We were working in a new lab that some of it is still being fitting up. Workers used scaffold to install facilities high on the ceiling. Suddenly I came up with an interesting idea that what if the scaffold is construct by protein. Then I realized that scaffold is such a regular network that we could carry something on it in the order we like. If the “something” were enzymes of co-catalyze system, we got a perfectly uniform distributed immobilized enzyme of co-catalysis system. If the “something” were cells the scaffold can provide a 3D microenvironment for them and we got a scaffold for tissue engineering.
So now the problem is how do we construct the scaffold. We knew that there was many protein self-assembly system. Usually these systems have a irregulate structure. Just like constructing a real scaffold we thought can use different linkage to link our materials altogether forming a regulate network. After research on literatures, we noticed two widely used self-assembly system in hydrogel: spy system and coiled coil system and our next problem is how to combine these two kinds of linkage.
Synthetic biology has long been working hard on gene circuits and switches ignoring another important biomacromolecule proteins. The modularization of proteins is more difficult than DNA, because of the complex interaction between different parts on their structure and function. But as technology developing, we are learning more and more about the relationship between protein’s structure and function. Now we can combine different proteins with special amino acid sequence like GS linker without hurting its function. So now it is time to get down to the modularization of proteins with which we will be able to design proteins itself for new function. For so long the combination of genes are rely on soldered dot of special restriction Enzyme cutting site. These soldered dots might influence to the function of protein “bricks” after combination. But now with assembly PCR and overlap PCR we can link gene sequences traceless and the proteins they expressed as well. Therefore, we have a way to combine to system that have been widely used in hydrogel to construct our “scaffold”.
Since the proteins in the system are hydrophilic molecules our scaffold is actually a hydrogel.
What is hydrogel?
A hydrogel is a network of polymer chains that are hydrophilic, sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent (they can contain over 90% water) natural or synthetic polymeric networks. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. The first appearance of the term 'hydrogel' in the literature was in 1894. Common uses for hydrogels include:
- Scaffolds in tissue engineering. When hydrogel is used as scaffolds, hydrogels may contain human cells to repair tissue. They mimic 3D microenvironment of cells
- Environmentally sensitive hydrogels (also known as 'Smart Gels' or 'Intelligent Gels'). These hydrogels have the ability to sense changes of pH, temperature, or the concentration of metabolite and release their load as result of such a change.
- Sustained-release drug delivery systems.
- Hydrogels that are responsive to specific molecules, such as glucose or antigens, can be used as biosensors, as well as in DDS.
How is our hydrogel special?
We used two self-assembling systems to construct four bio-parts which will finally construct a hydrogel when mixed. With our design, the hydrogel would construct a scaffold regularly carrying two proteins or even cells.
The systems we used was spy-tag spy-catcher system and coiled coil system. These two systems are widely studied and used in research of hydrogel. Spy-system consists of two protein molecules named spy-tag and spy-catcher which would construct a covalent bond between them and cling to each other. Coiled coli system is two special alpha helix of peptide which would twine around each other. Several pairs of peptide are found to be able to consist a coiled system. In our project, we used coiled coil systems between peptide RR and EE as well as the system between A and B. We combined these two systems by linking the coiled coil peptides to spy-catcher, spy-tag and two other proteins using GS linker to provide flexibility of the link. Therefore, we got four “bricks” of our project, which are
1.A—GS linker—spy-tag—GS linker—A
2.EE—GS linker—spy-catcher—GS linker—EE
3.B——GS linker—protein I—GS linker—B
4.RR—GS linker—protein II—GS linker—RR.
When our system work spy-tag and spy-catcher will form a covalent bond and part 1 and 2 becomes a cross. (fig 1.) The cross will specifically combine with part 2 and 4 by the coiled coil peptide; part 2 and 4
would also connect with other crosses and forming a peptide network. Finally, this network will construct a silicon dioxide like scaffold because of the flexibility of GS linker and forming a hydrogel. (fig 2.) The hydrogel is self-assembled. In this hydrogel, the two proteins carried are located in a uniform ditribution. What’s more he hydrogel is a homogenous structure.
In our experiment, the protein I and II was green fluorescent protein(GFP) and red fluorescent protein(RFP) in order to observe the regular configuration of the two proteins with confocal microscopy. In the future, protein I and II could be a co-catalysis system with two enzymes or one or two kinds of cells. What’s more, if we can add two more pairs of coiled coil peptides in our system we could achive the regulaly configuration of four diferent molecules or cells.
What’s more we can treat the cross formed with Spy-system as carbon atom in organics and the molecule with two coiled-coil peptide as oxigen atom and copy the world of organic chemistry on our protein scaffold. Similarly we can even make our “nitrogen atom”, “hydrogen atom” etc. with these two system. Therefore we can get variety of sturcture as we want carrying cells or molecule we like.
What is the use of our hydrogel?
(1). Immobolized co-catalyzed system: Co-cataliyze system has been widly used in industry. For example,degaration of cellulose require the cooperation of three enzymes: exo β-glucanase, inscribed β-glucanase, β-galactosidase. Traditionally an agitator tank is used for co-catalyze system to mix different enzymes. The problem is that an agitator can ensure a perfect uniform distribution of enzymes. Co-catalyze systems are also difficult to be immobilized for its difficult to get a regular configuration of the enzymes. In our system, we can carry two different enzymes perfect regularly in our hydrogel. For our hydrogel is a tight network of hydrophilic proteins, solution of materials could diffuse in the gel easily, therefore our system will be a great improve in the use co-catalyze system. Considering about bringing more coiled coil system into our hydrogel, we could even construct immobilized enzyme systems with three or four enzymes.
(2). 3D cell culture and tissue engineering. With the help of surface displaying we can display the coiled coil system mentioned above on the surface of cells. These cells would be able to link with the “cross” of spy-catcher and spy-tag and become a part of the hydrogel as the protein I and II in fig. 2. Since our hydrogel is a hydrophilic system the culture can be absorbed into the gel and reach the cells fixed in the hydrogel. Therefore, we can culture cells in three dimensions regardless of the adherent effect of animal cells improving the efficiency of cell culture. If we use different coiled coil system on the system we can carry at least two different cells regularly in the gel. What’s more with the help of 3D printing we can even decide the exact location of different cells, and turn the hydrogel into a scaffold for tissue engineering for developing artificial tissues.
How did we work?
After resaerching literatures on hydrogel we decided to use the coiled coil system between A and B aswell as EE and RR. We wanted link A with spy-tag, EE with spy-catcher, B with GFP and RR with RFP. So we designed for genes:
1.Nedl-A-GS linker-Spy-Tag-GS Linker-A-HindIII
2.Nedl-EE-GS linker-Spy-Catcher-GS Linker-EE-HindIII
3.Nedl-RR-GS linker-RFP-GS Linker-RR-HindIII
4.Nedl-B-GS linker-GFP-GS Linker-B-HindIII
Fistly we get our RR, B and the whole A-spy-tag-A sequences with assembly PCR. Then we used PCR to get RFP, GFP and spy-catcher from the plasmids of our laboratery. We asked Genescript® to synthesys the EEs sequence. Finally, we used overlap PCR to connecet these gene parts in proper order and expressed in E. coli BL21.With the proteins we got we constrct our hydrogel and measured its properties.
We worked in Professor Zhanglin Lin’s lab with mordern experimental facilities.