Difference between revisions of "Part:BBa K3089023"
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===Usage and Biology===
 
===Usage and Biology===
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(Zhong et al., 2014) and has been characterised by iGEM15_TU_Delft(<a href="https://parts.igem.org/Part:BBa_K1583104"target="_blank">BBa_K1583104</a>)
 
(Zhong et al., 2014) and has been characterised by iGEM15_TU_Delft(<a href="https://parts.igem.org/Part:BBa_K1583104"target="_blank">BBa_K1583104</a>)
 
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<span class='h3bb'><h3>Sequence and Features</h3></span>
 
<span class='h3bb'><h3>Sequence and Features</h3></span>
 
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<partinfo>BBa_K3089023 SequenceAndFeatures</partinfo>
  
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===Functional Parameters===
 
===Functional Parameters===
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Figure 1. Schematic view of mussel foot proteins (mfps) in a byssal plaque of Mytilus showing the approximate location of each of the major proteins (Lee, Messersmith et al. 2011).
 
Figure 1. Schematic view of mussel foot proteins (mfps) in a byssal plaque of Mytilus showing the approximate location of each of the major proteins (Lee, Messersmith et al. 2011).
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<h3>Characterization</h3>
 
<h3>Characterization</h3>

Revision as of 13:28, 21 October 2019


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

The csgA-linker-mfp5-mfp5-his fusion protein is the purified version of Csga-linker-Mfp5 recombinant protein by His-tag affinity purification (a -Histidine-Histidine-Histidine-Histidine-Histidine-Histidine-Histidine tag fused on the carboxyl-terminal). We designed this part by putting together two identical Mfp5 mussel proteins and hoped it shows higher adhesive features than Csga-Mfp5. CsgA is an amyloid-like protein encoded on genome of E.coli MG1655 providing cohesive mechanical 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 based on the paper "Strong underwater adhesives made by self-assembling multi-protein nanofibres" (Zhong et al., 2014) and has been characterised by iGEM15_TU_Delft(<a href="https://parts.igem.org/Part:BBa_K1583104"target="_blank">BBa_K1583104</a>)

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Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
    Illegal PstI site found at 400
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 400
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 585
    Illegal BamHI site found at 813
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
    Illegal PstI site found at 400
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
    Illegal PstI site found at 400
  • 1000
    COMPATIBLE WITH RFC[1000]

Figure 1. Schematic view of mussel foot proteins (mfps) in a byssal plaque of Mytilus showing the approximate location of each of the major proteins (Lee, Messersmith et al. 2011).

Characterization

This recombinant protein is our own new design which has a property of both cohesion (CsgA) and adhesion (Mfp5). By adding two Mfp5s to CsgA, we expect it to have a stronger adhesive property. The results show, this recombinant protein can adhere to plastics and glasses better than any other parts in our toolbox. Therefore, this design cannot only help with the design of underwater adhesives but can also be used to make underwater living material. We characterised this part using various qualitative and quantitative data from protein expression, purification, and functional analysis.

  • protein expression
  • protein purification
  • Surface coating analysis

Protein expression

Figure 2. The circuit of the protein BBa_K30889023

csgA-linker-mfp5-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 obvious protein bands of CsgA-mfp5-mfp5(~25 kDa) could be observed on lane WC compared with lane pET28b (pET28b empty vector), which means the expression of this protein is not well in BL21(DE3) Rosetta.

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

For we make producing underwater bio-adhesives as the final goal of our project, though no obvious protein bonds of interest could be observed, we straightly went on protein purification. CsgA is an amyloid-like protein characterised by β-strands, and CsgA monomers would form aggregates after expression inside cells. Therefore, denature protein purification methods were used. Weak bands presented on the lane E2. The mixed solutions were then loaded on the columns and dialysed with PBS buffer (PH=6.0) to wash away imidazole. Meanwhile, the protein was concentrated. After that, the concentrated protein was put under 4℃ for 72 hours to make it renature, protein concentrations of CsgA-linker-mfp5-mfp5 were measured by BCA assay, and its yield is 0.5mg/L.

Figure 4. Coomassie-stained SDS-PAGE gels confirm purification of the expressed protein Csg-linker-mfp5-mfp5 by cobalt-resin columns. Lanes: M, protein molecular weight marker; 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 5. Surface coating analysis of recombinant proteins on hydrophilic glass slides (left) and hydrophobic polystyrene (PS) plates (right).