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To examine whether the mutants detected were created de novo by adaptation, or were enriched from pre-existing mutants present in the natural HpSVd-grapevine population, four HpSVd-free hop plants were infected with a transcript from an infectious cDNA clone of HpSVd-grapevine.
Cultures are then plated on selective media to determine the number of mutants present in each culture.
This yield suggests that the screen has identified ~90% of the sporulation-defective mutants present in the collection.
LacZ transgene mutants present in the genomic DNA were identified using the Phenyl-β-D-galactopyranoside (P-gal) positive selection assay [ 4].
All the mutants pulled out as hits in the pso2-deleted cisplatin screen were subsequently re-made using the single mutants present in the deletion library.
Our screen identified >90% of the previously known sporulation-defective mutants present in the collection, suggesting that the screen has identified the majority of non-essential genes required for spore formation.
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The Chrnb4−/− mutants used were generated by a process similar to the Xu Chrnb2−/− mutants presented in this work [23].
However, the analysis of transposon-tagged Pseudomonas aeruginosa mutants presented in this article shows that this phenotype emerges from the action of numerous proteins from all functional categories.
A major new finding in the study of the PncA mutants presented in this report is the essential contribution of the studied residues not only to catalysis but also to protein folding and thermal stability.
Though, a full understanding of the complete mechanistic picture awaits a detailed three dimensional analysis of the wild type and the mutants presented in this paper, the results herein can serve as a good starting point for targeted drug development approaches.
The mutants presented in this work have shown to be stable over the course of 20 months.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com