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For 91 mutants, we used the protein design algorithm FoldX.
To identify the genes responsible for the heb mutants, we used the positional map-based approach with the short-root phenotype as an index.
To study the relation between phenotype and rhodopsin stability in disease mutants, we used a structure-based approach.
We chose to initially conduct cheating assays using only a 50% starting frequency, as both mutants we used have been reported to act as cheats under this condition in iron-limited CAA (Kümmerli et al., 2009b, 2015; Kümmerli and Brown, 2010).
To further investigate the relationship between EAT-6 function and phenotypes seen in eat-6 mutants, we used RNA interference (RNAi) to knock down eat-6 gene activity.
For construction of double mutants we used pHT315Ab-D136N harboring a point mutation D136N as template to introduce additional point mutations as E129K or T143D.
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To accurately estimate the expression differentiation between the wild type and the long-lived mutants, we use sub-array normalization followed by a variant of the median-polishing summarization.
To generate the models of wt hAID and N51A mutant we used structures that were available when we commenced our studies i.e. the structures of the C-terminal catalytic domain of APOBEC3G (PDB entry 3V4K and 3E1U).
To evaluate the soil-surface rooting phenotype of the mutant, we used four different methods: the pot method, the cup method, the glass tube method, and the basket method.
To provide an explanation for the residual activity in the hcpA mutant we used a flagella mutant of EDL933.
As previously described the 212-C-ter mutant we used is comprised of the fourth RBD and the GAR domains.
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