Difference between revisions of "Part:BBa K3089026"
 
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<partinfo>BBa_K3089026 short</partinfo>
 
<partinfo>BBa_K3089026 short</partinfo>
  
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T7 promoter+rBalcp19K-linker-mfp5-His
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<html>
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<p>
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This composite part is meant to express rBalcp19k-linker-mfp5 fusion genes under T7 promoter, and 7XHis-tag was fused on the C terminal to achieve affinity protein purification. This composite part is also expressed in Pichia pastoris( <a href="https://parts.igem.org/Part:BBa_K3089017"target="_blank">BBa_K3089017</a>)after codon optimization, and is an updated version from the basic part rBalcp19k( <a href="https://parts.igem.org/Part:BBa_K3089008"target="_blank">BBa_K3089008</a>) which is expressed in Pichia pastoris as well. Mfp5 is the mussel foot protein from Mytilus galloprovincialis responsible for interface adhesion. rBalcp19k, the barnacle adhesive protein from Balanus albicostatus, its homologous gene serves as adhesive proteins, also playing the role of interfacial adhesion in a way that mfp5 does in mussels(Figure 1.) We linked them together to achieve better adhesive ability, because rBalcp19k can self-assemble into aggregated nanofibers at acidic pHs. This composite part would be a promising new generation of bio-inspired adhesives for a wide range of applications.
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</p>
  
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<!-- Add more about the biology of this part here
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<center><figure>
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===Usage and Biology===
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<img width="450px" src="https://2019.igem.org/wiki/images/6/6d/T--Greatbay_SCIE--cross_sections_of_the_barnacle_cement.png">
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</figure></center>
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<center><figcaption>
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Figure 1. Schematic showing the cross-sections of the barnacle cement and various possible factors involved in barnacle adhesion.
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</figcaption></center>
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</html>
  
 
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'><h3>Sequence and Features</h3></span>
 
<partinfo>BBa_K3089026 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3089026 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3089026 parameters</partinfo>
 
<partinfo>BBa_K3089026 parameters</partinfo>
 
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<h3>Introduction</h3>
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<h3>Characterisation</h3>
 
<p>
 
<p>
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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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Three different experiments were done to characterise the BBa_K3089026 biobrick:
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</p>
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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 is combined with the formation of nanowires.
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<ul>
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<li>protein expression</li>
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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_K1583104 biobrick:
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<ul><li>protein expression</li>
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<li> protein purification</li>
 
<li> protein purification</li>
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<li> Surface coating analysis </li></ul>
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<li> Surface coating analysis </li>
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</p>
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</ul>
  
 
<h3> Protein expression </h3>
 
<h3> Protein expression </h3>
  
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<html>
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<center><Figure>
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<center>
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<img width="600px" src="https://static.igem.org/mediawiki/parts/1/16/T--Greatbay_SCIE--P--026-Figure_1.png">
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<Figure>
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</figure></center>
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<img width="450px" src="https://2019.igem.org/wiki/images/0/04/T--Greatbay_SCIE--The_circuit_of_T7_promoter+rBalcp19k-linker-mfp5-His.png">
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</figure>
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<figcaption> Figure 1 illustration of all reccombiant protein with linkers. </figcaption>
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<center><figcaption> Figure 2. The circuit of the protein BBa_K30889026 </figcaption></center>
  
 
<p>
 
<p>
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All 6 recombinant adhesive proteins with linkers were 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 they have different expression levels. fp1-linker-mfp5-linker-fp1-His(Fp1-mfp5-fp1) and Mfp5-linker-mfp3(Mfp5-mfp3) expressed better than other proteins. <span style="font-weight: bold;">Recombinant proteins with bands of interest in Figure 2A are in correct places as predicted, which means linker did help link two parts.</span>  
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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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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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</p>
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<center><Figure>
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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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</figure></center>
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<center><figcaption> Figure 3. 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>
  
 
<h3> Protein purification </h3>
 
<h3> Protein purification </h3>
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<p>
 
<p>
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For we make producing underwater bioadhesives as the final goal of our project, we straightly went on recombinant protein purification. CsgA is an amyloid-like protein characterized by β-strands and CsgA monomers would form aggregates after expression inside cells. Therefore, denature protein purification methods were used for CsgA containing proteins(Figure 3AB). Weak bands presented on the lane E2 for CsgA-linker-mfp5-mfp5 and CsgA-liner-mfp5, lane E1 for CsgA-linker-mfp5-linker-mfp3. Fp151 formed inclusion body after expression, so we also purified it under denature conditions. Bands of Fp1-linker-mfp5-fp1 presented on SDS-PAGE gel on lane E2 and E6. The mixed solutions after elution were then loaded on the columns and dialyzed with PBS buffer (PH=6.0) to wash away imidazole, meanwhile, protein was concentrated. After that concentrated protein was put under 4℃ for 72 hours to make it renature. rBalcp19k-linker-mfp5 were purified under native conditions, clear bands of interest were observed on lane E2 and E3 by Coomassie-blue-stained SDS-PAGE. Protein concentrations were measured by BCA assay and their yields were shown in Table 1. <span style="font-weight: bold;"> In conclusion, recombinant proteins with linker GGGGSGGGGGS can be successfully purified using different methods.</span>
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Barnacle cement proteins are very promising in making biomedical bio-glues. rBalcp19K from
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<i>Balanus albicostatus</i>
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had the properties of both self-assembly and adhesion.  
  
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<html>
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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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<center>
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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 3), which meant it was successfully expressed and purified under native condition(see details on our methods).  
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<Figure>
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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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<img width="450px" src="https://2019.igem.org/wiki/images/1/10/T--Greatbay_SCIE--Figure_2_Detection_of_expression_level_of_all_recombinant_proteins_by_SDS-PAGE..png">
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</figure>
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<figcaption> Figure 2 Detection of 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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<center><Figure>
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<img width="300px" src="https://2019.igem.org/wiki/images/f/f5/T--Greatbay_SCIE--SDS-PAGE_of_cp19k-mfp5.png">
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</figure></center>
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<center><figcaption>
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Figure 3.  
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SDS-PAGE of purified rBalcp19k-mfp5 by affinity chromatography under native conditions. 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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</figcaption></center>
  
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<table style="border-collapse: collapse;">
 
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  <thead><tr><td>Recombinant adhesive proteins</td><td>Yield (mg/L)</td></tr></thead>
 
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  <tbody>
 
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    <tr><td>CsgA-mfp5</td><td>0.5</td></tr>
 
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    <tr><td>CsgA-mfp5-mfp5</td><td>0.5</td></tr>
 
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    <tr><td>CsgA-mfp5-mfp3</td><td>0.7</td></tr>
 
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    <tr><td>Fp1-mfp5-fp1</td><td>4</td></tr>
 
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    <tr><td>rBalcp19K-mfp5</td><td>1</td></tr>
 
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  </tbody>
 
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</table>
 
 
<h3> Surface coating analysis </h3>  
 
<h3> Surface coating analysis </h3>  
 
<p>
 
<p>
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Next, we want to figure out if the linker would affect the functions of these fusion proteins. Surface coating analysis was conduct to test the coating ability of recombinant adhesive proteins on different materials. Glass and plastics were chosen for the test. Results showed that Mfp5 related proteins (unmodified) exhibited higher surface absorption abilities than other recombinant proteins, which meets our expectations. <span style="font-weight: bold;">It is suggested that linker doesn't affect the basic function of recombinant proteins.</span>
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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 Figure 4, 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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</p>
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<center><Figure>
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<img width="300px" src="https://2019.igem.org/wiki/images/3/39/T--Greatbay_SCIE--surface_coating_analysis_2.jpeg">
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</figure></center>
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<center><Figure>
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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></center>
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<center><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></center>

Latest revision as of 15:11, 21 October 2019

T7 promoter+rBalcp19K-linker-mfp5-His

This composite part is meant to express rBalcp19k-linker-mfp5 fusion genes under T7 promoter, and 7XHis-tag was fused on the C terminal to achieve affinity protein purification. This composite part is also expressed in Pichia pastoris( BBa_K3089017)after codon optimization, and is an updated version from the basic part rBalcp19k( BBa_K3089008) which is expressed in Pichia pastoris as well. Mfp5 is the mussel foot protein from Mytilus galloprovincialis responsible for interface adhesion. rBalcp19k, the barnacle adhesive protein from Balanus albicostatus, its homologous gene serves as adhesive proteins, also playing the role of interfacial adhesion in a way that mfp5 does in mussels(Figure 1.) We linked them together to achieve better adhesive ability, because rBalcp19k can self-assemble into aggregated nanofibers at acidic pHs. This composite part would be a promising new generation of bio-inspired adhesives for a wide range of applications.

Figure 1. Schematic showing the cross-sections of the barnacle cement and various possible factors involved in barnacle adhesion.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
    Illegal PstI site found at 411
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 411
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 720
    Illegal XhoI site found at 527
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
    Illegal PstI site found at 411
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
    Illegal PstI site found at 411
    Illegal AgeI site found at 390
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterisation

Three different experiments were done to characterise the BBa_K3089026 biobrick:

  • protein expression
  • protein purification
  • Surface coating analysis

Protein expression

Figure 2. The circuit of the protein BBa_K30889026

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℃. 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. We can get the same results using quantitative densitometry analysis of SDS-PAGE gels (Figure 1B).

Figure 3. 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

Barnacle cement proteins are very promising in making biomedical bio-glues. rBalcp19K from Balanus albicostatus had the properties of both self-assembly and adhesion. 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. 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 3), which meant it was successfully expressed and purified under native condition(see details on our methods). Protein concentrations of rBalcp19k-linker-mfp5 were measured by BCA assay, and its yield is 1mg/L.

Figure 3. SDS-PAGE of purified rBalcp19k-mfp5 by affinity chromatography under native conditions. 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

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 Figure 4, 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).