Part:BBa_K3853054

R0085-C0012-B2002-his-tag-SpyTag-HFB1-B0012

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.

2. Purification and Proof of the expression

We used Ni-NTA affinity column to obtain purified SpyTag-HFB1. Target bands could be observed at the position of about 15 kDa (Fig. 3), which means the protein of SpyTag-HFB1 was successfully expressed, and the related gene worked well.

Fig. 3 SDS-PAGE of purification products of SpyTag-HFB1. flow-through is the liquid flowing out of the column when loading the sample, 50 mM imidazole and 500 mM imidazation are eluates of different imidazole concentrations.</b>

3. Functional Verification

We dropped the liquid containing SpyTag-HFB1 and the liquid without SpyTag-HFB1 on a plastic dish, and observed the hydrophobic angle, shape and flattening state of different liquids to evaluate the effect of HFB1. The results were shown in Table 1, Fig. 4, Video 1. The contact angle of the liquid containing SpyTag-HFB1 was smaller, and the droplets were dispersed. Compared to SpyTag-HFB1-free liquid, the droplets containing SpyTag-HFB1 were not easy to move on the plastic surface, which showed that HFB1 effectively improved the hydrophilicity of the plastic surface.

Table 1. Contact angle measurement among different samples.

Significant difference analysis:^*** p < 0.001, ^**** p < 0.0001

Fig. 4 Contact angle of two types of liquid. A: Comparison between buffer and HFB1-buffer mixture after shaking on a PP surface. Contact angle on a PP surface (B), a HFB1-modified PP surface (C), a PE surface (D), and a HFB1-modified PE surface (E).

Video 1. Comparison of fluid movement status

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1170
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]