Counts selected
Binding
Count/Number
unitless
Illumina Sequencing
None
See Counts unselected assay.
Counts unselected details: Yeast-surface display is used to monitor binding activity. Both Ct and Cc cohesin domains are independently displayed on the surface of yeast. Dockerin domains are solubly expressed and chemically biotinylated. A C-terminal c-myc epitope tag is used to monitor protein display and a streptavidin-conjugated fluorophore is used to monitor cohesin–dockerin binding. Binding titration curves for the Ct cohesin– Ct dockerin interaction were determined by flow cytometry. Populations are discriminated by three sorting gates. The reference gate drawn around forward and side scatter channels captures all yeast cells passing through the flow cytometer. The displayed gate captures all yeast cells displaying the cohesin domain, while the bound gate captures approximately the top 5% of the binding population. Collected populations are grown overnight and the plasmid DNA containing cohesin variants is extracted, deep sequenced, and analyzed.
Deep sequencing followed the optimized protocol of Kowalsky et al. Briefly, yeast cells are lysed using zymolase, a freeze–thaw cycle, and alkaline lysis. Sheared genomic DNA and ssDNA are partially cleaned up from the plasmid DNA by an exonuclease processing step, and then the insert is amplified by PCR using a high-fidelity polymerase. PCR products are verified by agarose gel. Library DNA was sequenced using the 150 X 2 and 250 X 2 Illumina MiSeq kits (Illumina, San Diego, CA) at the Michigan State University Sequencing Core.