Difference between revisions of "Part:BBa K4765016"

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| '''Figure2 The dry weight of ''H.ex'' mtSSB, TDP, and the control after freeze-drying'''
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There is no statistically significant difference in the dry weights among the experimental groups, indicating that the number of ''E. coli'' is consistent between the bacterial tubes.
 
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Revision as of 09:20, 9 October 2023


Hypsibius exemplaris mitochondrial single-stranded DNA binding protein (H. ex mtSSB) contributed by Fudan iGEM 2023

Introduction

H. ex mtSSB is a type of mitochondrial single-stranded DNA binding protein derived from Hypsibius exemplaris . H. ex mtSSB is non-specific in binding single-stranded DNA. ssDNA is exposed by normal cellular functions like replication and transcription, as well as during genotoxic stress. DNA wrapped around H. ex mtSSB could be physically buffered against DNA damage and ensuing lethality. Under harsh conditions like desiccation heat, and radiation, H. ex mtSSB can maintain the stability of DNA through the above mentioned mechanism, thus enhance the survival rate of organisms in harsh environments[1].

Usage and Biology

We heterologously expressed H. ex mtSSB in E. coli, conferring improved resistance to desiccation and UV radiation. We also compared its desiccation resistance with that of SAHS 33020(BBa_K2306003) and tested the combined desiccation resistance of both proteins in BBa_K4765117

Characterization

Sequencing map

contributed by Fudan iGEM 2023
Figure1 Sequencing map of H. ex mtSSB

Sequencing was performed using the two primers：T7: 5-TAATACGACTCACTATAGG-3 and Kan-F: 5-ATTCTCACCGGATTCAGT-3.

Anti-desiccation capability

We attempt to compare the anti-desiccation capabilities of H. ex mtSSB and the pre-existing protein SAHS 33020BBa_K2306003 in order to identify a suitable desiccation-resistant protein for B-HOME. We've designed an experimental group with H. ex mtSSB and SAHS 33020, and used E. coli transformed with the empty PET28 vector as a control. All proteins are expressed via leakage in E. coli BL21 DE3.

All the groups are incubated overnight. After reaching an OD value of 1.0, liquid culture is centrifuged, and the supernatant is removed. Pellets are dried overnight in SpeedVac (Savant SpeedVac SC100) .Finally, the pellets are resuspended in LB medium and dilute 10^5-fold for CFU counting.

contributed by Fudan iGEM 2023
Figure2 The dry weight of H.ex mtSSB, TDP, and control after freeze-drying

There is no statistically significant difference in the dry weights among the experimental groups, indicating that the number of E. coli is consistent between the bacterial tubes.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 186
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 454
    Illegal BsaI.rc site found at 535
    Illegal SapI site found at 348


Reference

  1. ↑ Hibshman, J. D., Clark-Hachtel, C. M., Bloom, K. S., & Goldstein, B. (2023). A bacterial expression cloning screen reveals tardigrade single-stranded DNA-binding proteins as potent desicco-protectants (2023.08.21.554171). bioRxiv. https://doi.org/10.1101/2023.08.21.554171