Difference between revisions of "Part:BBa K3089021"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K3089021 short</partinfo> | <partinfo>BBa_K3089021 short</partinfo> | ||
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<html> | <html> | ||
− | This composite parts is meant to express csgA-linker-mfp5 fusion genes. 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 | + | <p> |
− | (<a href="https://parts.igem.org/Part:BBa_K1583002">BBa_K1583002</a>). | + | 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. |
+ | </p> | ||
</html> | </html> | ||
− | === | + | <partinfo>BBa_K3089021 SequenceAndFeatures</partinfo> |
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+ | <!-- Uncomment this to enable Functional Parameter display | ||
+ | ===Functional Parameters=== | ||
+ | <partinfo>BBa_K3089021 parameters</partinfo> | ||
+ | <!-- --> | ||
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+ | <html> | ||
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+ | <h3>Characterization</h3> | ||
<p> | <p> | ||
BBa_K3089021 was characterized in following experiments: | BBa_K3089021 was characterized in following experiments: | ||
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</p> | </p> | ||
+ | <ul> | ||
+ | <li>protein expression | ||
+ | <li> protein purification</li> | ||
+ | <li> Surface coating analysis</li> | ||
+ | </ul> | ||
+ | <div></div> | ||
− | + | <h3> Protein expression </h3> | |
− | + | ||
<html> | <html> | ||
+ | <Figure> | ||
<center> | <center> | ||
+ | <img width="600px" src="https://static.igem.org/mediawiki/parts/c/c2/T--Greatbay_SCIE--P--021-Figure_1.png"> | ||
+ | </figure> | ||
+ | </center> | ||
+ | |||
+ | <center><figcaption> Figure 1. The circuit of the protein BBa_K30889021 </figcaption></center> | ||
+ | |||
+ | <p> | ||
+ | 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> | ||
+ | </p> | ||
+ | |||
<Figure> | <Figure> | ||
− | <img width=" | + | <center> |
+ | <img width="600px" src="https://static.igem.org/mediawiki/parts/5/53/T--Greatbay_SCIE--Detection_of_expression_level.jpeg"> | ||
</figure> | </figure> | ||
− | < | + | |
− | </ | + | <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> |
</center> | </center> | ||
− | |||
+ | <br> | ||
+ | <h3> Protein purification </h3> | ||
+ | |||
+ | <p> | ||
+ | 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. | ||
<html> | <html> | ||
− | + | ||
<Figure> | <Figure> | ||
− | <img width=" | + | <center> |
+ | <img width="250px" src="https://static.igem.org/mediawiki/parts/6/6d/T--Greatbay_SCIE--SDS-PAGE_of_csgA-MFP-5.png"> | ||
</figure> | </figure> | ||
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− | |||
</center> | </center> | ||
− | |||
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− | |||
− | < | + | <figcaption> |
− | + | <center> | |
− | + | 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. | |
+ | </figcaption> | ||
+ | </center> | ||
+ | <br> | ||
+ | <h3> Surface coating analysis </h3> | ||
+ | <p> | ||
+ | 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. | ||
+ | </p> | ||
− | < | + | <Figure> |
− | == | + | <center> |
− | < | + | <img width="600px" src="https://static.igem.org/mediawiki/parts/8/8e/T--Greatbay_SCIE--P--Surface_coating.jpeg"> |
− | < | + | </figure> |
+ | </center> | ||
+ | <center> | ||
+ | <figcaption> | ||
+ | Figure 4. Surface coating analysis of recombinant proteins on hydrophilic glass slides (left) and hydrophobic polystyrene (PS) plates (right). | ||
+ | </figcaption> | ||
+ | </center> | ||
+ | |||
+ | <h3>References</h3> | ||
+ | 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.
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 314
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 314
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 516
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 314
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 314
- 1000COMPATIBLE WITH RFC[1000]
Characterization
BBa_K3089021 was characterized in following experiments:
- protein expression
- protein purification
- Surface coating analysis
Protein expression
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).
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.
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.