Difference between revisions of "Part:BBa K5258001"
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             <caption>Figure 2: Gene maps of 25#SBD</caption>
 
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             <caption>Figure 4: SDS-PAGE of SBD 25#</caption>
 
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Revision as of 08:48, 28 September 2024

25#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 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 581
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 81
  • 1000
    COMPATIBLE WITH RFC[1000]


25#SBD Gene Documentation

Profile

Name: 25#SBD

Base Pairs: 543 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 25#SBD
Figure 2: Gene maps of 25#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 achieve high-resolution separation of complex protein mixtures. The results showed that 25#SBD, induced by IPTG, was abundantly expressed in the precipitate, with no protein detected in the supernatant. As a result, the EMSA experiment could not be performed due to the lack of soluble viable protein.

In the future, we will explore methods to enhance the solubility of this protein, such as changing expression vectors or investigating protein denaturation techniques.

Figure 4: SDS-PAGE of SBD 25#
Figure 4: SDS-PAGE of SBD 25#

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).