Difference between revisions of "Part:BBa K4765018"
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| <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> | | <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> | ||
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| − | | '''Figure 2. SDS-PAGE electrophoresis of | + | | '''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. | 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. | ||
Revision as of 14:01, 12 October 2023
FEN1
Contents
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
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| Figure 1. Sequencing map of FEN1
Sequencing starts from the T7 terminator, with the primer 5-GCTAGTTATTGCTCAGCGG-3. |
Successful Protein Expression
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| 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. Anti-UV Survival AssayWe 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.
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.
Sequence and Features
Assembly Compatibility:
Reference |
- ↑ 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