The Integration Of Rational Design Of Biomolecules With Directed Evolution On An Experimental Perspective

Life is the highest form of material movement. Creating and artificial modification of life and its components will be helpful for us to understand and use them. The main purpose of synthetic biology is to use standardized and modular parts to construct the multi-levels of life (biology parts, biology devices, biosystems and so on) like industrial engineering; then design and construct more complicated systems by these parts or devices to understand life better and make them useful for human beings.However, there are only a few natural parts we can use, and most of them can’t work perfectly like electric parts. The biology switches are not totally on or off, the exogenetic parts can affect the host cells, the interactions among the biological parts are complicated, and there will always be unexpected results of the combination of biological parts. One way to solve this problem is to design new biology macromolecules to create more and better biological parts or devices. However, the success rate is limited. At the same time, directed evolution is also a powerful method to engineer biological macromolecules. The integration of rational design of biomolecules with directed evolution experimentally was discussed in this dissertation. Integrating these two methods more perfect biological parts or devices can be constructed, by which more complicated and large scale biological systems could be built. In this way we can understand life better and solve many important problems (e.g. energy, food, health and environment problems) by them.DNA is the carrier material of genetic information, and the protein is the undertaker of life activities. The protein design and engineering were mainly by synthesis or engineering the DNA.In the first part, de novo protein design and its modification were discussed. A de novo designed protein was manually mutated. According to the HSQC results, the structure of this protein was improved effectively.In the second part, the design of new proteins by combining natural modules and directed evolution was discussed. A protein designed by combining natural protein modules was successfully optimized by six rouds of directed evolution. According to physiochemical characterization, the stability and foldability of this protein were significantly improved. Particularly, the melting temperature was increased by more than 20℃. Moreover, a high rate appearance of a cysteine mutation to form intramolecular disulfide bond was found, which contributed significantly to the protein stability.In the third part, the designed protein-protein interaction and its experimental detection and directed evolution were discussed. A PCA (protein-fragment complementation assay) system based on murine DHFR (dihydrofolate reductase) was reconstructed, by which we detected the designed protein-protein interaction and improved it by directed evolution successfully. A highly tunable PCA system constructed based on Escherichia coli DHFR can be used for detecting more wide range of protein-protein interactions.In the fourth part, the specific tetramerization of a certain protein LacI (lactose repressor) and the design of low interference biological parts were studied. By using the transcriptional logic gates based on our LacI mutants to distinguish the homotetramerization and heterotetramerization of LacI, two genetic circuits containing double selection to select or screen the specific LacI heterotetramerization were contructed. One of the reporter parts was also knocked into E. coli genome.In the fifth part, the construction of an artificial genetic circuit to achieve a specific function was discussed. The quorum sensing system and the genetic toggle switch were conmbined by our transcriptional logic gates based on LacI mutants.The automatic differentiation of E. coli induced by the identity of cells was realized. In other words, the E. coli cells with the same genotype would differentiate into different phenotypes spontaneously.In this dissertation we discussed the experimental modification and optimization of de novo protein design, creating proteins by combining natural modules, designed protein-protein interactions and other rational design. This semi-rational design method is an efficient approach to create new biological parts, by which more complicated life components or life itself can be constructed to realize certain functions. The usage of artificial parts was also tried by constructing a genetic circuit to realize E. coli differentiation. All the plasmids were constructed according to the BioBrick Santdards, which will be convenient for further extension. The genomic integration of artificial devices was also studied, which is beneficial to construct large scale artificial systems. Most of the work needs further studies; we hope that these results will provide contributions to the design and understanding of life and their usage to practical applications.