The final product of our system is an in vivo library of the target sequence carrying different versions of mutations. Researchers can then continue to implement the selection process directly using this library, enabling the continuous evolution of our target towards its desired function. Moreover, as bacterial cells can express multiple genes in a polycistronic transcript, our system has the ability to evolve a series of genes at the same time, which opens up possible application in whole metabolic pathway evolution. The nature of our target sequence can be either protein or RNA, allowing for mutation library construction of a broader range. Our system also has the advantage of using parts orthogonal to native bacterial systems, thus could be applicated in various prokaryotic hosts.

Through application of our system, researchers would be able to evolve enzymes towards higher efficiency, higher precision and novel functionality, or to evolve metabolic pathways towards more balanced function, less toxic to host cell and higher total yield, or to evolve different functional RNAs. In addition, the mutagenesis system could be applied in different prokaryotic hosts.

Environmental resistance

Enzyme function greatly relies on stable and fitting environment. A small change in environmental factor such as temperature, pH, osmotic pressure or metal ion concentration could greatly affect the activity of a certain enzyme. The native environment is often moderate and cannot meet researchers’ divergent needs. Researchers have been applying directed evolution methods in generating enzymes which could function under their desired conditions, for example, heat-resisting enzymes. Employing our system could easily and efficiently create a mutation library that can be used for further selection.

Higher efficiency

Nature provides us with proteins exhibiting an almost endless diversity of functions. But we often find them acting less to satisfactory in heterologous systems or when our need exceeds the output of the native system. To meet our needs, we could associate the mutagenesis output with selection pressure such as antibiotic resistance, and only those who performs best will be able to survive. After gradient increase of selection pressure, the protein construct with highest efficiency can be easily selected out.

Higher precision

Off-targeting and crosstalk are not uncommon even in heterologous systems, let alone the complex intertwined relationship of proteins within the native system. To minimize or even eliminate crosstalk, researchers could employ our system to generate diverse mutations, and add selection pressure to obtain the desired result.

Novel function

In our effort to create orthogonal systems, expanding our genetic code, producing proteins with non-canonical amino acids, or generating novel compounds, researchers are in need of enzymes which does not exist in nature. Similar methods have already been used in generating orthogonal aminoacyl-tRNA synthetases, orthogonal ribosomes, and novel compounds such as organosilicons.

Our system is our sword penetrating the wall of natural world, through which we could not only take a glimpse, but also take a tentative step out and embrace the vastness of unknown. The existing world has its limitations, but out imagination does not, through in-lab evolution, we’re turning imagination into reality.

Metabolic pathway

Bacteria is a rapidly developing factory for biological and chemical synthesis. It is common for researchers to transfer multiple enzymes or even whole metabolic pathways at one time into the engineered bacteria. However, heterologous expression is often met with problems regarding metabolic pathway interference and differed expression profile in nonnative host.

Our system could mutate a sequence of a relatively long length (~10 kb) due to the outstanding processivity of our reverse transcriptase. Since our target sequence can be transcribed as a whole into RNA and go through the cycles of mutagenesis, no matter this sequence encodes protein or functions as regulatory component, our system has the potential of evolving full metabolic pathway together.

RNA

The nature of target sequence can vary. Apart from proteins, functional RNA can also be our target of mutagenesis. Cellular RNA has varied functions, including miRNA and riboswitch, both which are commonly used in synthetic biology. The RNA sequence could be inserted in the place of target sequence and be transcribed, then go through mutagenesis cycles of reverse transcription and recombination, which would output a mutation library of the RNA target. By utilizing our system, a mutation library of miRNA and riboswitch could be easily generated and be tested in later experiments.

Adenovirus associated virus (AAV) library

In our Integrated Human Practice, we interviewed Prof. Chen Ling, who expressed great interest in our system and conveyed to us that our system could be used in library construction for AAV’s capsid protein. The constructed library could be later used to generate gene delivery vehicles of enhanced function.

Multi-host directed evolution

In our system, we utilized parts that are orthogonal to native prokaryotic systems. The reverse transcriptase is of mammalian origin, and the priming tRNA sequence is orthogonal to that of prokaryotes or could be modified to align the target directly as the user wishes. Cre recombinase is originated from bacteriophage P1 and already widely applied in prokaryotic engineering. Our system has the ability of functioning in different bacteria and could enable directed evolution in different host species in parallel. This ability to build and test the target within the same system greatly increases the efficiency of desired part selection.