Method of nucleic acid cassette assembly

Inventors

Venter, J. Craig • Smith, Hamilton O. • Hutchison, III, Clyde A. • Gibson, Daniel G.

Assignees

Telesis Bio Inc

Abstract

Methods are provided for constructing a synthetic genome, comprising generating and assembling nucleic acid cassettes comprising portions of the genome, wherein at least one of the nucleic acid cassettes is constructed from nucleic acid components that have been chemically synthesized, or from copies of the chemically synthesized nucleic acid components. In one embodiment, the entire synthetic genome is constructed from nucleic acid components that have been chemically synthesized, or from copies of the chemically synthesized nucleic acid components. Rational methods may be used to design the synthetic genome (e.g., to establish a minimal genome and/or to optimize the function of genes within a genome, such as by mutating or rearranging the order of the genes). Synthetic genomes of the invention may be introduced into vesicles (e.g., bacterial cells from which part or all of the resident genome has been removed, or synthetic vesicles) to generate synthetic cells. Synthetic genomes or synthetic cells may be used for a variety of purposes, including the generation of synthetic fuels, such as hydrogen or ethanol.

Core Innovation

The invention provides a method for assembling a nucleic acid construct by generating a plurality of double-stranded nucleic acid cassettes from chemically synthesized oligonucleotides or copies thereof. The cassettes comprise adjacent regions of the nucleic acid construct to be assembled, and each cassette overlaps one or more other cassettes by at least 50 nucleotides. The cassettes are between about 4 kb and 7 kb in length, not including the overlaps.

The method further simultaneously assembles the plurality of nucleic acid cassettes in vitro in a chew back and repair step. The chew back and repair step uses an enzyme having 3′ or 5′ exonuclease activity, a DNA polymerase, polyethylene glycol (PEG) or a single-stranded binding protein, and a ligase. This chew back and repair process thereby assembles the nucleic acid construct.

Claims Coverage

The partial content provides one independent claim describing cassette generation from chemically synthesized oligonucleotides and simultaneous in vitro chew back and repair assembly with specified component classes and cassette overlap and size constraints. No other independent claims are explicitly provided in the partial content.

The claim coverage centers on an assembly approach that first generates overlapping double-stranded cassettes from chemically synthesized oligonucleotides with defined overlap and cassette length constraints, and then simultaneously assembles the construct in vitro via a chew back and repair step using an exonuclease-active enzyme, a DNA polymerase, PEG or a single-stranded binding protein, and a ligase.

Stated Advantages

Documented Applications

No documented applications found