Part:BBa_K3853010
his-tag-SpyTag-HFB1
Hydrophobin-1 (HFB1) is a kind of class Ⅱ HFBs derived from Trichoderma reesei. This is the version with SpyTag. In order to constructed our multi-enzyme complex, we introduced the SpyTag/SpyCatcher system. We added SpyTag to the N-terminus of each protein through 10 × ELP, which is a oligopeptide linker that does not affect the function of the protein it attaches to, as described in the literature[1]. So this part can be combined with dCas9-SpyCatcher through covalent isopeptide action, thereby being immobilized on dsDNA. His-tag was added to purify the protein. We use BBa_K3853054 to construct the expression system to express and purify the protein.
Biology
Hydrophobin (HFB) is a variety of secretory proteins rich in hydrophobic amino acids. As a type of biosurfactant, it possesses surface activity. By self-assembling at hydrophilic-hydrophobic interfaces, HFBs can enhance the affinity between hydrophilic proteins and hydrophobic materials. There is an opinion that some bacteria use similar biosurfactants to assist specific enzymes to degrade hydrocarbons. Besides, scientists have found that the degradation efficiency of polyethylene terephthalate (PET) can be significantly improved when several PET-degrading enzymes (PETase) are fused with certain types of class Ⅱ HFBs. This result is partly attributed to the surface activity provided by HFBs.
Usage
The SpyTag was fused to the N-terminus of AAO. The fusion protein could combined with dCas9-SpyCatcher (which is producted by BBa_K3853055 ) for multi-enzyme complex assembly. we obtained the composite part BBa_K3853054 (Fig. 1) and transformed the constructed plasmid into Pichia pastoris GS115 to verify its expression. The positive clones were cultivated.
Fig. 1 Gene circuit of SpyTag-HFB1.
Characterization
1. Identification
The plasmid carrying the gene of SpyTag-HFB1 was transformed into E.coli Rosetta (DE3) for heterogenous expression. Colony PCR was applied to screen monoclonal colonies that had positive transformation results for subsequent gene sequencing verification. The bands of target gene appeared at the normal position, the result was shown in Fig. 2. Sequencing verification results (
File 1
)
showed a successful transformation.
Fig. 2 Agarose gel electrophoresis of PCR products of monoclonal colonies of SpyTag-HFB1.
References
[1] Lim, S., Kim, J., Kim, Y., Xu, D. & Clark, D. S. CRISPR/Cas-directed programmable assembly of multi-enzyme complexes. Chemical communications (Cambridge, England) 56, 4950-4953, doi:10.1039/d0cc01174f (2020).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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