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US 12,180,487 B2Granted
Minicircle producing bacteria engineered to differentially methylate nucleic acid molecules therein
AssigneeFred Hutchinson Cancer Center
InventorsChristopher Johnston, Sean Cotton
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Abstract
Embodiments include engineered minicircle-producing bacterium with differential methylation capability, as well as kits and compositions comprising the bacterium. Further described are methods of using of the bacterium for producing differentially methylated minicircle DNA, and for improving transformation efficiency of exogenous DNA in intractable bacteria.
Claims12
An engineered, minicircle producing bacterium comprising:
- a parental plasmid comprising a minicircle nucleic acid sequence comprising an exogenous nucleic acid sequence and a plurality of restriction sites outside of the minicircle nucleic acid sequence,
- wherein at least one endogenous methyltransferase comprising at least one of a Dam methyltransferase, a Dem methyltransferase, a HsdM methyltransferase, or a combination thereof, is absent or non-functional in the engineered bacterium, such that the engineered bacterium has reduced DNA-methylation capability and the exogenous nucleic acid sequence lacks methylation at a plurality of methylation cites that would be methylated in a reference bacterium of the same species as the engineered bacterium.
A method, comprising:
- transforming a parental plasmid into an engineered bacterium,
- wherein the parental plasmid comprises a minicircle nucleic acid sequence comprising an exogenous nucleic acid sequence and a plurality of restriction sites outside of the minicircle nucleic acid sequence, and
- wherein at least one endogenous methyltransferase comprising at least one of a Dam methyltransferase, a Dem methyltransferase, a HsdM methyltransferase, or a combination thereof, is absent or non-functional in the engineered bacterium, such that the engineered bacterium has reduced DNA-methylation capability and the exogenous nucleic acid sequence lacks methylation at a plurality of methylation cites that would be methylated in a reference bacterium of the same species as the engineered bacterium;
- and
- producing a minicircle comprising the minicircle nucleic acid sequence, wherein the minicircle lacks methylation at a plurality of methylation sites that would be methylated in a reference bacterium of the same species as the engineered bacterium.
2 more elements
Dependencies are inferred from the claim text.