Figure 1 | Scientific Reports

Figure 1

From: Iterative screening methodology enables isolation of strains with improved properties for a FACS-based screen and increased L-DOPA production

Figure 1

Screening strategy. In vitro transposon-disruption library enables iterative screening for deletions that increase production of betaxanthin biosensor. (A) The biosensor gene cassette comprises 3 genes: tyrosine hydroxylase (CYP76AD1), DOPA dioxygenase (DOD), as well as an additional feedback-resistant mutant of DAHP synthase (ARO4 K229L); not shown. (B) The initial screen was performed using a pooled, transformed culture of the yeast deletion collection. (C) Subsequent screen iterations were performed using libraries generated through transposon-based shuttle mutagenesis. Details of cloning for transposon are in Supplementary Fig. S3. (D) Libraries generated using the protocols outlined in (B,C) were sorted first for size to control for the effects of size on fluorescence. Cells falling in the middle 30% of the size distribution were sorted for fluorescence at a threshold of 0.5% of the population. Sorted cells were plated and hits were later validated and sequenced.

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