Difference between revisions of "Part:BBa K5258000"
 
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             <caption>Figure 4: SDS-PAGE of 6# SBD protein expression</caption>
 
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Latest revision as of 04:11, 29 September 2024


6#SBD

SBD (Sulfur Binding Domain). After DNA is phosphorothioated, 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. The SBD recognizes PT-DNA by embedding sulfur into its hydrophobic cavity, hydrogen bonds, and electrostatic interactions between the SBD and the DNA, which also considerably contribute to binding. SBD proteins have a strong binding affinity on PT-DNA, especially 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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 279
  • 1000
    COMPATIBLE WITH RFC[1000]

6#SBD Gene Documentation

Profile

Name: 6#SBD

Base Pairs: 450 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 6# SBD
Figure 2: Gene maps of 6# 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

To verify successful protein expression, we conducted an SDS-PAGE test (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), a standard method to assess protein expression levels, purity, and mass. SDS and a reducing agent break bonds within the proteins, assisting in their linearization. The resulting gel image confirms that pET28a-6#SBD was successfully expressed with high purity. The first three lanes show the supernatant, precipitate, and flow-through, containing unwanted proteins. The rightmost lane displays the eluted liquid containing our target protein, with a single band at the predicted length, indicating a successful outcome.

Figure 4: SDS-PAGE of 6# SBD protein expression
Figure 4: SDS-PAGE of 6# SBD protein expression

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).