Difference between revisions of "Part:BBa K3089021"
 
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<partinfo>BBa_K3089021 short</partinfo>
 
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This composite parts is meant to express csgA-linker-mfp5 fusion genes under T7 promoter and purify CsgA-linker-Mfp5 recombinant proteins by His-tag affinity purification. 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).
 
  
 
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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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<h5>Figure. 2 Geraniol production analysis by gas chromatography. (A) GPPS and GES are arranged in operon regulated by pTac, placed on a high copy vector pUC20. (B) The MVA pathway is split into two clusters and placed on a low copy vector with the upper cluster containing three genes and the downstream containing four. (C) A combined plasmid with GPPS&GES operon and MVA pathway with a low copy p15A origin. (D) Gas chromatography for geraniol produced by E. coli expressing pMVA only (middle trace) or pMVA-GPPS-GES (bottom trace) whose peaks coincide with a geraniol standard (top trace). (E) Geraniol yield is noticeable with only the heterologous MVA pathway. Upon introduction of GPPS and GES, the yield doubled relative to the negative control pMVA only.</h5>
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===Usage and Biology===
 
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<span class='h3bb'>Sequence and Features</span>
 
 
<partinfo>BBa_K3089021 SequenceAndFeatures</partinfo>
 
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<partinfo>BBa_K3089021 parameters</partinfo>
 
<partinfo>BBa_K3089021 parameters</partinfo>
 
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<h3>Characterization</h3>
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<p>
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BBa_K3089021 was characterized in following experiments:
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</p>
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<ul>
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<li>protein expression
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<li> protein purification</li>
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<li> Surface coating analysis</li>
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</ul>
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<div></div>
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<h3> Protein expression </h3>
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<Figure>
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<img width="600px" src="https://static.igem.org/mediawiki/parts/c/c2/T--Greatbay_SCIE--P--021-Figure_1.png">
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<center><figcaption> Figure 1. The circuit of the protein BBa_K30889021 </figcaption></center>
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<p>
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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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<img width="600px" src="https://static.igem.org/mediawiki/parts/5/53/T--Greatbay_SCIE--Detection_of_expression_level.jpeg">
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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>
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<p>
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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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<img width="250px" src="https://static.igem.org/mediawiki/parts/6/6d/T--Greatbay_SCIE--SDS-PAGE_of_csgA-MFP-5.png">
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<figcaption>
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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>
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<p>
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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.
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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.
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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.
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<Figure>
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<center>
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<img width="600px" src="https://static.igem.org/mediawiki/parts/8/8e/T--Greatbay_SCIE--P--Surface_coating.jpeg">
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</figure>
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</center>
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<center>
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<figcaption>
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Figure 4. Surface coating analysis of recombinant proteins on hydrophilic glass slides (left) and hydrophobic polystyrene (PS) plates (right).
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</figcaption>
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</center>
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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