Difference between revisions of "Part:BBa K4765016"
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__TOC__
 
__TOC__
 
===Introduction===
 
===Introduction===
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''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.  
+
''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<ref>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</ref>.
+
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<ref>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</ref>.
 +
 
 
===Usage and Biology===
 
===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 [https://parts.igem.org/Part:BBa_K2306003 Secretory-abundant heat soluble protein 33020 (SAHS 33020)] and tested the combined desiccation resistance of both proteins in [https://parts.igem.org/Part:BBa_K4765117 ribozyme+RBS+CDS+stem-loop module:  ''H. ex'' mtSSB+SAHS 33020]
+
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( [https://parts.igem.org/Part:BBa_K2306003 BBa_K2306003]) and tested the combined desiccation resistance of both proteins in [https://parts.igem.org/Part:BBa_K4765117 BBa_K4765117]
 +
 
 
===Characterization===
 
===Characterization===
 +
====Sequencing map====
 +
{|
 +
| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/zsl/mtssb-sequence.png" alt="contributed by Fudan iGEM 2023"></html>
 +
|-
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| '''Figure 1. Sequencing map of ''H. ex'' mtSSB'''
 +
Sequencing starts from the T7 terminator, with the primer 5-GCTAGTTATTGCTCAGCGG-3.
  
 +
|}
  
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<!-- Add more about the biology of this part here
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====Successful Protein Expression====
−
===Usage and Biology===
+
{|
 +
| <html><img style="width:200px" src="https://static.igem.wiki/teams/4765/wiki/zsl/protein-gel/mtssb.png" alt="contributed by Fudan iGEM 2023"></html>
 +
|-
 +
| '''Figure 2. SDS-PAGE electrophoresis of ''H. ex'' mtSSB'''
 +
We constructed ''H. ex'' mtSSB into the pET28a plasmid and transformed it into ''E. coli'' BL21 DE3. Lanes 1 to 2 represent ''H. ex'' mtSSB, ''H. ex'' mtSSB + IPTG, as indicated by the red arrow, we successfully expressed ''H. ex'' mtSSB.
 +
 
 +
====Desiccation Survival Assay====
 +
We attempt to compare the anti-desiccation capabilities of ''H. ex'' mtSSB and the pre-existing protein [https://parts.igem.org/Part:BBa_K2306003 BBa_K2306003] in order to identify a suitable desiccation-resistant protein for B-HOME. Through the experiments, we found that H. ex mtSSB exhibits desiccation resistance comparable to SAHS.
 +
{|
 +
| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/zsl/anti-desiccation-protocol.png" alt="contributed by Fudan iGEM 2023"></html>
 +
|-
 +
| '''Figure 3. Workflow of Desiccation Survival Assay'''
 +
 
 +
|}
 +
 
 +
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 for 6.5 hr in  SpeedVac (Savant SpeedVac SC100) under 4 0 °C .Finally, the pellets are resuspended in LB medium and dilute 10^5-fold for CFU counting.
 +
{|
 +
| <html><img style="width:250px" src="https://static.igem.wiki/teams/4765/wiki/zsl/dry-weight-1-2.png" alt="contributed by Fudan iGEM 2023"></html>
 +
|-
 +
| '''Figure 4. Dry weight of ''H.ex'' mtSSB, TDP, and control after 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.
 +
|}
 +
 
 +
{|
 +
| <html><img style="width:250px" src="https://static.igem.wiki/teams/4765/wiki/zsl/mt-ssb-anti-desiccation-capability-1.png" alt="contributed by Fudan iGEM 2023"></html>
 +
|-
 +
| '''Figure 5. CFU colony count of ''H.ex'' mtSSB, TDP, and control after drying'''
 +
The ''E. coli'' expressing ''H.ex'' mtSSB and SAHS showed higher CFU counts after drying compared to the control group, indicating their desiccation resistance abilities. Mean values from three rounds of indepdent experiments are shown. Huge error bars suggest variations between rounds of experiments.
 +
|}
 +
 
 +
====Anti-UV Survival Assay====
 +
We employed the Colony-Forming Unit (CFU) assay. After plasmid transformation and plating, we shielded one/half of the agar plate from UV light using a black cloth, while the other one/half was exposed to UV irradiation (6W power) with wavelengths of 254 nm and 365 nm for 10 seconds.
 +
 
 +
{|
 +
| <html><img style="width:400px" src="https://static.igem.wiki/teams/4765/wiki/results-wyj/uv.jpg" alt="contributed by Fudan iGEM 2023"></html>
 +
|-
 +
| '''Figure 6. Anti-UV Assay.'''
 +
 
 +
|}
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 +
In our experimental findings, while mtSSB effectively guards against DNA damage caused by desiccation through its binding to single-stranded DNA, we did not observe any enhancement in UV resistance in ''E. coli'' expressing mtSSB. Surprisingly, the survival rate of ''E. coli'' expressing mtSSB was even lower than that of the control group without mtSSB.
 +
 
 +
 
 +
{|
 +
| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/results-wyj/uv-cfu.png" alt="contributed by Fudan iGEM 2023"></html>
 +
|-
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| '''Figure 7. Plates displaying transformed ''E. coli'' after anti-UV assay.'''
 +
|}
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 +
{|
 +
| <html><img style="width:300px" src="https://static.igem.wiki/teams/4765/wiki/results-wyj/uvresults.png" alt="contributed by Fudan iGEM 2023"></html>
 +
|-
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| '''Figure 8. Survival Rate after UV Exposure.'''
 +
Percentage of viable ''E. coli'' expressing proteins following UV radiation exposure<br> (Note: The quantitative graph is based on the whole plate CFU to avoid the blurriness at the boundaries of the cloth-shielded area from UV.)
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|}
  
 
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<partinfo>BBa_K4765016 parameters</partinfo>
 
<partinfo>BBa_K4765016 parameters</partinfo>
 
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==Reference==
 
==Reference==

Latest revision as of 15:21, 12 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
Figure 1. Sequencing map of H. ex mtSSB

Sequencing starts from the T7 terminator, with the primer 5-GCTAGTTATTGCTCAGCGG-3.

Successful Protein Expression

contributed by Fudan iGEM 2023
Figure 2. SDS-PAGE electrophoresis of H. ex mtSSB

We constructed H. ex mtSSB into the pET28a plasmid and transformed it into E. coli BL21 DE3. Lanes 1 to 2 represent H. ex mtSSB, H. ex mtSSB + IPTG, as indicated by the red arrow, we successfully expressed H. ex mtSSB.

Desiccation Survival Assay

We attempt to compare the anti-desiccation capabilities of H. ex mtSSB and the pre-existing protein BBa_K2306003 in order to identify a suitable desiccation-resistant protein for B-HOME. Through the experiments, we found that H. ex mtSSB exhibits desiccation resistance comparable to SAHS.

contributed by Fudan iGEM 2023
Figure 3. Workflow of Desiccation Survival Assay

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 for 6.5 hr in SpeedVac (Savant SpeedVac SC100) under 4 0 °C .Finally, the pellets are resuspended in LB medium and dilute 10^5-fold for CFU counting.

contributed by Fudan iGEM 2023
Figure 4. Dry weight of H.ex mtSSB, TDP, and control after 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.

contributed by Fudan iGEM 2023
Figure 5. CFU colony count of H.ex mtSSB, TDP, and control after drying

The E. coli expressing H.ex mtSSB and SAHS showed higher CFU counts after drying compared to the control group, indicating their desiccation resistance abilities. Mean values from three rounds of indepdent experiments are shown. Huge error bars suggest variations between rounds of experiments.

Anti-UV Survival Assay

We employed the Colony-Forming Unit (CFU) assay. After plasmid transformation and plating, we shielded one/half of the agar plate from UV light using a black cloth, while the other one/half was exposed to UV irradiation (6W power) with wavelengths of 254 nm and 365 nm for 10 seconds.

contributed by Fudan iGEM 2023
Figure 6. Anti-UV Assay.

In our experimental findings, while mtSSB effectively guards against DNA damage caused by desiccation through its binding to single-stranded DNA, we did not observe any enhancement in UV resistance in E. coli expressing mtSSB. Surprisingly, the survival rate of E. coli expressing mtSSB was even lower than that of the control group without mtSSB.


contributed by Fudan iGEM 2023
Figure 7. Plates displaying transformed E. coli after anti-UV assay.
contributed by Fudan iGEM 2023
Figure 8. Survival Rate after UV Exposure.

Percentage of viable E. coli expressing proteins following UV radiation exposure
(Note: The quantitative graph is based on the whole plate CFU to avoid the blurriness at the boundaries of the cloth-shielded area from UV.)

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