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US 11,111,507 B2Granted
Method for counterselection in microorganisms
AssigneeZymergen Inc.
InventorsTara F. Mahoney, Amanda Renae Reider Apel, Paloma Rueda Romero
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Abstract
The present disclosure is directed to methods of scarless genomic engineering in microorganisms, such as Bacillus, and provides for new molecular tools and methods which enable scarless genetic editing using at least one counterselectable marker that has been codon optimized for the microorganism. The disclosure allows for the high-throughput introduction of stable genetic edits to a genome using either plasmid or linear DNA constructs for genetic engineering.
Claims15
A high-throughput (HTP) method for generating at least one scarless genomic edit in a Bacillus species, comprising:
- providing a plasmid or linear DNA construct comprising a sequence of interest, a positive selectable marker, and two copies of the α-subunit of phenylalanyl-tRNA ligase (PheS) as counterselectable markers, wherein each of the counterselectable markers have been independently codon optimized for the Bacillus species and have a maximum continuous identity length of 500 base pairs when aligned with each other, and wherein each counterselectable marker is operably linked to at least one promoter;
- transforming the Bacillus species with the DNA construct;
- selecting for a Bacillus strain having integrated the DNA construct based on the positive selectable marker;
- selecting for a Bacillus strain having undergone a homologous recombination event excising the backbone of the plasmid containing the counterselectable markers to produce a loop-out strain;
- and
- screening the loop-out strain for the presence of the sequence of interest to produce a modified Bacillus strain having at least one scarless genomic edit.
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Dependencies are inferred from the claim text.