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
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 means for positive selection, and a counterselectable marker, wherein the counterselectable marker is an α-subunit of phenylalanyl-tRNA ligase, (PheS) that has been codon optimized for Bacillus and further comprises A309G/T255S mutations, and wherein the counterselectable marker is operably linked to at least one promoter;
- transforming a Bacillus species with the DNA construct;
- selecting for a Bacillus strain having integrated the DNA construct based on the means for positive selection;
- growing the Bacillus strain having integrated the DNA construct in the presence of 4-chlorphenylalanine to select for a Bacillus strain having undergone a homologous recombination event excising the backbone of the plasmid containing the counterselectable marker 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.
A high-throughput (HTP) method for generating at least one scarless genomic edit in a microorganism, comprising:
- providing a plasmid or linear DNA construct comprising a sequence of interest, a means for positive selection, and two counterselectable markers, wherein each of the counterselectable markers have been independently codon optimized for the microorganism and have a maximum tandem 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 microorganism with the DNA construct;
- selecting for a microorganism strain having integrated the DNA construct based on the means for positive selection;
- selecting for a microorganism 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 microorganism having at least one scarless genomic edit.
A DNA construct comprising at least one of a counterselectable marker comprising SEQ ID NO:
- 1, or a sequence at least 90% identical thereto, and a counterselectable marker comprising SEQ ID NO:
- 2, or a sequence at least 75% identical thereto.
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.
Dependencies are inferred from the claim text.