Difference between revisions of "Part:BBa K3089024"
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<h3>Characterization</h3>
 
<h3>Characterization</h3>
 
<p>
 
<p>
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Three different experiments were done to characterise the BBa_K3089026 biobrick:
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Three different experiments were done to characterise the BBa_K3089024 bio-brick:
 
<ul><li>protein expression</li>
 
<ul><li>protein expression</li>
 
<li> protein purification</li>
 
<li> protein purification</li>
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<p>
 
<p>
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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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The predicted size of Fp151 is 24.83 kDa and the isoelectric point is 10.58. Fp1-linker-mfp5-linker-fp1(Fp151) 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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In order to detect its expression, whole cells were collected after induction by centrifuging and prepared for SDS-PAGE. Results showed that no obvious protein bands of Fp151(~24.83 kDa) could be observed on lane fp1-mfp5-fp1 compared with lane pET28b (pET28b empty vector)(Figure 1A).
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We can get the same results using quantitative densitometry analysis of SDS-PAGE gels (Figure 1B).
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Interestingly, a higher nonspecific band between 25kDa and 36kDa presented, which may may the dimer form of Fp151. Quantitative densitometry analysis of SDS-PAGE indicated that Fp1-mfp5-fp1 expressed at the highest expression level under the same expression conditions(Figure 1B).
 
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</p>  
  
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</figure>
 
</figure>
  
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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>
  
 
<h3> Protein purification </h3>
 
<h3> Protein purification </h3>
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<html>
 
<Figure>
 
<Figure>
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<img width="300px" src="https://static.igem.org/mediawiki/parts/d/d9/T--Greatbay_SCIE--SDS-PAGE_of_rBalcp19k-mfp5.png">
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<img width="450px" src="https://static.igem.org/mediawiki/parts/7/77/T--Greatbay_SCIE--SDS-PAGE_of_FP-151.jpeg">
 
</figure>
 
</figure>
  
 
<figcaption>  
 
<figcaption>  
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Figure 3. SDS-PAGE of purified rBalcp19k-mfp5 by affinity chromatography under native conditions.  
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Figure 3.  
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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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(A) Coomassie-blue-stained SDS-PAGE analysis of Fp151 purification under through acetic acid extraction. Lanes: M, protein molecular weight marker; IS, insoluble cell debris fraction; AE, fraction extracted with 25% (v/v) acetic acid.
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(B) Coomassie-blue-stained SDS-PAGE analysis of Fp151 purification under denaturing conditions.  
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(C) Coomassie-blue-stained SDS-PAGE analysis of Fp151 His-tag affinity purification under denaturing conditions. Lanes: M, protein molecular weight marker; NC, whole-cell sample of pET28b empty vector; WC, whole-cell sample of recombinant proteins; FT, flow through after resin binding; E, eluted proteins.
 
</figcaption>
 
</figcaption>
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<p>
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  Recombinant hybrid fp-151 fused with histidine affinity ligand was successfully expressed in E. coli BL21(DE3) Rosetta.
 +
  Initially we tried to purified it with acetic acid(Choi et al., 2014) but  failed, since we simply couldn’t get rid of extra bands and obtain a single band of interest. The protein was successfully purified with the same denature protein purification methods for purifying CsgA-linker-mfp5.
 +
  Nonspecific bands in purified proteins were significant between 25-35 kDa（Figure 4C）,which may relate to polymers caused by self-assembly. Protein concentrations of CsgA-linker-mfp5-linker-mfp3 were measured by BCA assay and its yield is 4mg/L.
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</p>
  
 
<h3> Surface coating analysis </h3>  
 
<h3> Surface coating analysis </h3>  
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<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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<img width="450px" src="https://static.igem.org/mediawiki/parts/8/8e/T--Greatbay_SCIE--P--Surface_coating.jpeg">
 
</figure>
 
</figure>
  

Revision as of 03:27, 19 October 2019


T7 promoter+fp1-linker-mfp5-linker-fp1-His

fp151

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 368
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

Three different experiments were done to characterise the BBa_K3089024 bio-brick:

  • protein expression
  • protein purification
  • Surface coating analysis

Protein expression

The circuit of the protein BBa_K30889026

The predicted size of Fp151 is 24.83 kDa and the isoelectric point is 10.58. Fp1-linker-mfp5-linker-fp1(Fp151) 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 obvious protein bands of Fp151(~24.83 kDa) could be observed on lane fp1-mfp5-fp1 compared with lane pET28b (pET28b empty vector)(Figure 1A). Interestingly, a higher nonspecific band between 25kDa and 36kDa presented, which may may the dimer form of Fp151. Quantitative densitometry analysis of SDS-PAGE indicated that Fp1-mfp5-fp1 expressed at the highest expression level under the same expression conditions(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

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 2), 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. (A) Coomassie-blue-stained SDS-PAGE analysis of Fp151 purification under through acetic acid extraction. Lanes: M, protein molecular weight marker; IS, insoluble cell debris fraction; AE, fraction extracted with 25% (v/v) acetic acid. (B) Coomassie-blue-stained SDS-PAGE analysis of Fp151 purification under denaturing conditions. (C) Coomassie-blue-stained SDS-PAGE analysis of Fp151 His-tag affinity purification under denaturing conditions. Lanes: M, protein molecular weight marker; NC, whole-cell sample of pET28b empty vector; WC, whole-cell sample of recombinant proteins; FT, flow through after resin binding; E, eluted proteins.

Recombinant hybrid fp-151 fused with histidine affinity ligand was successfully expressed in E. coli BL21(DE3) Rosetta. Initially we tried to purified it with acetic acid(Choi et al., 2014) but failed, since we simply couldn’t get rid of extra bands and obtain a single band of interest. The protein was successfully purified with the same denature protein purification methods for purifying CsgA-linker-mfp5. Nonspecific bands in purified proteins were significant between 25-35 kDa（Figure 4C）,which may relate to polymers caused by self-assembly. Protein concentrations of CsgA-linker-mfp5-linker-mfp3 were measured by BCA assay and its yield is 4mg/L.

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).