Difference between revisions of "Part:BBa K4765019"

Latest revision as of 15:23, 12 October 2023

XRCC1

contributed by Fudan iGEM 2023

XRCC1 is a vital protein in DNA repair, particularly for single-strand breaks caused by radiation and alkylating agents. It collaborates with DNA ligase III, polymerase β, and poly (ADP-ribose) polymerase in base excision repair. It might also play a role in meiosis-related DNA processes. Although XRCC1 lacks enzymatic activity, it acts as a scaffold for repair enzymes, aiding in single-strand break repair, base excision repair, and nucleotide excision repair^[1]. XRCC1's structure includes three domains, enabling interactions with various repair proteins. Additionally, it is involved in error-prone microhomology-mediated end joining repair of double-strand breaks, often leading to mutation-inducing deletions.

Usage and Biology

We heterologously expressed codon-optimized XRCC1 in E. coli, endowing it with anti-UV capability.

Characterization

Sequencing map

Figure 1. Sequencing map of XRCC11 Sequencing starts from the T7 terminator, with the primer 5-GCTAGTTATTGCTCAGCGG-3.

Successful Protein Expression

Figure 2. SDS-PAGE electrophoresis of XRCC1

We constructed XRCC1 mtSSB into the pET28a plasmid and transformed it into E. coli BL21 DE3. Lanes 1 to 2 represent XRCC1, XRCC1 + IPTG, as indicated by the red arrow, we successfully expressed XRCC1.

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
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 685
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1735

Reference

↑ London R. E. (2015). The structural basis of XRCC1-mediated DNA repair. DNA repair, 30, 90–103. https://doi.org/10.1016/j.dnarep.2015.02.005

[1] London R. E. (2015). The structural basis of XRCC1-mediated DNA repair. DNA repair, 30, 90–103. https://doi.org/10.1016/j.dnarep.2015.02.005

[1]

@@ Line 18: / Line 18: @@
 | '''Figure 1. Sequencing map of XRCC11''' Sequencing starts from the T7 terminator, with the primer 5-GCTAGTTATTGCTCAGCGG-3.
 |}
+====Successful Protein Expression====
+{|
+| <html><img style="width:200px" src="https://static.igem.wiki/teams/4765/wiki/zsl/protein-gel/x.png" alt="contributed by Fudan iGEM 2023"></html>
+|-
+| '''Figure 2. SDS-PAGE electrophoresis of XRCC1'''
+We constructed XRCC1 mtSSB into the pET28a plasmid and transformed it into ''E. coli'' BL21 DE3. Lanes 1 to 2 represent XRCC1, XRCC1 + IPTG, as indicated by the red arrow, we successfully expressed XRCC1.
 ====Anti-UV Survival Assay====
@@ Line 25: / Line 31: @@
 | <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 2. Anti-UV Assay.'''
+| '''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.
+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.
 {|
 | <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>
 |-
-| '''Figure 3. Plates displaying transformed E. coli after anti-UV assay.'''
+| '''Figure 4. Plates displaying transformed E. coli after anti-UV assay.'''
 |}
@@ Line 40: / Line 46: @@
 | <html><img style="width:400px" src="https://static.igem.wiki/teams/4765/wiki/results-wyj/uvresults.png" alt="contributed by Fudan iGEM 2023"></html>
 |-
-| '''Figure 4. Survival Rate after UV Exposure.'''
+| '''Figure 5. 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.)
 |}