Part:BBa_K5286008

CAHS106094+pT7max

This part expresses tardigrade protein under T7max promoter and it when coexpressed with the autolysate pAD-LyseR plasmid according to Dydovik et al.^[1] and Sun et al.^[2], the lysate produced can be desiccated and will last stored at a room temperature for weeks.

This part was originally added by team TUM Straubing in 2024. This construct was used to heterologously express Tardigrade CAHS proteins in the E. coli BL21 (DE3) strain co-expressing p-AD-LyseR. This construct significantly enhances desiccation tolerance in the lysate produced from the strain. For more information about this part and how it was used in the project, including protocols, visit the team's wiki.

Usage and Biology

Tardigrade-derived proteins, specifically intrinsically disordered proteins (IDPs) undergo a conformational change upon desiccation, forming a non-crystalline, amorphous solid state through a process known as vitrification. This vitrified layer encapsulates cellular components, including proteins, nucleic acids, and organelles, which protects them against extreme desiccation conditions, and has already made them helpful in preserving different biological components, such as biopharmaceuticals ^[3].

Proposed mechanism of action

Tardigrade proteins protect cellular components during desiccation and extreme conditions through three key mechanisms. Vitrification involves forming an amorphous, glass-like state that immobilizes biomolecules, preventing aggregation or degradation. Water replacement occurs as these proteins mimic water’s hydrogen bonding with macromolecules, maintaining structural integrity in its absence. Water retention enables these proteins to stabilize cellular hydration, reducing osmotic stress and delaying desiccation effects. Together, these mechanisms ensure cellular stability and functionality under extreme environmental stress. Three Figures

CAHS 107838 Structure — **Figure 2:** First mechanism of action: vitrification .

CAHS 106094 Structure — **Figure 3:** Second mechanism of action: Water replacement

CAHS 107040 Structure — **Figure 4:** Third mechanism of action: Water retention

Validation of functionality

This part was transformed into BL21 (DE3) to create stabilized E. coli lysate, resistant to desiccation.

CAHS“ width=

Figure 5:Relative fluorescence (% of fresh lysate) after 2 weeks post-lyophilization (storage at room temperature), Gene 1 (CAHS107838 ,BBa_K5286000) and Gene 2 (CAHS106094), untreated, normalized to fresh lysate. Error bars: SD (n=3), biol. dupl.

This effect was characterized for long term-stability. Lyophilized lysate containing CAHS107838 can be stored at room temperature for a minimum of 6 weeks.

CAHS“ width=

Figure 6:Relative fluorescence (% of fresh lysate) post-lyophilization (storage at room temperature for different timeframes), Gene 1 (CAHS107838, BBa_K5286000) normalized to fresh lysate. Error bars: SD (n=3)

This lysate can also be desiccated using a conventional vaccuum desiccator. CAHS107838 also confers stabilizing properties when processing the lysates using this low-cost method (figure 7).

CAHS“ width=

Figure 7:Normalised fluorescence of rehydrated vaccuum dessicated lysate following 2 weeks storage at room temperature. Gene 1 (CAHS107838, BBa_K5286000),control (Ctrl) ( lysate produced from autolysate strain ) Error bars: SD (n=3), Background corrections was performed without template.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 1269
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Functional Parameters

References

↑ Boothby, T. C., Tapia, H., Brozena, A. H., Piszkiewicz, S., Smith, A. E., Giovannini, I., Rebecchi, L., Pielak, G. J., Koshland, D., & Goldstein, B., “Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression“, ACS Synth Biol. 2017 Dec 15;6(12):2198-2208 [1].
↑ Sun ZZ, Hayes CA, Shin J, Caschera F, Murray RM, Noireaux V., “Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology“, J Vis Exp. 2013 Sep 16;(79):e50762 [2].
↑ Boothby, T. C., Tapia, H., Brozena, A. H., Piszkiewicz, S., Smith, A. E., Giovannini, I., Rebecchi, L., Pielak, G. J., Koshland, D., & Goldstein, B., “Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation“, Mol. Cell, vol. 65, issue 6, p. 975-984 [3].

[Dydovik-1] Boothby, T. C., Tapia, H., Brozena, A. H., Piszkiewicz, S., Smith, A. E., Giovannini, I., Rebecchi, L., Pielak, G. J., Koshland, D., & Goldstein, B., “Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression“, ACS Synth Biol. 2017 Dec 15;6(12):2198-2208 [1].

[Sun-2] Sun ZZ, Hayes CA, Shin J, Caschera F, Murray RM, Noireaux V., “Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology“, J Vis Exp. 2013 Sep 16;(79):e50762 [2].

[General_Tardigrade_Proteins-3] Boothby, T. C., Tapia, H., Brozena, A. H., Piszkiewicz, S., Smith, A. E., Giovannini, I., Rebecchi, L., Pielak, G. J., Koshland, D., & Goldstein, B., “Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation“, Mol. Cell, vol. 65, issue 6, p. 975-984 [3].

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