Difference between revisions of "Part:BBa K3089013"
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<partinfo>BBa_K3089013 short</partinfo> | <partinfo>BBa_K3089013 short</partinfo> | ||
| − | <span class='h3bb'>Sequence and Features</span> | + | <span class='h3bb'><h3>Sequence and Features</h3></span> |
<partinfo>BBa_K3089013 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3089013 SequenceAndFeatures</partinfo> | ||
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This recombinant protein is our own new design which has a property of both cohesion (CsgA) and adhesion (Mfp5). CsgA is an amyloid-like protein encoded on genome of E.coli MG1655 providing mechanical cohesive strength. Mfp5 is mussel foot proteins from Mytilus galloprovincialis responsible for interface adhesion. By adding two Mfp5s to CsgA, we expect it to have a stronger adhesive property. The results show, this recombinant protein can adhere to plastics and glasses better than any otherparts in our toolbox. The sequence of this composite part is optimized to achieve better expression in Pichia pastoris. | This recombinant protein is our own new design which has a property of both cohesion (CsgA) and adhesion (Mfp5). CsgA is an amyloid-like protein encoded on genome of E.coli MG1655 providing mechanical cohesive strength. Mfp5 is mussel foot proteins from Mytilus galloprovincialis responsible for interface adhesion. By adding two Mfp5s to CsgA, we expect it to have a stronger adhesive property. The results show, this recombinant protein can adhere to plastics and glasses better than any otherparts in our toolbox. The sequence of this composite part is optimized to achieve better expression in Pichia pastoris. | ||
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| + | <span class='h3bb'>Sequence and Features</span> | ||
| + | <partinfo>BBa_K3089013 SequenceAndFeatures</partinfo> | ||
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| + | <figure> | ||
| + | <center> | ||
| + | <img width="450px" src="https://2019.igem.org/wiki/images/d/d7/T--Greatbay_SCIE--distribution_in_barnacle_cements.png"> | ||
| + | </center> | ||
| + | </figure> | ||
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| + | <figcaption> | ||
| + | <center> | ||
| + | Figure 1. Protein distribution in barnacle cements | ||
| + | </center> | ||
| + | </figcaption> | ||
<h3>Characterization</h3> | <h3>Characterization</h3> | ||
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<h3> Molecular cloning </h3> | <h3> Molecular cloning </h3> | ||
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<img width="600px" src="https://2019.igem.org/wiki/images/d/d4/T--Greatbay_SCIE--molecular_cloning_of_csgA-linker-mfp5-mfp5.jpeg"> | <img width="600px" src="https://2019.igem.org/wiki/images/d/d4/T--Greatbay_SCIE--molecular_cloning_of_csgA-linker-mfp5-mfp5.jpeg"> | ||
| + | </center> | ||
</figure> | </figure> | ||
| − | <figcaption> | + | <figcaption> |
| − | Figure | + | <center> |
| + | Figure 2. Illustration of gene insertion into Pichia pastoris GS115 genome. Box in blue indicates the recombinant protein CsgA-linker-mfp5-mfp5. | ||
| + | </center> | ||
</figcaption> | </figcaption> | ||
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<img width="450px" src="https://2019.igem.org/wiki/images/a/aa/T--Greatbay_SCIE--gene_insertion_gel_csga-mfp5-mfp5.jpeg"> | <img width="450px" src="https://2019.igem.org/wiki/images/a/aa/T--Greatbay_SCIE--gene_insertion_gel_csga-mfp5-mfp5.jpeg"> | ||
| + | </center> | ||
</figure> | </figure> | ||
| − | <figcaption> | + | <figcaption> |
| − | Determination of gene insertion into the Pichia genome by gel electrophoresis. | + | <center> |
| + | Figure 3. Determination of gene insertion into the Pichia genome by gel electrophoresis. | ||
| + | </center> | ||
</figcaption> | </figcaption> | ||
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Proteins were expressed in small scale induced by methanol. Unfortunately, no proteins of interest were found in culture medium of CsgA-Mfp5-Mfp5 after induced expression for 48 hours at 30℃(Figure 3). | Proteins were expressed in small scale induced by methanol. Unfortunately, no proteins of interest were found in culture medium of CsgA-Mfp5-Mfp5 after induced expression for 48 hours at 30℃(Figure 3). | ||
</p> | </p> | ||
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| + | <figure> | ||
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<img width="450px" src="https://2019.igem.org/wiki/images/1/17/T--Greatbay_SCIE--SDS-PAGE_of_CsgA-mfp5-mfp5.jpeg"> | <img width="450px" src="https://2019.igem.org/wiki/images/1/17/T--Greatbay_SCIE--SDS-PAGE_of_CsgA-mfp5-mfp5.jpeg"> | ||
| + | </center> | ||
</figure> | </figure> | ||
<figcaption> | <figcaption> | ||
| − | SDS-PAGE confirm expression(30℃，48h) of CsgA-mfp5-mfp5 and rBalcp19k-mfp5(4 colonies) expression. P, cell pellets. S, supernatant. | + | <center> |
| + | Figure 4. SDS-PAGE confirm expression(30℃，48h) of CsgA-mfp5-mfp5 and rBalcp19k-mfp5(4 colonies) expression. P, cell pellets. S, supernatant. | ||
| + | </center> | ||
</figcaption> | </figcaption> | ||
| + | </html> | ||
Latest revision as of 11:54, 21 October 2019
csgA-linker-mfp5-mfp5-His fusion protein for yeast expression
Sequence and Features
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 274
Illegal PstI site found at 23 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 274
Illegal PstI site found at 23 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 274
Illegal XhoI site found at 244 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 274
Illegal PstI site found at 23 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 274
Illegal PstI site found at 23 - 1000COMPATIBLE WITH RFC[1000]
This recombinant protein is our own new design which has a property of both cohesion (CsgA) and adhesion (Mfp5). CsgA is an amyloid-like protein encoded on genome of E.coli MG1655 providing mechanical cohesive strength. Mfp5 is mussel foot proteins from Mytilus galloprovincialis responsible for interface adhesion. By adding two Mfp5s to CsgA, we expect it to have a stronger adhesive property. The results show, this recombinant protein can adhere to plastics and glasses better than any otherparts in our toolbox. The sequence of this composite part is optimized to achieve better expression in Pichia pastoris.
Sequence and Features
Characterization
Three different experiments were done to characterise the BBa_K3089013 biobrick:
- Molecular cloning
- Protein expression
Molecular cloning
CsgA-linker-mfp5-mfp5-His was synthesized and cloned to yeast Pichia pastoris and 1 strain was verified by gel electrophoresis and sequencing (Figure 2).
Protein expression
Proteins were expressed in small scale induced by methanol. Unfortunately, no proteins of interest were found in culture medium of CsgA-Mfp5-Mfp5 after induced expression for 48 hours at 30℃(Figure 3).
