system and used red fluorescent protein (RFP) to test if the system could work functionally. First, we chose the restriction enzymes to cut the plasmid. Then we ligated the fragments together and transfered the reagent into competent cells. However, we didn’t get the transformation result often. After that, we increased the concentration of the DNA fragments and changed the T4 ligament. Finally, the rate of successful transformation increased. Next, we used the GIBSON assembly to connect multiple fragments together and accomplish our preliminary construct. This construct has an RFP gene regulated by pTetR. Yet, the bacteria didn’t fluoresce red, which is out of our expectation. Consequently, we use modeling to analyze the possible cause. We suspect that it’s the leakage of TetR that we use to control the pTetR that makes our construct not work properly. Finally, we designed another construct with a tandem promoter so as to avoid the problem of leakage. For more information, please visit our
Engineering Success page.
Collaborations
We hosted the 2021 iGEM International Optogenetics Virtual Conference. We posted the invitation on iGEM collaboration page, and sent the meeting materials via email. The participant was Team KUAS_Korea. Both teams presented their projects, actively participated in the Q&A session, and exchanged ideas on each other’s optogenetic designs.
We also collaborated with Team TAS_Taipei. After receiving our protocol, they conducted a confirmative experiment for us - measuring the expression rate of Red Fluorescent Protein (RFP) of our genetically modified bacteria under different concentrations of L-arabinose, and provided us with
E. coli Nissle 1917, which we use to make our final product. In return, we provided them with the plate reader they needed, and invited them to conduct experiments in our laboratory.
Moreover, we joined the 2021 iGEM Taiwan Virtual Meetup, and discussed our projects with Team NCHU_Taichung, NCKU_Tainan, NCTU_Formosa, CCU_Taiwan, NTHU_Taiwan, and our host, CSMU_Taiwan. For more information, please visit our
Collaborations page.
Human Practices
According to WHO statistics, a person suffers from cardiovascular disease (CVDs) every 16 seconds, and a person dies from thrombus every 37 seconds. Also, the overall mortality rate of Deep Vein Thrombosis (DVT) is 37% one year after being diagnosed. There is no doubt that CVDs, thrombosis, and DVT affect human health so severely that the consequences mustn’t be ignored. Therefore, by building an effective prevention method for DVT, we aim to positively impact the health of the general public.
Building up our project, we have consulted 3 biotech companies, 2 cardiology professionals, 1 expert on elderly care, 4 professors and 1 researcher on biomedical related subjects. On the social aspect, we believe that our project can potentially improve public health by lowering thrombosis risks with our Nattokinase
E. coli and our detection device; and raise public awareness on CVDs, thrombosis and DVT through our public engagements - including a physical lecture in Taipei First Girls High School, an online public survey, scientific educations through social media platforms, and the health education leaflets we designed and gave out to local elderly care institutions.
On the scientific aspect, we believe that the remote control system that we designed has the potential of bringing changes to the medical field, transforming long-term medication into just one take of capsule, where patients can be given precise amounts of medicine with manipulation from outside the body.
Secondly, we designed a kill switch system requiring fewer components than previous iGEM team designs, in other words, an efficient plasmid construction for a kill switch system, which is potentially useful for other biosafety designs in synthetic biology.
Thirdly, with our dry lab, we proposed a software model aiming to predict the cleavage site recognized by the serine protease, Nattokinase, by integrating machine learning algorithms and feature selection based on sequences data of the S8 family. Since Nattokinase cuts not only fibrin but also other substrates, it brings about health benefits other than breaking down thrombosis. We therefore hope to implement our software on not only the prediction of thrombolysis by Nattokinase, but also studying other health effects of it through finding out potential substrates of the protein, by bringing in other protein databases of the human body. For more information, please visit our
Human Practices page.
Proposed Implementation
In addition to conducting experiments, we participated in expert visiting, doctor visiting, public surveys, IHP, sponsorship, education, and professors’ meetings to implement our project. To put it simply, we designed a home D-dimer detection device, a convenient App, and an optogenetics control system. Besides, we have put the safety aspects into consideration as well by meeting with experts and doctors. During the implementation of our project, we have been faced with several challenges. We are confident that our project is “more than just a concept” but a feasible design. For more information, please visit our
Implementation page.
Gold
Integrated Human Practices
With a framework designed by NYCU-Taipei team members, our project has integrated opinions from stakeholders, experts, and the public.
A series of both virtual and physical meetings was organized, a public survey was conducted; lively discussions with the scientific community were made, and the system-wide impact of our project in medical fields and the society was thoroughly reevaluated. As we gained inspirations from human practices events, the core of our project had gradually taken shape - the use of Nattokinase, the concept of Preventive Medicine, and the idea of Live Biotherapeutic Product (LBP).
Moreover, parts of our wet lab design have undergone significant changes. The MazEF kill switch design was proposed; oral delivery system has been simplified from complicated ideas; and Deep Vein Thrombosis (DVT) was singled out as the main target of our thrombosis detection kit. For more information, please visit our
Integrated Human Practices page.
Improvement of an Existing Part
In our kill switch design, we hope to generate a stronger pBad promoter with a greater expression rate and wider inducing range. With the regulation of both constitutive promoter and L-arabinose induced pBad promoter, we hope it can help the pBad to increase its expression ability. In other words, we are trying to improve BioBrick_BBa_K206000. We want to ligate pBad promoter with a constitutive promoter to form a tandem promoter. We hypothesized that by adding a constitutive promoter beside pBad, the expression of the tandem promoter will be better than the normal pBad promoter (BioBrick_BBa_K206000). We compared the intensity of RFP gene constructed behind the pBad promoter and pBad-J23106 tandem promoter to see if the tandem promoter has better expression rate. For more information, please visit our
Parts page.
Project Modeling
We aim to construct an engineered
E. coli stably adhered to the small intestine which can release Nattokinase by optogenetic control and safely removed with the least impact on gut microbiota. First, we’ve modelled the production of the adhesive protein, FimH, and the adhesion ability of
E. coli introduced by FimH. We got the amount of OmpA-FimH protein and its mRNA in 45 minutes production. Furthermore, the adhesion ability shows that how many FimH produced can let the
E. coli stably adhere to epithelial cells. Secondly, we use the protein docking server, pydock, to confirm our optogenetic system experiment design. Lastly, since the constitutive inhibition of MazE to MazF prevents cell death and could be disrupted by tetR, we constructed the model from tetR expression to MazE and MazF. We found out that MazF could inactivate the tetR due to the ACA sequence cutting ability. Avoiding using upstream regulators might be a better way to this kill switch system. For more information, please visit our
Model page.
Proof of Concept
To verify the function of our Nattokinase live biotherapeutic product, we used step-by-step experiments to verify that our bacteria are effective and useful in the treatment of DVT patients in the future. The proof of concept is divided into 5 parts. From diagnosis, we created a home D-dimer detection device connected to an app to show the risk of thrombosis. Moving on to therapy, we established an optogenetic system to control the release of Nattokinase inside our body. Besides, we constructed a kill switch system that functions both in vivo and the environment through chemical inducement or temperature decline. Finally, we modeled parts that were inaccessible through wet lab experiments and established a novel software with the algorithm for S8-family protease-substrate relationship by only sequence data and protease properties input. We believe that, “Natto it out” is an integrated project from diagnosis to therapy, which is more than just a concept but feasible in the future.
Education and Communication
We have engaged in the education and communication with the general public, high school students, and the elderly community.
For the public, we have provided health education on DVT and CVDs on our Facebook and Instagram page, hashtagged “sciencefacts.” We’ve also promoted synthetic biology, and have broken down our projects step-by-step for better understanding. Also, we cherish the voice of the public. We conducted a public survey on the use of Nattokinase, cardiovascular diseases, and the preference of capsules, and have received over 160 responses.
For high schoolers, we wanted to provoke their interest and encourage their participation in scientific research. We held a physical lecture for the Biology Club in Taipei First Girls High School. We shared our experiences on genetic engineering, experimental designs and iGEM participation.
For the elderly community, we wanted to inform them of the risk of thrombosis and potentially improve the quality of life in their later years. We communicated with the local elderly long-term care center, Yi-Ching Yuan, and provided them with health education leaflets on the negative effects of DVT. For more information, please visit our
Education & Communication page.
Special Prize
Best Integrated Human Practices
“Natto It Out” is a strategic approach aiming at the prevention and detection of thrombosis. With a framework designed by NYCU-Taipei team members, it has integrated opinions from stakeholders, experts, and the public.
A series of both virtual and physical meetings were organized, lively discussions with the scientific and public community were made, and the system-wide impact of our project in medical fields and the society were thoroughly reevaluated. As we gained inspiration from human practice events, the core of our project had gradually taken shape - the use of Nattokinase, the concept of Preventive Medicine, and the idea of Live Biotherapeutic Product (LBP).
Moreover, parts of our wet lab design have undergone significant changes. The MazEF kill switch design was proposed; the oral delivery system has been simplified from complicated ideas; and Deep Vein Thrombosis (DVT) was singled out as the main target of our thrombosis detection kit. For more information, please visit our
Integrated Human Practices page.
Best Measurement
NYCU-Taipei’s 2021 iGEM project is titled “Natto It Out”. Nattokinase is an enzyme that was discovered in a Japanese food, and we are going to ues it to fight against thrombus, or fatal blood clots. As you will find out in this video, there is still room for improvement for thrombosis detection and treatment, especially in the pandemic world nowadays. We present an effective approach incorporating a thrombosis risk detection system and genetically modified bacteria to make timely medical care no longer an unreachable dream! For more information, please visit our
Measurement page.
Best Model
We aim to construct an engineered
E. coli stably adhered to the small intestine which can release Nattokinase by optogenetic system and safely removed with the least impact on gut microbiota. First, we’ve modelled the production of adhesive protein, FimH, and the adhesion ability of
E. coli introduced by FimH. We got the amount of OmpA-FimH protein and its mRNA in 45 minutes production. Furthermore, the adhesion ability shows that how many FimH produced can let the
E. coli stably adhere to epithelial cells. Second, we use the protein docking server, pydock, to confirm our optogenetic system experiment design. Last, since the constitutive inhibition of MazE to MazF prevents cell death and could be disrupted by tetR, we constructed the model from tetR expression to MazE and MazF. We found out that MazF could inactivate the tetR due to the ACA sequence cutting ability. Avoiding using upstream regulators might be a better way to this kill switch system. For more information, please visit our
Model page.
Best Software Tool
Currently, the cleavage sites of Nattokinase remain unelucidated. We developed a method to predict the cleavage sites of Nattokinase. Since Nattokinase is one of the S8-family proteases, we exploit the database of the S8-family for our model. The past research for S8-family protease-substrate relationship is highly related to its folding structures. However, learning the features of the folding structures is computationally expensive. Our contribution is that we find the hidden features in sequences that represent the critical structure in protease. Our algorithms have great performances on analyzing S8-family protease-substrate relationships and also mitigate the computational cost. For more information, please visit our
Software page.
Safety and Security Award
Our team developed a complete on-and-off control system. From thrombosis-risk detector triggering IR to optogenetic Nattokinase production, we aim to precisely release NK in the small intestine. For cleaning up the
E. coli and minimizing the gene burden, we designed a simplest and easily-controlled kill switch system. L-arabinose or room temperature could disrupt the constitutive inhibition of MazE to MazF. MazF could inactivate mRNA in
E. coli leading to cell death. We verified the system with our wet lab experiments and modeling. It could successfully achieve our safety and security expectations. For more information, please visit our
Safety page.
