Team:Greatbay SCIE/Design

Project Design

An Underwater Adhesive Toolbox - SticKit

A toolbox is defined as an organization or storage of tools; in our project, it is a collection of multiple types of proteins from various marine organisms and bacteria. Since most of these proteins came from oceanic or waterborne organisms, they possess strong underwater adhesive abilities. We named this toolbox SticKit.

After identifying all the parts in our toolbox, the tools – the proteins – can then be assembled in different arrangements to provide them with extended functions. In other words, the proteins act as modules or parts, that can be combined to make recombinant proteins.

These recombinant proteins have higher performance on adhesion compared to their parts. Also, these proteins are biodegradable. These qualities enable them to be used in various fields.

Inside SticKit

The proteins in our toolbox consist of mainly two types: adhesive proteins and cohesive proteins. Cohesive proteins can self-assemble into fibers, while adhesive protein is responsible for the interfacial adhesion. Combining these two types of proteins forms the basis of bioadhesives, as the cohesive protein possesses interior forces and is responsible for holding protein molecules together for fibril formation, which will then create crosslinks, a process essential for hydrogel formation.

Table 1. Full list of proteins we have investigated.

We investigated a wide range of adhesive and cohesive proteins in waterborne organisms (Table 1), but only a few are chosen to constitute our toolbox.

The selecting criteria are listed below:

1. The sequence must be obtainable;
2. The protein must be charachterized by previous studies;
3. The protein must have potential to be modularized and standardized.

All selected proteins that can be further incorporated into synthetic adhesives are listed below.

Table 2. List of proteins we selected for our toolbox.

Adhesive proteins

All of the chosen adhesive proteins (Table 2) are crucial members of underwater adhesion. Their durable and waterproof abilities made them perfect candidates for our project.

Mfp3, Mfp5

There are six main types of mussel foot proteins (Mfp) identified in the mussel byssal. Among all, Mfp3 and Mfp5 are the two most well-studied adhesive proteins. They both come from Mytilus edulis , also called the sea mussels, and they are found at the bottom of the mussel byssal plaque, a fortress where interface adhesion is carried out (Figure 1).

Figure 1. Mussel foot proteins found in mussel byssal plaque.

Both Mfp3 and Mfp5 have coiled structures and are rich in DOPA residue, which is a post-translationally modified tyrosine that forms bidentate hydrogen bonds with various surfaces to provide adhesion. Therefore, in order to obtain functionable Mfp3 and Mfp5, the amino acid tyrosine must be modified into DOPA by tyrosinase, which we will discuss later.

Cp19k

‘Cp’ stands for cement protein, obtained from the barnacle cement. Similar to mussel foot proteins, barnacle cement proteins also form a complex that can adhere to various surfaces. What is different is that these cement proteins do not need any post-translational modification, allowing them to be more available.

Figure 2. Barnacle cement proteins.

A large number of cement proteins have been identified from the species Balanus albicostatus. Cp19k, Cp20k, and Cp68k are responsible for barnacles’ surface adhesion, while Cp52k and Cp100k play a bulk cohesion role in holding all the proteins together in a matrix.

Among all, cp19k is our top candidate. It is the most crucial protein for surface adhesion with a relatively small size of 19kDa, making it suitable for molecular cloning.

Cohesive proteins

Cohesive proteins provide internal forces by protein-protein interaction. These proteins are essential for self-assembly into fibers, which will then form crosslinks, a network that triggers the formation of biological adhesives. (Table 2).

CsgA

Out of all the cohesive proteins we investigated, CsgA is among the top. Along with CsgB, they make up the major components of curli fibers on the E. coli cell surface membrane. Its original task was to self-assemble and self-polymerize into a fiber to form curli. Thus, CsgA have a very specific function of providing cohesive forces for self-assembly, and this meet our demands.

Mfp1

Mfp1 is a cohesive protein found on the surface of mussel byssal plaque. It is a large collagen-like protein containing 75 decapeptide repeats (Hwang & Waite, 2012) that plays a protective role by forming a collagen-like layer on the plaque’s surface.

Cp19k

Cp19k was discussed above as an adhesive protein. However, in addition to that, Cp19k can also provide cohesion. Though shorter, it can also self-assemble into a fiber similar to that of CsgA. Therefore, this protein is expected to function as both adhesively and cohesively parts in our Toolbox.

Designs based on SticKit

Different arrangements were formed from the fusion of either the full length or segmented domains of adhesive or cohesive proteins. These proteins with their integrated functions are known as recombinant proteins.

Through our investigation and the follow-up of the results of the previously mentioned research groups, we found that all of the recombinant proteins follow a general pattern: cohesive proteins fuse with adhesive proteins, and this design had become one of our core designs throughout our project.

Another core of our design is that, since Mfp5 is the most extensively studied adhesive protein, and its strong underwater adhesion fits the requirements of our project perfectly; therefore, most of our designs include Mfp5 as part of the recombinant protein.

Figure 3. Toolbox of recombinant proteins

CsgA-Mfp series

In Zhong’s design of CsgA-Mfp3/5, a CsgA molecule is combined with a Mfp3 and a Mfp5 respectively. Fusing CsgA to the adhesive protein will enable the recombinant proteins is able to hierarchically self-assemble into higher order structures (Zhong et al., 2014). Dynamic simulations have predicted the structures of these self-assembled recombinant proteins: the CsgA proteins formed an amyloid core, whereas the Mfp3 and Mfp5 are exposed to the exterior surface (Figure 4). These recombinant proteins significantly outperform the adhesion and cohesion of Mfps and CsgA molecules on their own.

Figure 4. Diagram of CsgA-Mfp5.

Based on Zhong’s design, we extended two of our own recombinant proteins from our toolbox: CsgA-Mfp5-Mfp5 and CsgA-Mfp5-Mfp3.

Figure 5. Diagram of CsgA-Mfp5-Mfp5, CsgA-Mfp5-Mfp3.

The reason for the design of CsgA-Mfp5-Mfp5 is because, the more the number of Mfp5 connected in series, the stronger the adhesion. (Kim et al., 2018) Therefore, we connected two Mfp5s together, and some very interesting experimental results suggested that Mfp5 can indeed provide a very significant improvement. (See Results.)

On the other hand, CsgA-Mfp5-Mfp3 is an inspiration we got from an interview with Zhong Chao. He suggested an exciting fact that Mfp3 and Mfp5 can naturally form crosslinks inside the mussel byssal plaque. Therefore, we can create a bio mimicking design that may enhance the interfacial adhesion based on this fact. (See Integrated Human Practice.)

Fp-151

Though the design of CsgA-Mfp3/5 showed very promising properties, their relatively low yields remained problematic and was challenging to overcome. On the other side, fp-151 (Hwang, Gim, Yoo, & Cha, 2007) obtained a high yield by connecting part of Mfp1 at the N and C terminus.

Figure 6. Diagram of fp-151.

Mfp1 contains 75 decapeptide repeats, each repeat is a peptide containing ten amino acids. In this recombinant protein, only 6 of the 75 repeats are added to both the N terminus and the C terminus of Mfp5. This protein is very adhesive and its yields increase significantly.

Cp19k-Mfp5

As mentioned before, Cp19k is a protein that can be adhesive and cohesive. In this design, we combined Cp19k with Mfp5, which means that Cp19k is used as a cohesive protein, for the recombinant protein to increase its adhesiveness.

Figure 7. Diagram of Cp19k-Mfp5.

Besides, we also wanted to know 1) how well Cp19k can function as a cohesive protein, and 2) wether Mfp5 can significantly alter the adhesion of the recombinant protein compared to Cp19k alone (Without Mfp5). This result is also demonstrated on the Results page. (See Results.)

Dopa modification

Since all of our designs are base on Mfp5, a post-translational modification of DOPA from the amino acid tyrosine is crucial for our project because mussel foot proteins must be modified before they can carry out surface adhesion.

During DOPA modification, tyrosine was first hydroxilized to 3,4-dihydoxyphenylalanine (DOPA), which is then modified into Dopaquinone either through auto-oxidation or in the presence of tyrosinase (Cui et al., 2017) (Figure 8).

Figure 8. Process of DOPA modification.

The adhesive property of Mfp-containing recombinant protein is mainly dependent on the first step of modification from tyrosine to DOPA. It is the bidentate hydrogen bond between DOPA and the substratum that induce adhesion.

However, the catalytic efficiency of mushroom tyrosinase used in provious studies is often not satisfactory for large-scale production of adhesive proteins. (Choi, Yang, Yang, & Cha, 2012) Therefore, we adopted a tyrosine from a marine archaeon Candidatus Nitrosopumilus koreensis called mTyr-CNK (Figure 9).

Figure 9. mTyr-CNK from Candidatus Nitrosopumilus koreensis (Do, Kang, Yang, Cha, & Choi, 2017.

This protein contained key tyrosinase domains, such as copper-binding domains and an O2-binding motif; phylogenetic analysis revealed that it was distinct from other known bacterial tyrosinases. (Do et al., 2017).

mTyr-CNK had several advantages:

- Enhance catalytic efficiency against L-tyrosine;
- Highly active in different conditions;
- High levels of expression in E. coli.

In vitro and in vivo modification

For in vitro modifications, we used purified mTyr-CNK to hydrolyze tyrosine into DOPA. However, the high amount of basic and aromatic amino acids in mussel adhesive proteins make the proteins easy to aggregate, while tyrosinase oxidizes free tyrosines more efficiently than those in aggregates.

Therefore, a co-expression system in a dual vector system was designed to improve the conversion efficiency by modifying the protein right after the protein is produced.

Figure 10. Co-expression system.

For example, the purified in vivo modified fp-151 from the co-expression system showed a 4-fold higher bulk adhesive strength compared to the in vitro modified fp-151 (Choi, Yang et al. 2012).

In conclusion, SticKit is a toolbox with a collection of various adhesive and cohesive proteins from different organisms. Besides our own designs derived from the toolbox, there can still be many diverting inspirations for future iGEMers to discover.

Reference

Choi, Y. S., Yang, Y. J., Yang, B., & Cha, H. J. (2012). In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli. Microb Cell Fact, 11, 139. doi:10.1186/1475-2859-11-139

Cui, M., Ren, S., Wei, S., Sun, C., & Zhong, C. (2017). Natural and bio-inspired underwater adhesives: Current progress and new perspectives. APL Materials, 5(11). doi:10.1063/1.4985756

Do, H., Kang, E., Yang, B., Cha, H. J., & Choi, Y. S. (2017). A tyrosinase, mTyr-CNK, that is functionally available as a monophenol monooxygenase. Sci Rep, 7(1), 17267. doi:10.1038/s41598-017-17635-0

Hofman, A. H., van Hees, I. A., Yang, J., & Kamperman, M. (2018). Bioinspired Underwater Adhesives by Using the Supramolecular Toolbox. Adv Mater, 30(19), e1704640. doi:10.1002/adma.201704640

Huang, J., Liu, S., Zhang, C., Wang, X., Pu, J., Ba, F., . . . Zhong, C. (2019). Programmable and printable Bacillus subtilis biofilms as engineered living materials. Nat Chem Biol, 15(1), 34-41. doi:10.1038/s41589-018-0169-2

Hwang, D. S., Gim, Y., Yoo, H. J., & Cha, H. J. (2007). Practical recombinant hybrid mussel bioadhesive fp-151. Biomaterials, 28(24), 3560-3568. doi:10.1016/j.biomaterials.2007.04.039

Hwang, D. S., & Waite, J. H. (2012). Three intrinsically unstructured mussel adhesive proteins, mfp-1, mfp-2, and mfp-3: analysis by circular dichroism. Protein Sci, 21(11), 1689-1695. doi:10.1002/pro.2147

Kim, E., Dai, B., Qiao, J. B., Li, W., Fortner, J. D., & Zhang, F. (2018). Microbially Synthesized Repeats of Mussel Foot Protein Display Enhanced Underwater Adhesion. ACS Appl Mater Interfaces, 10(49), 43003-43012. doi:10.1021/acsami.8b14890

Lee, B. P., Messersmith, P. B., Israelachvili, J. N., & Waite, J. H. (2011). Mussel-Inspired Adhesives and Coatings. Annu Rev Mater Res, 41, 99-132. doi:10.1146/annurev-matsci-062910-100429

Liang, C., Ye, Z., Xue, B., Zeng, L., Wu, W., Zhong, C., . . . Messersmith, P. B. (2018). Self-Assembled Nanofibers for Strong Underwater Adhesion: The Trick of Barnacles. ACS Appl Mater Interfaces, 10(30), 25017-25025. doi:10.1021/acsami.8b04752

Wang, Y. J., Sanai, K., & Nakagaki, M. (2009). A Novel Bioadhesive Protein of Silk Filaments Spun Underwater by Caddisfly Larvae. Advanced Materials Research, 79-82, 1631-1634. doi:10.4028/www.scientific.net/AMR.79-82.1631

Wunderer, J., Lengerer, B., Pjeta, R., Bertemes, P., Kremser, L., Lindner, H., . . . Ladurner, P. (2019). A mechanism for temporary bioadhesion. Proc Natl Acad Sci U S A. doi:10.1073/pnas.1814230116

Zhong, C., Gurry, T., Cheng, A. A., Downey, J., Deng, Z., Stultz, C. M., & Lu, T. K. (2014). Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nat Nanotechnol, 9(10), 858-866. doi:10.1038/nnano.2014.199