NheI (10 U/µL)

Protocol tips

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	For the bacterial expression of glutathione S-transferase (GST) fusion products, ORF fragments were excised from the phagemid DNA with the restriction endonucleases PteI and NheI (Thermo Fischer Scientific), subcloned into a custom-designed pGEX-Flag expression vector (36), and grown in a minifermenter as previously described in Deantonio et al. (50).

Protocol tips
For the bacterial expression of glutathione S-transferase (GST) fusion products, ORF fragments were excised from the phagemid DNA with the restriction endonucleases PteI and NheI (Thermo Fischer Scientific), subcloned into a custom-designed pGEX-Flag expression vector (36), and grown in a minifermenter as previously described in Deantonio et al. (50).

Publication protocol

"Phagemids clones were rescued from the selected libraries by inverse PCR as previously described (36, 37). Briefly, a pair of specific
back-to-back outward primers was designed for each of the tested
genes, centering on the nucleotide region identified by the overlapping reads. For each sample, 50 ng of the phagemid DNA minipreps were used as template, and inverse PCR reactions were
performedwith a PhusionHigh-FidelityDNA Polymerase (Thermo
Fisher Scientific). PCR products were purified from agarose gel,
phosphorylated with T4 polynucleotide kinase, ligated by T4 DNA
ligase, and transformed into E. coli DH5aF9. Transformants were
screened by colony PCR and verified by Sanger sequencing.
For the bacterial expression of glutathione S-transferase (GST)
fusion products, ORF fragments were excised from the phagemid
DNA with the restriction endonucleases PteI and NheI (Thermo
Fischer Scientific), subcloned into a custom-designed pGEX-Flag
expression vector (36), and grown in a minifermenter as previously
described in Deantonio et al. (50). The vector harbors a Flag epitope
tag (DYKDDDDK) for the C-terminal tagging of expressed proteins."

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