Common Storage Methods

    There are several bio-preservation methods for bacteria. Nevertheless, at research institutes, ultra-low temperature freezing is the most commonly used method for long-term storage. However, the storage relies on specialized equipments, including the ultralow temperature refrigerators (-70℃~-80℃). Ultralow temperature refrigerators are expensive, noisy, and consumes lots of electricity, so there is usually no such equipment outside of laboratories.

    Though there are some biopreservation means besides ultralow temperature, each method has shortcomings. This promoted us to initiate our project.

Freeze-drying / Lyophilization

    The metabolism slows down if the cells lose water. Indeed, there are 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. Firstly, researchers tried to improve the procedure of freeze-drying; Secondly, they tried to optimize the constituent of protectants and excipients in the solution before lyophilization, but 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 and is famous for its strong vitality. It can survive extreme environments, like freezing, desiccation, vacuuming, extreme temperatures, high pressure, and radiation.

     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].

    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.

Presented by iGEM_20 QHFZ-China

Authors:
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!

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].

(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 since they are expensive and easy to malfunction. Our method can allow the transportation of engineered bacteria without the usage of cold chains so that transportation is easier. The engineered bacteria can be used more widely around the world.

Acknowledgement

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.

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