Difference between revisions of "Part:BBa K3853010"
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===Usage=== | ===Usage=== | ||
The SpyTag was fused to the N-terminus of AAO. The fusion protein could combined with dCas9-SpyCatcher (which is producted by <partinfo>BBa_K3853055</partinfo> ) for multi-enzyme complex assembly. we obtained the composite part <partinfo>BBa_K3853054</partinfo> (<b>Fig. 1</b>) and transformed the constructed plasmid into <i>Pichia pastoris</i> GS115 to verify its expression. The positive clones were cultivated. | The SpyTag was fused to the N-terminus of AAO. The fusion protein could combined with dCas9-SpyCatcher (which is producted by <partinfo>BBa_K3853055</partinfo> ) for multi-enzyme complex assembly. we obtained the composite part <partinfo>BBa_K3853054</partinfo> (<b>Fig. 1</b>) and transformed the constructed plasmid into <i>Pichia pastoris</i> GS115 to verify its expression. The positive clones were cultivated. | ||
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| + | <img src="https://2021.igem.org/wiki/images/7/7f/T--CPU_CHINA--BBa_K3853010_fig_1.png" width="90%" style="float:center;"> | ||
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| + | <p style="font-size:1rem"> | ||
| + | </p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </html> | ||
| + | <p style="text-align:center"><b>Fig. 1 Gene circuit of SpyTag-HFB1.</b></p> | ||
===References=== | ===References=== | ||
Revision as of 08:58, 21 October 2021
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.
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]