Composite Parts

Parts Overview

Our project aims to build a modularized adhesive platform consisting of the E. coli biofilm protein CsgA and adhesive proteins. CsgA protein has the ability to self-assemble into a network of amyloid nanofibers outside the cell. Therefore, we designed composite parts that adhesive proteins are linked to CsgA with a flexible linker to enable the display of recombinant proteins on the surface of E. coli. These parts were designed based on previous studies. [1]

Nature adhesive recombinant proteins

First, we found adhesive proteins of mussels (Mfp3, Mfp5) and barnacles (Bamcp20k-1, cp19k, Mrcp20k, Aacp19k). Next, we constructed composite parts of these adhesive proteins with lac promoter, RBS, CsgA, and GS linker.

Mussel adhesive recombinant proteins

We chose adhesive proteins from the Mediterranean mussel (Mytilus galloprovincialis) as part of our biobrick design because mussel foot proteins have excellent adhesion properties. Mussels can secrete special proteins to adhere to surfaces under turbulent environments.

BBa_K4854014: CsgA+Mfp3
Figure 1.The composite part BBa_K4854014 contains Lac promoter, RBS, CsgA, GS linker and Mfp3.
BBa_K4854015: CsgA+Mfp5
Figure 2.The composite part BBa_K4854015 contains Lac promoter, RBS, CsgA, GS linker and Mfp5.

Barnacle adhesive recombinant proteins

We chose adhesive proteins from the barnacles (Amphibalanus amphitrite, Balanus albicostatus, Megabalanus rosa) as part of our biobrick design because barnacle cement proteins have excellent adhesion properties. At the cyprid stage before a barnacle becomes mature, the cyprid releases cement protein to anchor itself to substrates like rocks.

BBa_K4854016: CsgA+Bamcp20k-1
Figure 3.The composite part BBa_K4854016 contains Lac promoter, RBS, CsgA, GS linker and Bamcp20k-1.
BBa_K4854019: CsgA+cp19k
Figure 4.The composite part BBa_K4854019 contains Lac promoter, RBS, CsgA, GS linker and cp19k.
BBa_K4854020: CsgA+Mrcp20k
Figure 5.The composite part BBa_K4854020 contains Lac promoter, RBS, CsgA, GS linker and Mrcp20k
BBa_K4854021: CsgA+Aacp19k
Figure 6.The composite part BBa_K4854021 contains Lac promoter, RBS, CsgA, GS linker and Aacp19k.

Potential adhesive recombinant proteins

We found potential adhesive proteins (ecpA, Nid1, epd2, zig-4) from Uniprot picked by the drylab model. Next, we constructed composite parts of these adhesive proteins with lac promoter, RBS, CsgA, and GS linker.

BBa_K4854022: CsgA+ecpA

ecpA is a fimbrial protein from Eikenella corrodens. It belongs to the a pilin family and shows features for transmembrane.

Figure 7.The composite part BBa_K4854022 contains Lac promoter, RBS, CsgA, GS linker and ecpA.
BBa_K4854023: CsgA+Nid1

Nidogen-1 (Nid1) is a sulfated glycoprotein from rat (Rattus norvegicus). It distributes in basement membranes and probably plays a role in cell-extracellular matrix interactions.

Figure 8.The composite part BBa_K4854023 contains Lac promoter, RBS, CsgA, GS linker and Nid1.
BBa_K4854024: CsgA+epd2

Ependymin-2 protein (epd2) is a glycoprotein from rainbow trout (Oncorhynchus mykiss). It probably plays a role in neural plasticity and axon regeneration.

Figure 9.The composite part BBa_K4854024 contains Lac promoter, RBS, CsgA, GS linker and epd2.
BBa_K4854025: CsgA+zig-4

Zwei Ig domain protein zig-4 from the roundworm (Caenorhabditis elegans) plays a role in maintaining axon position.

Figure 10.The composite part BBa_K4854025 contains Lac promoter, RBS, CsgA, GS linker and zig-4.

Functional recombinant protein

We designed GFP as the functional protein linked to the N-terminus or C-terminus of the CsgA+Mfp5 fusion protein by a flexible linker. GFP was chosen because of its unique fluorescent properties, which can be used to test CoPlat's functionality and ensure that our experimental aims can be clearly verified.

BBa_K4854026: CsgA+Mfp5+GFP
Figure 11.The composite part BBa_K4854026 contains Lac promoter, RBS, CsgA, GS linker, a flexible linker, GFP and Mfp5.
BBa_K4854027: GFP+CsgA+Mfp5
Figure12: The composite part BBa_K4854027 contains Lac promoter, RBS, CsgA, GS linker, a flexible linker, GFP and Mfp5.