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These first-step mutants contained single, non-synonymous mutations to gyrA and no other mutations (e.g. in efflux pumps) linked to ciprofloxacin resistance (Supplementary Data 1).
By this criteria, the majority of mutants contained single insertions.
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In the pyelonephritogenic isolate CFT073, mutants containing single deletions of ΔhofQ or ΔyheF were able to infect and colonize bladder and kidney tissues similar to the WT strain.
The mutants containing single intron insertion were determined as target mutants with no off-target integration, and the number of target mutants was counted to calculate the target specificity by dividing the number of all tested mutants.
All of the mutants identified contained single base insertions or deletions leading to frameshift mutations, resulting in receptor truncations within the hormone-binding domain between amino acids (aa) 324-351.
Both single mutants contained a single intron insertion in the chromosome with expected sizes of 4.1 or 4.9 kbp.
Southern blot analysis of GUM1 and GUM2 showed that both mutants contained a single-copy T-DNA integrated into the genome (Figure 3A).
Sequence of the 10 mutant plasmids indicated that each plasmid contained single or multiple mutations in the rep gene or the flanking regions.
Both the wild-type and several mutant proteins containing single- or double-point mutations changing individual cysteine residues to alanine were soluble and expressed efficiently, unless stated otherwise.
These mutants contained catalytically deleterious single amino acid substitutions either in the AAA+ (pBAD18-yeaG K116A and pBAD18-yeaG R232A) or STK (pBAD18-yeaG K426A) domain.
In temperature-shift and acid washing studies with G-CSFR mutants containing either single or double leucine to alanine substitutions at residues 753 and 754, no significant differences in internalized ligand were observed between the WT and L→A transfectants (Figure 2).
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