Difference between revisions of "Part:BBa K3089021"
 
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<h3>Introduction</h3>
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We used this fusion protein linker GGGGSGGGGS to link all bioglue-related parts in our toolbox, making them to be recombinant adhesive proteins. For example, the linker was added between csgA(From E.coli MG1655) and mfp5 (from Mytilus galloprovincialis)to get csgA-linker-mfp5 (BBa_K3089021). Other recombinant protein, csgA-linker-mfp5-linker-mfp3-His (BBa_K3089022), csgA-linker-mfp5-mfp5-His(BBa_K3089023),fp1-linker-mfp5-linker-fp1-His(BBa_K3089024), mfp5-linker-mfp3-His(BBa_K3089025),rBalcp19K-linker-mfp5-His(BBa_K3089026) were also designed following the same method by inserting this fusion protein linker between different parts to preventing disruption.
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This composite parts is meant to express <i>csgA</i>-linker-<i>mfp5</i> fusion genes under T7 promoter. CsgA is an amyloid-like protein encoded on genome of <i>E.coli</i> 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 designed based on the core part——<i>mfp5</i>, which has been submitted into parts registry by iGEM2015 Tu_delft(<a href="https://parts.igem.org/Part:BBa_K1583002"target="_blank">BBa_K1583002</a>). We hoped that fusion of CsgA on the N-terminal of mfp5 would improve its expression and make it become a bioadsive composed of both adhesion and cohesion.  
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<p>CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms taken from <i>E.coli K-12 MG1655</i>. Inspired by mussels, the Mfp5 (mussel foot protein) has high adhesive properties towards wet polar surfaces. CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms of E.coli. This protein is transported as an unfolded protein out of the cell. Outside the cell, CsgA proteins self-assemble into nanowires after nucleation on the membrane protein CsgB. By creating a fusion protein of Mfp5 and CsgA, the adhesive properties of the mussel foot protein are combined with the formation of nanowires.
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<partinfo>BBa_K3089021 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K3089021 parameters</partinfo>
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<h3>Characterization</h3>
 
<h3>Characterization</h3>
 
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BBa_K3089011 was characterized in following experiments:
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BBa_K3089021 was characterized in following experiments:
 
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<h3> Protein expression </h3>
 
<h3> Protein expression </h3>
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<figcaption> The circuit of the protein BBa_K30889026 </figcaption>
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<center><figcaption> Figure 1. The circuit of the protein BBa_K30889021 </figcaption></center>
  
 
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The predicted size of rBalcp19k-linker-mfp5 is 28.17 kDa, and the isoelectric point is 10.41. rBalcp19k-linker-mfp5 was cloned into pET28b and expressed in E.coli BL21(DE3) Rosetta by 500μM IPTG for 5h at 37℃.  
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csgA-linker-mfp5 was cloned into pET28b and expressed in <i>E.coli BL21(DE3) Rosetta by 500μM IPTG for 5h at 37℃. In order to detect its expression, whole cells were collected after induction by centrifuging and prepared for SDS-PAGE. Results (Figure 1)showed that no obvious protein bands of CsgA-mfp5(~24 kDa) could be observed on lane csgA-linker-mfp5 compared with lane NC (pET28b empty vector) and <i>mfp5</i>(<a href="https://parts.igem.org/Part:BBa_K1583002"target="_blank">BBa_K1583002</a>) , which means the expression of this protein is not well in BL21(DE3) Rosetta (Figure 1A). <b>Quantitative densitometry of SDS-PAGE gel analysis revealed that csgA-linker-mfp5 expressed better than mfp5 alone (Figure 1B).</b>
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In order to detect its expression, whole cells were collected after induction by centrifuging and prepared for SDS-PAGE. Results showed that no protein bands of rBalcp19k-linker-mfp5(~28 kDa) could be observed on lane rBalcp19k-mfp5 compared with lane pET28b (pET28b empty vector)(Figure 1A), which means the expression of this protein is not well in BL21(DE3) Rosetta.  
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We can get the same results using quantitative densitometry analysis of SDS-PAGE gels (Figure 1B).
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<figcaption> Figure 2 Detection of the expression level of all recombinant proteins by SDS-PAGE.(A) SDS-PAGE of whole-cell lysates of all recombinant proteins. Red arrows show the predicted place of certain proteins. (B) Protein SDS-PAGE bands optical densities were measured by quantitative densitometry of SDS-PAGE of whole-cell aliquots. </figcaption>
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<figcaption> Figure 2. Detection of the expression level of all recombinant proteins by SDS-PAGE.(A) SDS-PAGE of whole-cell lysates of all recombinant proteins. Red arrows show the predicted place of certain proteins. (B) Protein SDS-PAGE bands optical densities were measured by quantitative densitometry of SDS-PAGE of whole-cell aliquots. </figcaption>
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<h3> Protein purification </h3>
 
<h3> Protein purification </h3>
  
 
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Barnacle cement proteins are very promising in making biomedical bio-glues. rBalcp19K from Balanus albicostatus had the properties of both self-assembly and adhesion.
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For we make producing underwater bio-adhesives as the final goal of our project, we straightly went on protein purification of CsgA-linker-mfp5 with the methods used for Mfp5 purification. Weak bands presented on the lane E2 and its size is larger than predicted which is resulting from high isoelectric point value (9.76). Protein concentrations of CsgA-mfp5 were measured by BCA assay and its yield is 0.5mg/L. In conclusion, putting CsgA on the N-terminal of Mfp5 increase its expression level.
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It also could function in more basic condition than Mfps. Thus we also designed a novel recombinant protein by combining it with Mfp5. We expected rBalcp19k-Mfp5 would perform better adhesive ability to solidify our idea of modularisation of Mfp5.
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We tried to purify it under native conditions, and we found bands of rBalcp19K-linker-mfp5 appeared between 25kDa and 35kDa on 12% SDS-PAGE gel(Figure 2), which meant it was successfully expressed and purified under native condition(see details on our methods).  
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Protein concentrations of rBalcp19k-linker-mfp5 were measured by BCA assay, and its yield is 1mg/L.
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Figure 3. SDS-PAGE of purified rBalcp19k-mfp5 by affinity chromatography under native conditions.  
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Lanes: M, protein molecular weight marker; NC, whole-cell sample of pET28b empty vector; WC, whole-cell sample of recombinant protein rBalcp19K; S, soluble cell fraction; W1, fraction.
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Figure 3. Coomassie-stained SDS-PAGE gels confirm purification of the expressed protein CsgA-mfp5 by cobalt-resin columns. Lanes: M, protein molecular weight marker; NC, whole-cell sample of pET28b empty vector; WC, whole-cell sample of recombinant proteins; E, eluted proteins. 12% SDS-PAGE gels were used for the analyses.
 
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<h3> Surface coating analysis </h3>  
 
<h3> Surface coating analysis </h3>  
 
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Figure 4. Surface coating analysis of recombinant proteins on hydrophilic glass slides (left) and hydrophobic polystyrene (PS) plates (right).
 
Figure 4. Surface coating analysis of recombinant proteins on hydrophilic glass slides (left) and hydrophobic polystyrene (PS) plates (right).
 
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<h3>References</h3>
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Zhong, C. et al., 2014. Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nature nanotechnology, 9(10), pp.858–66

Latest revision as of 14:12, 21 October 2019

T7 promoter+csgA-linker-mfp5-His fusion protein


This composite parts is meant to express csgA-linker-mfp5 fusion genes under T7 promoter. 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 designed based on the core part——mfp5, which has been submitted into parts registry by iGEM2015 Tu_delft(BBa_K1583002). We hoped that fusion of CsgA on the N-terminal of mfp5 would improve its expression and make it become a bioadsive composed of both adhesion and cohesion.


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 314
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 314
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 516
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 314
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 314
  • 1000
    COMPATIBLE WITH RFC[1000]



Characterization

BBa_K3089021 was characterized in following experiments:

  • protein expression
  • protein purification
  • Surface coating analysis

Protein expression

Figure 1. The circuit of the protein BBa_K30889021

csgA-linker-mfp5 was cloned into pET28b and expressed in E.coli BL21(DE3) Rosetta by 500μM IPTG for 5h at 37℃. In order to detect its expression, whole cells were collected after induction by centrifuging and prepared for SDS-PAGE. Results (Figure 1)showed that no obvious protein bands of CsgA-mfp5(~24 kDa) could be observed on lane csgA-linker-mfp5 compared with lane NC (pET28b empty vector) and mfp5(BBa_K1583002) , which means the expression of this protein is not well in BL21(DE3) Rosetta (Figure 1A). Quantitative densitometry of SDS-PAGE gel analysis revealed that csgA-linker-mfp5 expressed better than mfp5 alone (Figure 1B).

Figure 2. Detection of the expression level of all recombinant proteins by SDS-PAGE.(A) SDS-PAGE of whole-cell lysates of all recombinant proteins. Red arrows show the predicted place of certain proteins. (B) Protein SDS-PAGE bands optical densities were measured by quantitative densitometry of SDS-PAGE of whole-cell aliquots.

Protein purification

For we make producing underwater bio-adhesives as the final goal of our project, we straightly went on protein purification of CsgA-linker-mfp5 with the methods used for Mfp5 purification. Weak bands presented on the lane E2 and its size is larger than predicted which is resulting from high isoelectric point value (9.76). Protein concentrations of CsgA-mfp5 were measured by BCA assay and its yield is 0.5mg/L. In conclusion, putting CsgA on the N-terminal of Mfp5 increase its expression level.

Figure 3. Coomassie-stained SDS-PAGE gels confirm purification of the expressed protein CsgA-mfp5 by cobalt-resin columns. Lanes: M, protein molecular weight marker; NC, whole-cell sample of pET28b empty vector; WC, whole-cell sample of recombinant proteins; E, eluted proteins. 12% SDS-PAGE gels were used for the analyses.

Surface coating analysis

After obtaining a small number of recombinant proteins, surface coating analysis for qualitatively assessing the surface adsorption ability of recombinant proteins was conducted on two of most commonly used bio-related surfaces: hydrophilic glass slides and hydrophobic polystyrene tissue culture plates. As shown in Figure3, rBalcp19k-linker-mfp5 recombinant protein showed higher surface absorption abilities on both different substrates than rBalcp19k without fusion of mfp5 on its C-terminal. It’s suggested that Mfp improves the coating ability of rBalcp19k-linker-mfp5 fusion proteins. The In-vitro DOPA modification by mTyr-CNK tyrosinase significantly improved its surface absorption abilities, which suggested the positive contribution of DOPA in adhesive protein performances.

Figure 4. Surface coating analysis of recombinant proteins on hydrophilic glass slides (left) and hydrophobic polystyrene (PS) plates (right).

References

Zhong, C. et al., 2014. Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nature nanotechnology, 9(10), pp.858–66