Difference between revisions of "Part:BBa K3853009"
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The synthetic plasmid was linearized and electrotransformed into <em>Pichia pastoris</em> GS115. Colony PCR was applied to screen monoclonal colonies that had positive transformation results for subsequent gene sequencing verification. The target bands appeared in the normal position, and the result was shown in <strong>Fig. 3</strong>. Sequencing results (<strong>File 1</strong>) showed a successful transformation. | The synthetic plasmid was linearized and electrotransformed into <em>Pichia pastoris</em> GS115. Colony PCR was applied to screen monoclonal colonies that had positive transformation results for subsequent gene sequencing verification. The target bands appeared in the normal position, and the result was shown in <strong>Fig. 3</strong>. Sequencing results (<strong>File 1</strong>) showed a successful transformation. | ||
| + | <p><b>2. qRT-PCR</b></p> | ||
===References=== | ===References=== | ||
Revision as of 05:09, 21 October 2021
his-tag-SpyTag-AAO
Aryl alcohol oxidase (AAO) is an enzyme containing flavin-adenine-dinucleotide (FAD). 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_K3853053 to construct the expression system to express and purify the protein.
Biology
Aryl alcohol oxidase (AAO, EC 1.1.3.7), a member of the glucose-methanol-choline oxidase / dehydrogenase (GMC) superfamily, is an enzyme containing flavin-adenine-dinucleotide (FAD) that catalyze the oxidation of aromatic and aliphatic allylic primary alcohols to the corresponding aldehydes while reducing molecular oxygen to H2O2(The mechanism has shown in Fig. 1).
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_K3853052 (Fig. 2) and transformed the constructed plasmid into Pichia pastoris GS115 to verify its expression. The positive clones were cultivated.
Characterization
1. Identification
The synthetic plasmid was linearized and electrotransformed into Pichia pastoris GS115. Colony PCR was applied to screen monoclonal colonies that had positive transformation results for subsequent gene sequencing verification. The target bands appeared in the normal position, and the result was shown in Fig. 3. Sequencing results (File 1) showed a successful transformation.
2. qRT-PCR
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]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 699
- 21INCOMPATIBLE WITH RFC[21]Unknown
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Unknown
- 1000COMPATIBLE WITH RFC[1000]