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.
Claims30
An isolated nucleic acid comprising SEQ ID NO:
- 1, or a sequence at least 90% identical thereto, or SEQ ID NO:
- 2, or a sequence at least 75% identical thereto.
A 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 a 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.
Dependencies are inferred from the claim text.