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Using this strategy, we selected 6 mutants for further studies, in which their productivities were evaluated by fermentation in shaken flasks and bioreactor.
Or, at the opposite extreme, they may lethally affect plant development, resulting in no viable mutants for further analyses.
DOI: http://dx.doi.org/10.7554/eLife.05868.017 We, hence, have chosen vti11 mutants for further investigation and tested if VTI11 function is required for the auxin effect on vacuoles.
These two mutations could thus be useful as GAP-insensitive mutants for further in vitro and in vivo studies, assuming that effector-binding capacity is not perturbed.
In this first round of screening, we dissected 3257 independent lines among which we kept 225 mutants for further analysis in a secondary screen.
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Since these two mutant alleles showed basically the same phenotype (data not shown), we chose kog1-105 mutant for further study.
We concluded that Leu/Ile residues 461, 463, 468 and 470 contributed to NES function in vivo and therefore chose the Mia1p-MutNES4 mutant for further characterization.
In order to generate a Δ cps2E mutant for further study, the high-frequency approach to allele exchange was applied.
We therefore used this mutant for further filtering; we modeled the V71E mutant of each model in F, performing 500 Modeller iterations per V71E side-chain replacement.
We chose the Pep2-K145A mutant for further experimentation, verifying by size exclusion chromatography that this mutant still formed a complex with TreS.
We therefore chose this mutant for further purification for quantitative analysis of phospholipid transfer and membrane binding in a liposome floatation assay.
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