Difference between revisions of "Part:BBa K3089013"
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===Functional Parameters===
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<partinfo>BBa_K3089013 parameters</partinfo>
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<h3>Introduction</h3>
 
<h3>Introduction</h3>
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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
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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.
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  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
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  parts in our toolbox. The sequence of this composite part is optimized to achieve better expression in Pichia pastoris.
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   Three different experiments were done to characterise the BBa_K3089013 biobrick:
 
   Three different experiments were done to characterise the BBa_K3089013 biobrick:
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<ul>
 
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   <li>Molecular cloning</li>
 
   <li>Molecular cloning</li>
 
   <li>Protein purification</li>
 
   <li>Protein purification</li>
 
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<h3> Molecular cloning </h3>
 
<h3> Molecular cloning </h3>
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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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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> Determination of gene insertion into the Pichia genome by gel electrophoresis. </figcaption>
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Determination of gene insertion into the Pichia genome by gel electrophoresis.  
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<h3> Protein purification </h3>
 
<h3> Protein purification </h3>

Revision as of 10:42, 20 October 2019

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 otherparts 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:

  • Molecular cloning
  • Protein purification

Molecular cloning

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

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).

Determination of gene insertion into the Pichia genome by gel electrophoresis.

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).

SDS-PAGE confirm expression(30℃，48h) of CsgA-mfp5-mfp5 and rBalcp19k-mfp5(4 colonies) expression. P, cell pellets. S, supernatant.