Designing of new bionanomaterials represents a very important field in nanotechnology, which is rapidly developing. Molecular self-assemby enables design of new artificial protein nanomaterials that are smart, adaptable, biocompatible and biodegradable. The new way of designing protein structures that self-assemble from a single polypeptide chain has high potential for many different applications. A single polypeptide chain is composed of many interconnected peptide modules. Golden Gate method is very useful for assembling modular structures of this kind, as it enables ligation of several modules in the same reaction. It uses type IIs restriction endonucleases and T4 DNA-ligase. Restriction enzymes of this type cut DNA outside of ther recognition site, which enables cutting different DNA sequences using only one restriction enzyme. The construction of DNA sequences is therefore faster, as the whole process takes place in a single reaction. With correct designing of restriction endonuclease recognition sites we can also determine the order in which the modules are assembled.
We designed a sequence for a triangle-shaped protein with a fluorescent protein at the vertices. The designed protein is composed of nine modules that are interconnected in a defined order. Six modules are forming three pairs of coiled-coils that represent sides of the triangle. The remaining three modules contain sequences encoding fluorescent proteins. Each side represents a 15 heptad long coiled-coil homodimer. We used slightly modified sequences from natural homodimers tropomyosin, Rho-kinase and cortexillin. Coiled-coil structure ensures structural stability of the protein and enables in vivo self-assembly to the designed geometrical shape. We designed nucleotide sequence of nine modules, each between 300 and 700 nucleotides in length, that contain restriction endonuclease BsaI recognition sites. With the use of Golden Gate method we successfully cut and ligated in final vector on around 4000 nucleotides long sequence, that is composed of 9 modules arranged in a defined order.
Additionally, we optimized the Golden Gate assembly method so that it could be used for assembly of more than 12 modules. We optimised the reaction prameters, which included the concentration of enzymes (DNA ligase and BsaI), and the concentration of ATP. We tested different approaches and the most successfull one involved preliminary assembly of shorter sequences in separate reactions. These partial constructs are in a second step joined together and ligated into the target vector. Using this approach we succeded to assemble the sequence encoding proteins composed of 12 and 18 modules, respectively.
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