Difference between revisions of "Part:BBa K5258002"

 
 
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A typical SBD is monomeric, composed of ~160 amino acids, and contains a hydrophobic surface cavity. The SBD recognizes PT-DNA by embedding sulfur into its hydrophobic cavity, hydrogen bonds, and electrostatic interactions between the SBD and the DNA, significantly contributing to binding. SBD proteins have a binding solid affinity on PT-DNA, primarily when binding to PT-double-strand (ds)DNA. This allows SBD to be utilized as a targeting tool in which synthetic PT-modified oligos were used as probes to anneal with target single-strand DNA and form a hemi PT-modified double strand to be recognized by SBD.
 
A typical SBD is monomeric, composed of ~160 amino acids, and contains a hydrophobic surface cavity. The SBD recognizes PT-DNA by embedding sulfur into its hydrophobic cavity, hydrogen bonds, and electrostatic interactions between the SBD and the DNA, significantly contributing to binding. SBD proteins have a binding solid affinity on PT-DNA, primarily when binding to PT-double-strand (ds)DNA. This allows SBD to be utilized as a targeting tool in which synthetic PT-modified oligos were used as probes to anneal with target single-strand DNA and form a hemi PT-modified double strand to be recognized by SBD.
  
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===Usage and Biology===
 
  
 
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===Functional Parameters===
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<partinfo>BBa_K5258002 parameters</partinfo>
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<head>
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<!-- -->
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    <meta charset="UTF-8">
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    <meta name="viewport" content="width=device-width, initial-scale=1.0">
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    <title>46#SBD Gene Documentation</title>
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</head>
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<body>
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    <!-- Profile Section -->
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    <h2>Profile</h2>
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    <p><strong>Name:</strong> 46#SBD</p>
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    <p><strong>Base Pairs:</strong> 546 bp</p>
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    <p><strong>Origin:</strong> Streptomyces coelicolor A3(2), Streptomyces lividans [1], synthesized</p>
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    <!-- Properties Section -->
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    <h3>Properties</h3>
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    <p>Sulfur atoms are cleverly embedded in a shallow hydrophobic pit (binding pocket) in one of the conserved domains (SBD). The sulfur atom has a larger atomic radius than the oxygen atom. As a result, the attraction effect of the outer shell electrons is weakened, showing a better effect. The weak electronegativity gives the sulfur atoms hydrophobic properties. SBD uses this subtle difference to distinguish sulfur-modified DNA from ordinary DNA.</p>
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    <!-- Figure 1 -->
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    <div style="text-align:center;">
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        <img src="https://static.igem.wiki/teams/5258/bba-k5258002/1.jpg" width="30%" alt="Figure 1: The 3D-version structure of the sulfur binding domain (SBD)">
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        <div style="text-align:center;">
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            <caption>Figure 1: The 3D-version structure of the sulfur binding domain (SBD)</caption>
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        </div>
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    </div>
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    <!-- Usage and Biology Section -->
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    <h3>Usage and Biology</h3>
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    <p>SBD contains a hydrophobic surface cavity formed by the aromatic ring of Y164, the pyrrolidine ring of P165, and the nonpolar side chains of four other residues, which serve as the cavity's lid, base, and wall. The SBD and PT-DNA undergo conformational changes upon binding.</p>
 +
    <p>The SBD recognizes PT-DNA by embedding sulfur into its hydrophobic cavity; hydrogen bonds and electrostatic interactions between the SBD and the DNA also significantly contribute to binding. SBD proteins have a strong affinity for PT-DNA, especially when binding to PT-double-strand (ds)DNA. This allows SBD to be utilized as a targeting tool, where synthetic PT-modified oligos are used as probes to anneal with target single-strand DNA and form a hemi PT-modified double strand to be recognized by SBD [2].</p>
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    <!-- Figure 2 -->
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    <div style="text-align:center;">
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        <img src="https://static.igem.wiki/teams/5258/bba-k5258002/2.png" width="70%" alt="Figure 2: Gene maps of 46#SBD">
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        <div style="text-align:center;">
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            <caption>Figure 2: Gene maps of 46#SBD</caption>
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        </div>
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    </div>
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    <!-- Cultivation Section -->
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    <h3>Cultivation</h3>
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    <p>After the recombinant plasmid enters the <i>E.coli</i> DH5α by heat shock, the <i>E.coli</i> DH5α needs to be renewed for 1 hour at 37°C, followed by the spread plate method to evenly place the bacteria fluid on the LB medium. The medium is reversed, and the bacteria are cultivated for 12 hours at 37°C.</p>
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    <!-- Figure 3 -->
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    <div style="text-align:center;">
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        <img src="https://static.igem.wiki/teams/5258/bba-k5258002/3.jpg" width="70%" alt="Figure 3: Result of gel electrophoresis of gene extracted from the E.coli">
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        <div style="text-align:center;">
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            <caption>Figure 3: Result of gel electrophoresis of gene extracted from the E.coli</caption>
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        </div>
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    </div>
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    <!-- Protein Purification and SDS-PAGE Section -->
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    <h3>Protein Purification and SDS-PAGE</h3>
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    <p>SDS-PAGE was conducted to assess protein purity. Samples included the supernatant, sediment, flow-through liquid, and eluent containing SBD46# (from left to right). A significant band at 20 kDa in the eluent confirmed that the protein was successfully expressed.</p>
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    <!-- Figure 4 -->
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    <div style="text-align:center;">
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        <img src="https://static.igem.wiki/teams/5258/bba-k5258002/4.jpg" width="30%" alt="Figure 4: Result of the SDS-PAGE">
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        <div style="text-align:center;">
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            <caption>Figure 4: Result of the SDS-PAGE</caption>
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        </div>
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    </div>
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    <!-- References Section -->
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    <h3>References</h3>
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    <p>[1] Liu, G., Fu, W., Zhang, Z., He, Y., Yu, H., Zhao, Y., Deng, Z., Wu, G., He, X. Sulfur binding domain of ScoMcrA complexed with phosphorothioated DNA PDB.</p>
 +
    <p>[2] Liu, G., Fu, W., Zhang, Z. et al. Structural basis for the recognition of sulfur in phosphorothioated DNA. Nat Commun 9, 4689 (2018).</p>
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 +
</body>
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</html>

Latest revision as of 05:12, 29 September 2024

46#SBD


A typical SBD is monomeric, composed of ~160 amino acids, and contains a hydrophobic surface cavity. The SBD recognizes PT-DNA by embedding sulfur into its hydrophobic cavity, hydrogen bonds, and electrostatic interactions between the SBD and the DNA, significantly contributing to binding. SBD proteins have a binding solid affinity on PT-DNA, primarily when binding to PT-double-strand (ds)DNA. This allows SBD to be utilized as a targeting tool in which synthetic PT-modified oligos were used as probes to anneal with target single-strand DNA and form a hemi PT-modified double strand to be recognized by SBD.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 217
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


<!DOCTYPE html> 46#SBD Gene Documentation

Profile

Name: 46#SBD

Base Pairs: 546 bp

Origin: Streptomyces coelicolor A3(2), Streptomyces lividans [1], synthesized

Properties

Sulfur atoms are cleverly embedded in a shallow hydrophobic pit (binding pocket) in one of the conserved domains (SBD). The sulfur atom has a larger atomic radius than the oxygen atom. As a result, the attraction effect of the outer shell electrons is weakened, showing a better effect. The weak electronegativity gives the sulfur atoms hydrophobic properties. SBD uses this subtle difference to distinguish sulfur-modified DNA from ordinary DNA.

Figure 1: The 3D-version structure of the sulfur binding domain (SBD)
Figure 1: The 3D-version structure of the sulfur binding domain (SBD)

Usage and Biology

SBD contains a hydrophobic surface cavity formed by the aromatic ring of Y164, the pyrrolidine ring of P165, and the nonpolar side chains of four other residues, which serve as the cavity's lid, base, and wall. The SBD and PT-DNA undergo conformational changes upon binding.

The SBD recognizes PT-DNA by embedding sulfur into its hydrophobic cavity; hydrogen bonds and electrostatic interactions between the SBD and the DNA also significantly contribute to binding. SBD proteins have a strong affinity for PT-DNA, especially when binding to PT-double-strand (ds)DNA. This allows SBD to be utilized as a targeting tool, where synthetic PT-modified oligos are used as probes to anneal with target single-strand DNA and form a hemi PT-modified double strand to be recognized by SBD [2].

Figure 2: Gene maps of 46#SBD
Figure 2: Gene maps of 46#SBD

Cultivation

After the recombinant plasmid enters the E.coli DH5α by heat shock, the E.coli DH5α needs to be renewed for 1 hour at 37°C, followed by the spread plate method to evenly place the bacteria fluid on the LB medium. The medium is reversed, and the bacteria are cultivated for 12 hours at 37°C.

Figure 3: Result of gel electrophoresis of gene extracted from the E.coli
Figure 3: Result of gel electrophoresis of gene extracted from the E.coli

Protein Purification and SDS-PAGE

SDS-PAGE was conducted to assess protein purity. Samples included the supernatant, sediment, flow-through liquid, and eluent containing SBD46# (from left to right). A significant band at 20 kDa in the eluent confirmed that the protein was successfully expressed.

Figure 4: Result of the SDS-PAGE
Figure 4: Result of the SDS-PAGE

References

[1] Liu, G., Fu, W., Zhang, Z., He, Y., Yu, H., Zhao, Y., Deng, Z., Wu, G., He, X. Sulfur binding domain of ScoMcrA complexed with phosphorothioated DNA PDB.

[2] Liu, G., Fu, W., Zhang, Z. et al. Structural basis for the recognition of sulfur in phosphorothioated DNA. Nat Commun 9, 4689 (2018).