• •       Description
  •       Design
  •       Proof Of Concept
  •       Results
  •       Implementation
  •       Experiments
  •       Notebook
• •     Modeling
  •     Protein
• • Intergrated
  • Education
• •                         Parts
  •                         Contribution
  •                         Engineering
  •                         Collection
• •                  Safety
• •            Judging
• •           Team
  •           Attributions
  •           Collaboration
  •           Partnership
  •           Acknowledgement

   TOP

Part Collection

Introduction

We try to apply for Best Part Collection Award because we measured a series of parts that can protect bacteria from freeze-drying. As far as we found, no one studied the parts in live cells druing freeze-drying. We will gave details in this page and thank you for attention.

Description

    There is no ultra-low temperature freezer for bacteria storage in our daily life, so the practical application of engineered bacteria is limited. This year, we tried to introduce a new biopreservation method. We used freeze-drying to make the engineered into dry powder. Then the powder can be stored at room temperature for a long time. This method can store bacteria get rid of ultra-low temperature freezers to promote the practical application of engineered bacteria out of the laboratory. However, the stresses during freeze-drying and subsequent dry storage, including freeze, dry, and vacuum, are lethal to bacteria. We use Tardigrade intrinsically Disordered Protein (TDPs), including Cytosolic-abundant heat soluble protein (CAHS) 89226, CAHS 94205, CAHS 106094, CAHS 107838 and secreted abundant heat soluble protein (SAHS) 33020, to help bacteria survive the situation. We also measured two other parts besides TDPs, which are LEA and OtsBA.

    TDP is a kind of heat soluble protein found from Tardigrade Hypsibius dujardini in 2017 ^[1]. Tardigrade, also called water bear, is a kind of tenacious organism. It can survive extreme environment, such as desiccation, freeze and vacuum. The super capacity of Tardigrade partially owes to TDPs. Thus, we designed to let bacteria express TDPs to help itselves survive the freeze-drying and subsequent dry storage. Before TDPs were found, people used two elements to enhance the stress resistance, LEA and trehalose. Thus, we also tried the effect of the two parts, (1) Late Embryogenesis Abundant (LEA) protein, LEA3 protein, from soybean (Glycine max), and OtsBA (OtsB and OtsA), two enzymes that can dimerize glucose into trehalose.

Overview of the part collection

Overview of our contribution

(1) CAHS 89226 was not existed in the iGEM library before and we are the first to register it.
(2) For CAHS 107838, it has been registered by others but they did not offer any data. We modified the part and we are the first to add experimental data.
(3)For other basic parts, there exist some data, but none data is about freeze-drying live cells. We modified the parts and we are the first to add data about freeze-drying live cells.
(4) For all parts, we optimized the sequence to make it suitable to be expressed in E. coli. For all parts other than OtsBA, we added a 6x His tag, for easy detection by Western blot and purification by Ni-chelating affinity chromatography.

For the data and results, please check the parts above.

Reference:

[1] Boothby, T.C., Tapia, H., Brozena, A.H., Piszkiewicz, S., Smith, A.E., Giovannini, I., Rebecchi, L., Pielak, G.J., Koshland, D., and Goldstein, B. (2017). Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation. Mol Cell 65, 975-984 e975.

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