Part:BBa_K3089026

T7 promoter+rBalcp19K-linker-mfp5-His

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]

Introduction

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.

CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms taken from E.coli K-12 MG1655. 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.

Characterization

Three different experiments were done to characterise the BBa_K3089026 biobrick:

protein expression
protein purification
Surface coating analysis

Protein expression

Figure 1 illustration of all reccombiant protein with linkers.

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).

Protein purification

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. In conclusion, recombinant proteins with linker GGGGSGGGGGS can be successfully purified using different methods.

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.

Recombinant adhesive proteins	Yield (mg/L)
CsgA-mfp5	0.5
CsgA-mfp5-mfp5	0.5
CsgA-mfp5-mfp3	0.7
Fp1-mfp5-fp1	4
rBalcp19K-mfp5	1

Surface coating analysis

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. It is suggested that linker doesn't affect the basic function of recombinant proteins.