Difference between revisions of "Part:BBa K4765018"

 
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FEN1 plays a pivotal role in DNA maintenance, addressing 5' overhanging "flaps" formed when DNA strands can't bind correctly.
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<html><img style="float:right;width:128px" src="https://static.igem.wiki/teams/4765/wiki/2023-b-home.png" alt="contributed by Fudan iGEM 2023"></html>
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__TOC__
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===Introduction===
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FEN1 plays a pivotal role in DNA maintenance, addressing 5' overhanging "flaps" formed when DNA strands can't bind correctly. It also manages the 5' ends of Okazaki fragments in lagging strand DNA synthesis. This protein interacts directly with AP endonuclease 1 during long-patch base excision repair, enabling seamless substrate transfer between enzymes. Belonging to the XPG/RAD2 endonuclease family, it's one of ten essential proteins for cell-free DNA replication<ref>Hiraoka, L. R., Harrington, J. J., Gerhard, D. S., Lieber, M. R., & Hsieh, C. L. (1995). Sequence of human FEN-1, a structure-specific endonuclease, and chromosomal localization of the gene (FEN1) in mouse and human. ''Genomics, 25''(1), 220–225. https://doi.org/10.1016/0888-7543(95)80129-a</ref>. However, certain DNA structures can hinder its flap processing at trinucleotide repeats, concealing the crucial 5' end. This obstruction can compromise its protective function, potentially causing site-specific trinucleotide expansions linked to genetic disorders.
  
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===Usage and Biology===
 
===Usage and Biology===
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We heterologously expressed codon-optimized FEN1 in ''E. coli'', endowing it with anti-UV capability.
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===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-fen1.jpg" alt="contributed by Fudan iGEM 2023"></html>
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|-
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| '''Figure 1. Sequencing map of FEN1'''
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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/f.png" alt="contributed by Fudan iGEM 2023"></html>
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|-
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| '''Figure 2. SDS-PAGE electrophoresis of FEN1'''
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We constructed FEN1 mtSSB into the pET28a plasmid and transformed it into ''E. coli'' BL21 DE3. Lanes 1 to 2 represent FEN1, FEN1 + IPTG, as indicated by the red arrow, we successfully expressed FEN1.
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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 demonstrated that most DNA repair and binding proteins exhibited **a higher survival rate** compared to plain ''E. coli'', indicating improved anti-UV tolerance, especially XRCC1 and FEN1. We hypothesized that these proteins function by aiding in DNA repair or binding to DNA, thus 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.
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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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==Reference==

Latest revision as of 15:22, 12 October 2023


FEN1

contributed by Fudan iGEM 2023

Introduction

FEN1 plays a pivotal role in DNA maintenance, addressing 5' overhanging "flaps" formed when DNA strands can't bind correctly. It also manages the 5' ends of Okazaki fragments in lagging strand DNA synthesis. This protein interacts directly with AP endonuclease 1 during long-patch base excision repair, enabling seamless substrate transfer between enzymes. Belonging to the XPG/RAD2 endonuclease family, it's one of ten essential proteins for cell-free DNA replication[1]. However, certain DNA structures can hinder its flap processing at trinucleotide repeats, concealing the crucial 5' end. This obstruction can compromise its protective function, potentially causing site-specific trinucleotide expansions linked to genetic disorders.

Usage and Biology

We heterologously expressed codon-optimized FEN1 in E. coli, endowing it with anti-UV capability.

Characterization

Sequencing map

contributed by Fudan iGEM 2023
Figure 1. Sequencing map of FEN1

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 FEN1

We constructed FEN1 mtSSB into the pET28a plasmid and transformed it into E. coli BL21 DE3. Lanes 1 to 2 represent FEN1, FEN1 + IPTG, as indicated by the red arrow, we successfully expressed FEN1.

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 demonstrated that most DNA repair and binding proteins exhibited **a higher survival rate** compared to plain E. coli, indicating improved anti-UV tolerance, especially XRCC1 and FEN1. We hypothesized that these proteins function by aiding in DNA repair or binding to DNA, thus 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
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 556
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1134
    Illegal AgeI site found at 240
  • 1000
    COMPATIBLE WITH RFC[1000]


Reference
  1. ↑ Hiraoka, L. R., Harrington, J. J., Gerhard, D. S., Lieber, M. R., & Hsieh, C. L. (1995). Sequence of human FEN-1, a structure-specific endonuclease, and chromosomal localization of the gene (FEN1) in mouse and human. Genomics, 25(1), 220–225. https://doi.org/10.1016/0888-7543(95)80129-a