Modified yeast two-hybrid assays were performed as described (Bartel and Fields, 1997 ). Briefly, sygl-1 cDNA encoding wild-type, full-length SYGL-1 (aa 1-206), or full-length SYGL-1 carrying PIM mutations were cloned into the NcoI site in pACTII (Gal4 activation domain plasmid), generating pJK1580, pJK1581, pJK1582, pJK2094, pJK2095 and pJK2096 using the Gibson assembly method. The PUF repeat region of FBF-2 (aa 121-632) was cloned into the NdeI site in pBTMknDB (LexA-binding domain plasmid) to generate pJK2046. Activation and binding domain plasmids were co-transformed into the L40-ura strain using the Te-LiAc method (Gietz and Schiestl, 2007 (link)). lacZ reporter activity was assayed in defined media (SD) supplemented with -Leu-Trp using the Beta-Glow® Assay System following commercially available protocols (Promega, E4720). In short, yeast cultures were grown to mid-log phase, diluted to the same optical density (0.1), and added to equal volumes of Beta-Glow® reagent. Yeast clones were then incubated for 1 h at room temperature, and luminescence was quantitated using a Biotek Synergy 4 Hybrid plate reader and Gen5 software. A complete list of plasmids used in yeast two-hybrid assays is given in Table S3 .
Beta glow assay system
Manufactured by Promega
The Beta-Glow® Assay System is a luminescence-based solution for the quantitative determination of beta-lactamase enzyme activity. It provides a simple, sensitive, and rapid method for measuring beta-lactamase levels in biological samples.
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2 protocols using beta glow assay system
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Modified Yeast Two-Hybrid Assays
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Modified Yeast Two-Hybrid Assay for SYGL-1 and FBF-2
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Modified yeast two-hybrid assays were performed as described (Bartel & Fields, 1997). Briefly, sygl-1 cDNA encoding wild-type full-length SYGL-1 (a.a. 1–206), or full-length SYGL-1 carrying PIM mutations were cloned into the Nco I site in pACTII (Gal4 activation domain plasmid), generating pJK1580, pJK1581, pJK1582, pJK2094, pJK2095, and pJK2096 respectively, using the Gibson assembly method. The PUF repeat region of FBF-2 (a.a. 121–632) was cloned into the Nde I site in pBTMknDB (LexA binding domain plasmid) to generate pJK2046. Activation and binding domain plasmids were co-transformed into the L40-ura strain using the Te-LiAc method (Gietz & Schiestl, 2007). LacZ reporter activity was assayed in defined media (SD) supplemented with -Leu-Trp using the Beta-Glow® Assay System following commercially available protocols (Promega #E4720). In short, yeast cultures were grown to mid-log phase, diluted to the same optical density (0.1), and added to equal volumes of Beta-Glow® reagent. Yeast clones were then incubated for 1 hr at room temperature, and luminescence was quantitated using a Biotek Synergy 4 Hybrid plate reader and Gen5 software (Winooski, VT). A complete list of plasmids used in yeast two-hybrid assays is available in Table S3 .
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