Difference between revisions of "Team:QHFZ-China/Poster"

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                             &nbsp;&nbsp;&nbsp;&nbsp;Our project is a continuation of the project created by QHFZ-2019. Last year, we made a portable household uric acid detector, which is a genetically engineered bacterium. It would facilitate the recovery of gout patients. However, we realized that there are still some problems hindering its practical application. Among them, we wanted to solve the issue of room-temperature storage before anything else.<br>
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                             &nbsp;&nbsp;&nbsp;&nbsp;Our project is a continuation of the project created by QHFZ-2019. Last year, we made a portable household uric acid detector, which contains a genetically engineered bacterium. It would facilitate the recovery of gout patients. However, we realized that there are still some problems hindering its practical application. Among them, we wanted to solve the issue of room-temperature storage before anything else.<br>
 
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Revision as of 15:35, 10 November 2020

Poster: QHFZ-China

Super-Serum for Engineered Bacteria

Presented by iGEM_20 QHFZ-China

Authors:
Poster design: Bohan Zhang1, 2
Wiki code: Xinyuan Wang1, 2
Content: Yixian Yang1, 2, Zhandong Jiao1, 2
Proofread: Bert Shan1, 2, Shao Chang1, 2
Instructor: Xing (Leaves) Zhang1, 3
1. iGEM team QHFZ-China 2020, China, Asia.
2. Tsinghua University High School, Beijing, China.
3. Beijing ZENO Co., Ltd., Beijing, China.


Abstract

    Various engineered bacteria displayed useful functions. However, these bacteria's storage usually requires -80℃ refrigerators, which substantially limited their transportation and usages in daily life. Therefore, we decided to utilize TDPs from tardigrades (water bears) to supply a new storage method. We could produce dry life-paused bacteria powder by introducing the TDPs as protectants into bacteria during lyophilization (freeze-drying). The powder can then be stored at room temperature for a long time without any professional equipment usage.
    This year, we confirmed that certain TDPs could maintain the survival rate of E. coli. We also optimized the method by regulating the expression levels and combining different TDPs. Also, we proved the modularity and tried to regulate the degradation of TDPs after freeze-drying. Considering that many iGEM teams and researchers are designing engineered bacteria for various application scenarios out of the laboratory, we hope our improvement in bio-preservation methods would promote their practical applications.

Team



    This year, QHFZ-China is composed of 15 students. It is the second year for QHFZ-China to attend iGEM Jamboree.

Leaders: Yixian Yang, Zhandong Jiao
Members: Celine Siu Ham Zhang, Jiangshan Gao, Sizhe Duan, Marco Xue, Yeqi Wong, Xinyuan Wang, Bert Shan, James Haorui Wang, Junfei (Sophie) Li, Yiming Tang, Shao Chang, Siyuan Wang, Bohan Zhang
Primary PI: Shiqi Wang
Instructors: Xing (Leaves) Zhang, Tianze Zhu
Advisors: Zhimeng Xu, Jun Ma, Aiqi Zhao, Li TianHong, Luo Shaowei, Weizhao Chen

    All of us welcome you to visit and critique our poster!

Introduction & Inspiration
    Our project is a continuation of the project created by QHFZ-2019. Last year, we made a portable household uric acid detector, which contains a genetically engineered bacterium. It would facilitate the recovery of gout patients. However, we realized that there are still some problems hindering its practical application. Among them, we wanted to solve the issue of room-temperature storage before anything else.

    Engineered bacteria need to be stored for an extended period before being used at room temperature. They should stay alive while their metabolism paused. In laboratories, we use ultralow temperature to achieve this. That is why -80℃ refrigerator is necessary for molecular biology labs. However, it is hard to utilize such professional equipment from laboratories since they cannot be found daily. In general, household refrigerators can only achieve about -20℃, which is insufficient. Besides, it is not ideal to use the refrigerators to store bacteria, which we primarily use to store food. However, many engineered bacteria are designed for our daily life. We interviewed ten iGEM teams, and most of them believed that a suitable method to solve the problem would benefit the practical application of their products.

    As a result, we decided to develop a biopreservation method, which could help store the engineered bacteria without professional equipment.
The Limitations of Current Methods and Our Solution
Common Storage Methods

    Ultra-low temperature freezing is the most commonly used long-term bio-preservation method at research institutes. However, the storage relies on specialized equipment, including the ultralow temperature refrigerators, but there is usually no such equipment outside of laboratories.
    Though there are some other biopreservation means, each method has its own shortcomings.
Freeze-drying / Lyophilization

    The metabolism slows down if the cells lose water. Indeed, we can find some dried bacteria products on the market. Thus, we decided to make engineered bacteria into dry powder. Then the powder can be stored at room temperature without any professional equipment.


    The direct drying method is time-consuming, and the metabolism will not stop during the process. However, freeze-drying is very fast. Indeed, freeze-drying has been widely used to produce vaccines, medicaments, and food because it can maintain the materials' original state.
    Nonetheless, during the process, at least three stresses affect cells' survival: freeze, desiccation and vacuum. Several studies have tried to improve the survival rate of bacteria: (1) to improve the procedure of freeze-drying; (2) to optimize the constituent of protectants and excipients in the solution before lyophilization. Yet, the survival rate still stayed at about 10%. Thus, we hope to introduce another element to elaborate on previous studies.

Tardigrade (water bear) / TDPs

    To apply lyophilization/freeze-drying, we need to utilize the proteins of a very special organism. Tardigrade (water bear) is a miraculous organism found in 1773. It can survive extreme environments, like freezing, desiccation, vacuuming, extreme temperatures, high pressure, radiation, etc.
     The strong vitality of tardigrades at least partially owes to TDPs (Tardigrade intrinsically Disordered Proteins), which are exceptionally resistant to desiccation.
     TDPs are found in 2017, mainly including several Cytosolic-abundant heat soluble proteins (CAHS) and secreted massive heat soluble proteins (SAHS). TDPs were proven to protect living cells from drying[1] and maintain the activeness of proteins after freeze-drying[2,3], but no one studied whether they can protect living cells from freeze-drying or not. Therefore, we tried to allow bacteria to express TDPs and tested whether they can survive freeze-drying and the subsequent storage processes.

    In this project, we studied 5 TDPs: CAHS 89226, CAHS 94205, CAHS 106094, CAHS 107838, and SAHS 33020, as well as two gene parts that are used before TDPs were found: LEA (Late Embryogenesis Abundant protein) from Glycine max [4] and OtsBA (producing trehalose)[5].

Methodology / Protocol
    During most experiments, we used a modified pet28a vector to express TDPs in E. coli BL21 (DE3) strain.

    To test the survival rate, we used the following protocols to conduct our experiments:

    We used lyophilization to make dry powder so that the cells can be stored at room temperature for a long time. To accelerate our experiments, we stored the cells at room temperature for two days before testing the survival rate. Simultaneously, we also conducted some extra experiments trying to prolong the storage time to more than ten days and received similar results.

Result 1 st: Functional verification of TDPs.
    By SDS-PAGE as well as other techniques, we confirmed that TDPs were normally expressed.

    We verified all the gene parts we constructed.
    Compared with the control group (sfGFP), several gene parts enhanced the bacteria's survival rate during freeze-drying and subsequent dry storage phases at room temperature. Among these, CAHS 106094 showed the best effect.

    Subsequently, we focused on CAHS 106094 and continued to experiment.

Super-Serum for Engineered Bacteria

Presented by iGEM_20 QHFZ-China

Authors:
Poster design: Bohan Zhang1, 2
Wiki code: Xinyuan Wang1, 2
Content: Yixian Yang1, 2, Zhandong Jiao1, 2
Proofread: Bert Shan1, 2, Shao Chang1, 2
Instructor: Xing (Leaves) Zhang1, 3
1. iGEM team QHFZ-China 2020, China, Asia.
2. Tsinghua University High School, Beijing, China.
3. Beijing ZENO Co., Ltd., Beijing, China.


Abstract

    Various engineered bacteria displayed useful functions. However, these bacteria's storage usually requires -80℃ refrigerators, which substantially limited their transportation and usages in daily life. Therefore, we decided to utilize TDPs from tardigrades (water bears) to supply a new storage method. We could produce dry life-paused bacteria powder by introducing the TDPs as protectants into bacteria during lyophilization (freeze-drying). The powder can then be stored at room temperature for a long time without any professional equipment usage.
    This year, we confirmed that certain TDPs could maintain the survival rate of E. coli. We also optimized the method by regulating the expression levels and combining different TDPs. Also, we proved the modularity and tried to regulate the degradation of TDPs after freeze-drying. Considering that many iGEM teams and researchers are designing engineered bacteria for various application scenarios out of the laboratory, we hope our improvement in bio-preservation methods would promote their practical applications.

Team



    This year, QHFZ-China is composed of 15 students. It is the second year for QHFZ-China to attend iGEM Jamboree.

Leaders: Yixian Yang, Zhandong Jiao
Members: Celine Siu Ham Zhang, Jiangshan Gao, Sizhe Duan, Marco Xue, Yeqi Wong, Xinyuan Wang, Bert Shan, James Haorui Wang, Junfei (Sophie) Li, Yiming Tang, Shao Chang, Siyuan Wang, Bohan Zhang
Primary PI: Shiqi Wang
Instructors: Xing (Leaves) Zhang, Tianze Zhu
Advisors: Zhimeng Xu, Jun Ma, Aiqi Zhao, Li TianHong, Luo Shaowei, Weizhao Chen

    All of us welcome you to visit and critique our poster!

Result 2 nd: Optimization & Modularity & Long-term storage
    Rather than qualitative research, we wanted to conduct a deep study to optimize the method. First, we tried to find the optimal expression level of CAHS 106094. We used different promoters to regulate the expression of CAHS 106094. Then, as we expressed the TDPs in vector, we measured the relative strengths of the promoters.

    The survival rate displayed a gradient ascent as the expression levels of CAHS 106094 increased, indicating that the improvement of TDP’s expression in a suitable range can achieve our ideal effects.

    Tardigrade co-expresses different TDPs to protect itself. We combined different TDPs to study whether they have a synergistic effect. Thus, we combined CAHS 106094 and SAHS 33020. The co-expression of the two gave a higher survival rate.

    In addition, (1) We prolonged the storage time. Bacteria expressing TDPs still showed a higher survival rate; (2) We changed the vector and E. coli strain, and proved that CAHS 106094 also worked in E. coli DH5alpha strain.

    The information above implied that TDPs could protect bacteria regardless of vectors, strains, and storage time at room temperature. Additionally, the method can still be further optimize.

Modeling
    To avoid the influence of TDPs on the functions of the engineered bacteria, we designed a gene circuit. Before lyophilization, the left part is activated by inducer 1 (iPTG), promoting the expression of TDPs. After rehydration, the right part is activated by inducer 2 (arabinose), TDPs with a pdt tag are specifically degraded by mf-Lon, a protease from mycoplasma[6].

Basic model

    With reasonable assumptions, we constructed a basic model to simulate the process.
    If the left part is activated, TDP (CAHS 106094) is expressed and finally reaches the equilibrium. We can change the strengths of Pc and RBS 1 to achieve different TDP levels[7,8].

    If the right part is activated, TDP (CAHS 106094) is degraded in a short time. We can change pdt tag and the strength of RBS 2 to regulate the time needed.

    In our wet lab this year, we tested several parts of the gene circuit, including the TDP expression module and arabinose-inducible promoter.

Advanced model

    Indeed, we further constructed an advanced model to simulate the process more accurately. In the model, we carefully considered the state of the repressors (LacI, acaC) when the inducer is added. The model based on stereochemistry gave a deeper theoretical direction.

Conclusions & Future
Conclusions

(1) TDPs helped bacteria during freeze-drying and subsequent storage phases.
(2) CAHS 106094 was the best!
(3) The effect could be improved by regulating the expression levels and through co-expression.
(4) TDPs were suitable for long-term storage and different E. coli strains.
(5) In summary, the method based on freeze-drying and TDPs functions, and we created a part collection for different strains, with various effects and different inducers.

Future work / Next plan

(1) Confirm the results by repeating previous experiments.
(2) Test more TDP expression levels and more combinations.
(3) Test the effects on various kinds of microorganisms.
(4) Combine the method of TDP expression with the previous bacterium-protecting methods. We hope that TDP expression+ advanced lyophilization protocols + excellent protectants/excipients will comprehensively protect engineered bacteria during lyophilization and subsequent storage phases at room temperature.
(5) Complete analysis and verification of the safety of the method[9,10].

Part collection
Application scenarios
(1) Our project aims to improve on the project of our team last year. QHFZ-China 2019 + 2020 will provide a kit to detect uric acid at home without any professional equipment for gout patients.

(2) the bio-preservation method can also help other iGEM teams/researchers store their engineered bacteria at various places. For example, in 2020, researchers made a paper-based bio-battery, and the bacteria are freeze-dried on the paper[11]. Our method can improve the bacteria's activity so that they can produce more electricity.


(3) There are some dry microbiological products, such as dry yeast for fermentation, probiotics tablets for health and nitrobacteria capsule for fish culture. The method can enhance the survival rate of the microorganisms to reduce the production costs.


(4) There is a lack of cold chains. Our method can allow the transportation of engineered bacteria without the usage of cold chains. Thus, the engineered bacteria can be used more widely around the world. Meanwhile, NASA has an imagination to transport organisms to other planets. Our method would offer help.

Human practices
    We used a picture to show how we came up with and improved our project via human practices this year.

Communications with iGEM teams and the public
Collaborations / Partnerships

    We interacted with many iGEM teams this year. For example, we had deep cooperation with Jilin_China in many fields.

    We also held a meetup called "Beijing International iGEMer Collaboration Seminar."


Science communication / Education

    We spread the knowledge about synthetic biology via lessons, large synthetic biology festival events, blogs, video platforms, etc. We showed an example here. We contacted Beijing Bayi School Affiliated Yuquan Middle School and made a real-time online lesson for eighth grade students.


Acknowledgement & References & Sponsors
Acknowledgement

    To accomplish the project, we did most of the work. However, without others help, we cannot reach this achievement.
We thank
Our teachers: Shiqi Wang, Xing (Leaves) Zhang, Tianze Zhu, Zhimeng Xu, Jun Ma, Aiqi Zhao, Li TianHong, Luo Shaowei and Weizhao Chen
Our interviewees: Guangyuna Song, Jiajia Liu, Miatta Momoh, Shan Jiang, Shaojie Li, Tianqi Zhang, Yikai Bao, ZhiZhong Gong;
Cooperating teams: Jilin_China, ZJUT_China_B, NEFU-China, XMU-China, ASTWS-China, BNDS_China, NAU-China, SHSBNU_China, SCU-China, NEU_China, ECUST_China, KEYSTONE, BHSF, BJ101HS, NFLS, Tsinghua, BUCT-China, JNFLS, Nanjing_NFLS, RDFZ-China, QHFZ-China (2019), BNU-China (2019) and BEAS_China (2019)
The organizations helping us: iGEM Foundation, Tsinghua University High School, ZENO CO.,LTD, BEIJING TIDE PHARMACEUTICAL CO.,LTD, YOUCARE PHARMACEUTICAL GROUP CO.,LTD, MathsWorks, Snapgene, Geneious, Beijing Haidian vocational school, Beijing Bayi School Affiliated Yuquan Middle School, Tsinghua University
and all people who gave us help.
Please accept our deepest thanks to everyone.

References

[1] G.J., Koshland, D., and Goldstein, B. (2017). Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation. Mol Cell 65, 975-984 e975.
[2] iGEM Team TUDelft 2017
[3] iGEM Team XMU-China 2018
[4] iGEM Team Valencia 2010
[5] iGEM Team Imperial_College_London 2009
[6] iGEM Team William_and_Mary 2017
[7] Registey of Standard Biological Parts, Part:BBa_J23100
[8] Registey of Standard Biological Parts, Part:BBa_B0034
[9] Di Stasi, A., Tey, S. K., Dotti, G., Fujita, Y., Kennedy-Nasser, A., Martinez, C., . . . Brenner, M. K. (2011). Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med, 365(18), 1673-1683. doi: 10.1056/NEJMoa1106152
[10] iGEM Team BEAS_China 2019
[11] Cho, J.H., Gao, Y., Ryu, J., and Choi, S. (2020). Portable, Disposable, Paper-Based Microbial Fuel Cell Sensor Utilizing Freeze-Dried Bacteria for In Situ Water Quality Monitoring. ACS Omega 5, 13940-13947.

Sponsors