Difference between revisions of "Part:BBa K4765018"

Revision as of 14:01, 12 October 2023

FEN1

contributed by Fudan iGEM 2023

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

Figure 1. Sequencing map of FEN1

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

Successful Protein Expression

Figure 2. SDS-PAGE electrophoresis of FEN1

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 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.

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.

Figure 4. Plates displaying transformed E. coli after anti-UV assay.

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

↑ 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

[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

[1]

@@ Line 22: / Line 22: @@
 ====Successful Protein Expression====
 {|
-| <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>
+| <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 FEN1'''
-We constructed FEN1 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.
+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 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.