Difference between revisions of "Part:BBa K4765025"

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===Characterization===
 
===Characterization===
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====Sequencing map====
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{|
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| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/zsl/sequencing-map-of-rv-dsup.jpg" alt="contributed by Fudan iGEM 2023"></html>
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|-
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| '''Figure 1. Sequencing map of Rv Dsup''' Sequencing starts from the T7 terminator, with the primer 5-GCTAGTTATTGCTCAGCGG-3.
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|}
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====Successful Protein Expression====
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{|
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| <html><img style="width:200px" src="https://static.igem.wiki/teams/4765/wiki/zsl/protein-gel/d.png" alt="contributed by Fudan iGEM 2023"></html>
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|-
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| '''Figure 2. SDS-PAGE electrophoresis of Rv Dsup'''
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We constructed Rv Dsup into the pET28a plasmid and transformed it into ''E. coli'' BL21 DE3. Lanes 1 to 2 represent Rv Dsup, Rv Dsup + IPTG, as indicated by the red arrow, we successfully expressed Rv Dsup.
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|}
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====Anti-UV Survival Assay====
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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.
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{|
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| <html><img style="width:400px" src="https://static.igem.wiki/teams/4765/wiki/results-wyj/uv.jpg" alt="contributed by Fudan iGEM 2023"></html>
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|-
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| '''Figure 3. Anti-UV Assay.'''
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|}
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Our experimental results revealed that the majority of DNA repair and binding proteins exhibited significantly increased survival rates when compared to plain ''E. coli,'' indicating enhanced resistance to UV radiation, with XRCC1 and FEN1 being particularly noteworthy. Although ''E. coli'' expressing Dsup showed a slightly higher survival rate, this difference wasn't statistically significant. We hypothesized that these proteins operate by facilitating DNA repair or binding to DNA, thereby shielding chromatin from hydroxyl radicals induced by UV radiation.<br>
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Interestingly, we observed that the expression of green fluorescence **(stayGold)** in *E. coli*, intended as a negative control, significantly enhanced the survival rate. We suspected that this effect may be due to fluorescent protein absorbing a certain amount of UV radiation through structural changes.
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{|
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| <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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|-
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| '''Figure 4. Plates displaying transformed ''E. coli'' after anti-UV assay.'''
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|}
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{|
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| <html><img style="width:400px" src="https://static.igem.wiki/teams/4765/wiki/results-wyj/uvresults.png" alt="contributed by Fudan iGEM 2023"></html>
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|-
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| '''Figure 5. Survival Rate after UV Exposure.'''
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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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===Usage and Biology===
 
  
 
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<partinfo>BBa_K4765025 parameters</partinfo>
 
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==Reference==
 
==Reference==

Latest revision as of 15:24, 12 October 2023


Rv Dsup codon optimized

contributed by Fudan iGEM 2023

Introduction

Rv Dsup is a damage suppressor protein from the Tardigrade Species Ramazzottius Varieornatus [1] . This is a nucleosome-binding protein that protects chromatin from hydroxyl radicals[2] , thereby inhibiting DNA molecule breakage. This protein can reduce X-ray-induced damage in human cells by 40%.

Usage and Biology

We optimized the codons of wildtype Rv Dsup for E. coli K12 and heterologously expressed it in E. coli to enhance its survival rate under UV radiation.

Characterization

Sequencing map

contributed by Fudan iGEM 2023
Figure 1. Sequencing map of Rv Dsup 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 Rv Dsup

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

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 3. Anti-UV Assay.

Our experimental results revealed that the majority of DNA repair and binding proteins exhibited significantly increased survival rates when compared to plain E. coli, indicating enhanced resistance to UV radiation, with XRCC1 and FEN1 being particularly noteworthy. Although E. coli expressing Dsup showed a slightly higher survival rate, this difference wasn't statistically significant. We hypothesized that these proteins operate by facilitating DNA repair or binding to DNA, thereby shielding chromatin from hydroxyl radicals induced by UV radiation.

Interestingly, we observed that the expression of green fluorescence **(stayGold)** in *E. coli*, intended as a negative control, significantly enhanced the survival rate. We suspected that this effect may be due to fluorescent protein absorbing a certain amount of UV radiation through structural changes.

contributed by Fudan iGEM 2023
Figure 4. Plates displaying transformed E. coli after anti-UV assay.


contributed by Fudan iGEM 2023
Figure 5. 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 AgeI site found at 169
    Illegal AgeI site found at 240
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 166
    Illegal SapI.rc site found at 895


Reference

  1. ↑ Hashimoto, T., Horikawa, D. D., Saito, Y., Kuwahara, H., Kozuka-Hata, H., Shin-I, T., Minakuchi, Y., Ohishi, K., Motoyama, A., Aizu, T., Enomoto, A., Kondo, K., Tanaka, S., Hara, Y., Koshikawa, S., Sagara, H., Miura, T., Yokobori, S. I., Miyagawa, K., Suzuki, Y., … Kunieda, T. (2016). Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein. Nature communications, 7, 12808. https://doi.org/10.1038/ncomms12808
  2. ↑ Chavez, C., Cruz-Becerra, G., Fei, J., Kassavetis, G. A., & Kadonaga, J. T. (2019). The tardigrade damage suppressor protein binds to nucleosomes and protects DNA from hydroxyl radicals. eLife, 8, e47682. https://doi.org/10.7554/eLife.47682