Self-Selection Using Artificial Networks: An Adaptive Tool for Directed Evolution

Selection-based strategy proved to be very efficient for directed evolution of enzymes but relies on the possibility of finding a case-specific selection tool that links the enzyme activity to its gene survival. Our self-selection process involves the use of a simple artificial molecular network. Introduced as a feedback loop it links directly the activity of the enzyme with the PCR amplification of its own gene. Molecular networks, such as the one using the DNA toolbox (1), are designed to produce short DNA strands interacting within each other’s. They can then generate short oligonucleotides at the output. Assessing enzyme activity via the network allows to produce a correlated amount of primers and therefore will link enzyme activity to a PCR amplification yield.

In an approach inspired by the compartmentalized self-replication of Ghadessy and al. (2) we aim at using microdroplets as compartments to perform the self-selection process. Microfluidics allows us to generate a large amount of water-in-oil microdroplet. Up to 10⁸ parallel tests on individual copy of the gene can be run simultaneously with few amount of space and reagents.

 After generation of a mutant library, bacteria carrying and expressing the mutant are separated in individual droplets containing the molecular program. The program is next launched in the droplets before the PCR step. Then droplets are lysed to retrieve mutant gene copies and a new evolution cycle can be run. Promising results have been obtained with the nicking enzyme Nt.Bst.NBI used as a first model with this adaptive selection tool for directed evolution.

(1) K. Montagne, R. Plasson, Y. Sakai, T. Fujii, and Y. Rondelez, “Programming an in vitro DNA oscillator using a molecular networking strategy,” Mol. Syst. Biol., vol. 7, p. 466, Feb. 2011.

(2) F. J. Ghadessy, J. L. Ong, and P. Holliger, “Directed evolution of polymerase function by compartmentalized self-replication,” Proc. Natl. Acad. Sci. U. S. A., vol. 98, no. 8, pp. 4552–4557, Apr. 2001.