Difference between revisions of "Part:BBa K3089024"

Revision as of 01:33, 21 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]

Introduction

This composite part is meant to express fp1-linker-mfp5-linker-fp1 fusion genes under T7 promoter, and 7XHis-tag was fused on the C terminal to achieve affinity protein purification. Fp1 is one kind of mussel foot protein, from Mytilus edulis, in the adhesion plaque at the end of each thread that enables it to anchor to various wet surfaces. Mfp5 is the mussel foot protein from Mytilus galloprovincialis responsible for interface adhesion(Figure 1). In this part, we have used a fusion protein comprising six fp-1 decapeptide repeats at each fp-5 terminus. Strong and water-insoluble mussel adhesives like this have attracted interest for potential uses in biotechnological applications because they could be used as cell, tissue, or medical adhesives and have the added advantage of being environmentally friendly.

Characterization

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

protein expression
protein purification
Surface coating analysis

Protein expression

Figure 1. 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).

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 <html>
-<!-- There is no introduction here
 <h3>Introduction</h3>
 <p>
-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.
+This composite part is meant to express fp1-linker-mfp5-linker-fp1 fusion genes under T7 promoter, and 7XHis-tag was fused on the C terminal to achieve affinity protein purification. Fp1 is one kind of mussel foot protein, from Mytilus edulis, in the adhesion plaque at the end of each thread that enables it to anchor to various wet surfaces. Mfp5 is the mussel foot protein from Mytilus galloprovincialis responsible for interface adhesion(Figure 1).
+In this part, we have used a fusion protein comprising six fp-1 decapeptide repeats at each fp-5 terminus. Strong and water-insoluble mussel adhesives like this have attracted interest for potential uses in biotechnological applications because they could be used as cell, tissue, or medical adhesives and have the added advantage of being environmentally friendly.
+</p>
+<figure>
+<img width="450px" src="https://static.igem.org/mediawiki/parts/9/99/T--Greatbay_SCIE--P--022-Introdcution.png">
+</figure>
-<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 are combined with the formation of nanowires.
--->
 <h3>Characterization</h3>