Journal: Nature Communications

Article Title: Deep mutational scanning reveals a correlation between degradation and toxicity of thousands of aspartoacylase variants

doi: 10.1038/s41467-024-48481-0

Figure Lengend Snippet: A Schematic representation of the expression system. HEK293T cells, carrying a landing pad for Bxb1-catalyzed site-specific integration are transfected with the expression vector and a Bxb1 expression plasmid (not shown). Upon integration at the landing pad locus, the BFP-iCasp9-Blast R gene is displaced downstream, and the cells therefore become resistant to AP1903, while GFP-ASPA and mCherry are expressed from the tetracycline/doxycycline regulated promoter. The same mRNA leads to both GFP-ASPA and mCherry protein production, which in turn allows flow sorting of cells based on the GFP:mCherry ratio. Finally, variants in each bin can be identified by sequencing the barcodes. Figure adapted from refs. , , . Figure created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. B Fluorescence microscopy of cells transfected with either wild-type ASPA (WT) or ASPA C152W variant. Note the reduced amount of the C152W variant. Scale bar = 20 μm. C Cells were transfected with either WT or C152W ASPA variants fused to GFP in the N-terminus or C-terminus as indicated. A mock transfection was included as a control. Whole-cell lysates were then resolved by SDS-PAGE and analyzed by Western blotting using antibodies to GFP, Cherry, or, as a loading control, GAPDH. Note the reduced level of the C152W variant. D Scatter plots of flow cytometry analyses of the WT (blue) and C152W (red) ASPA variants, along with the site-saturated ASPA library (gray). Note that the mCherry levels are similar, while the GFP levels differ approximately 10-fold. E Histograms of the GFP:mCherry ratio based on WT (blue) and C152W (red) ASPA variants, and the ASPA variant library (gray). F The ASPA library was sorted into four separate bins (1–4) as indicated, with each bin containing 25% of the total population.

Article Snippet: The Gibson reaction was then cleaned and concentrated with the Zymo Research kit and transformed by electroporation into NEB-10β electro-competent E. coli cells with 2 kV.

Techniques: Expressing, Transfection, Plasmid Preparation, Sequencing, Fluorescence, Microscopy, Variant Assay, Control, SDS Page, Western Blot, Flow Cytometry