Difference between revisions of "Part:BBa K3089013"

 
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<partinfo>BBa_K3089013 parameters</partinfo>
 
<partinfo>BBa_K3089013 parameters</partinfo>
 
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<h3>Introduction</h3>
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<p>
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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 other parts in our toolbox. The sequence of this composite part is optimized to achieve better expression in Pichia pastoris.
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</p>
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<h3>Characterization</h3>
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<p>
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Three different experiments were done to characterise the BBa_K3089013 biobrick:
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<p>
 +
<ul>
 +
<li>Molecular cloning</li>
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<li>Protein purification</li>
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</ul>
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<h3> Molecular cloning  </h3>
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<Figure>
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<img width="450px" src="https://2019.igem.org/wiki/images/d/d4/T--Greatbay_SCIE--molecular_cloning_of_csgA-linker-mfp5-mfp5.jpeg">
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</figure>
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<figcaption> Figure 1. Illustration of gene insertion into Pichia pastoris GS115 genome. Box in blue indicates the recombinant protein CsgA-linker-mfp5-mfp5.
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</figcaption>
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<p>
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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).
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</p>
 +
 +
<Figure>
 +
<img width="450px" src="https://2019.igem.org/wiki/images/a/aa/T--Greatbay_SCIE--gene_insertion_gel_csga-mfp5-mfp5.jpeg">
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</figure>
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<figcaption> Determination of gene insertion into the Pichia genome by gel electrophoresis.  </figcaption>
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<h3> Protein purification </h3>
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 +
<p>
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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).
 +
 +
<Figure>
 +
<img width="450px" src="https://2019.igem.org/wiki/images/1/17/T--Greatbay_SCIE--SDS-PAGE_of_CsgA-mfp5-mfp5.jpeg">
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</figure>
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<figcaption>
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SDS-PAGE confirm expression(30℃，48h) of CsgA-mfp5-mfp5 and rBalcp19k-mfp5(4 colonies) expression. P, cell pellets. S, supernatant.
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</figcaption>

Revision as of 10:30, 20 October 2019


csgA-linker-mfp5-mfp5-His fusion protein for yeast expression

Csga-linker-mfp5-mfp5-His fusion protein is the purified version of Csga-linker-Mfp5 recombinant protein by His-tag affinity purification (a -Histidine-Histidine-Histidine-Histidine-Histidine-Histidine-Histidine tag fused on the carboxyl terminal). We designed this part by putting together two identical Mfp5 mussel proteins and hoped it shows higher adhesive features than Csga-Mfp5. CsgA is an amyloid-like protein encoded on genome of E.coli MG1655 providing mechanical cohesive strength and Mfp5 is a mussel foot protein from Mytilus galloprovincialis responsible for interface adhesion. This recombinant protein would self-assemble into fibrous bundles or films with adhesive properties by displaying the mussel adhesion domains on the surface of amyloid scaffolds, which would be a promising new generation of bio-inspired adhesives for a wide range of applications. This part was based on the paper "Strong underwater adhesives made by self-assembling multi-protein nanofibres" (Zhong et al, 2014) and has been characterized by iGEM15_TU_Delft (BBa_K1583104). We decided to use another chassis for the same protein rather than solely using E.coli BL21(DE3) because of relatively low and harder protein expression.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 274
    Illegal PstI site found at 23
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 274
    Illegal PstI site found at 23
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 274
    Illegal XhoI site found at 244
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 274
    Illegal PstI site found at 23
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 274
    Illegal PstI site found at 23
  • 1000
    COMPATIBLE WITH RFC[1000]


Introduction

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 other parts in our toolbox. The sequence of this composite part is optimized to achieve better expression in Pichia pastoris.

Characterization

Three different experiments were done to characterise the BBa_K3089013 biobrick: <p>

  • Molecular cloning
  • Protein purification

Molecular cloning


<Figure> <img width="450px" src="T--Greatbay_SCIE--molecular_cloning_of_csgA-linker-mfp5-mfp5.jpeg"> </figure>

<figcaption> Figure 1. Illustration of gene insertion into Pichia pastoris GS115 genome. Box in blue indicates the recombinant protein CsgA-linker-mfp5-mfp5. </figcaption>

<p> 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).

<Figure> <img width="450px" src="T--Greatbay_SCIE--gene_insertion_gel_csga-mfp5-mfp5.jpeg"> </figure>

<figcaption> Determination of gene insertion into the Pichia genome by gel electrophoresis. </figcaption>

Protein purification

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). <Figure> <img width="450px" src="T--Greatbay_SCIE--SDS-PAGE_of_CsgA-mfp5-mfp5.jpeg"> </figure> <figcaption> SDS-PAGE confirm expression(30℃，48h) of CsgA-mfp5-mfp5 and rBalcp19k-mfp5(4 colonies) expression. P, cell pellets. S, supernatant. </figcaption>