Protein Engineering Protocols - Mycobacteriology research center

102 Koide and Koide4. Discard the supernatant. Suspend cells in 10 mL HEPES buffer and transfer thesuspension to a 50-mL centrifuge tube. Rinse the first bottle with the buffer andrecover the cell suspension to the 50-mL tube.5. Centrifuge at 4300g (6 krpm in a SS-34 rotor) for 10 min at 0 to 2°C. Discardsupernatant. Suspend cells in 10 mL of 10% glycerol by knocking the tube on ice,then add 20 mL of 10% glycerol and mix the suspension by inverting the tubeseveral times.6. Centrifuge at 4300g for 10 min at 0 to 2°C. Discard supernatant. Add 300 µL of10% glycerol and suspend cells by knocking the tube on ice. Check the volumeand add 10% glycerol to a final volume of 700 µL. Aliquot the suspension into two350-µL suspensions in microcentrifuge tubes.3.1.6. Electroporation for Phagemid DNA1. Add chilled DNA (Subheading 3.1.4., step 4) to 350 µL of electrocompetent cellsin a microcentrifuge tube and incubate on ice for 5 min. Transfer the mixture to aprechilled electroporation cuvette (2-mm gap).2. Electroporate the cells, e.g., using BTX ECM395 electroporator in the highvoltagemode at 2.5 kV.3. Immediately add 1 mL SOC into the cuvet and suspend the cells. Transfer the cellsto a 250-mL flask. Add 1 mL of SOC into the cuvet and wash the remaining cellsand transfer to the flask. Repeat this process three more times.4. Add 20 mL SOC into the flask. Shake the flask at 180 rpm, 37°C for 30 min, andcheck the titer by plating a diluted portion of the suspension on LB-Ap plate.5. Add the entire suspension to 500 mL of prewarmed 2xYT-Ap. For a plasmid preparationfor the yeast two-hybrid screening, shake at 37°C overnight and prepare plasmid.For phagemid preparation, add 10 10 pfu/mL of helper phage KO7, shakeovernight at 37°C, and prepare phagemids as described in Subheading 3.1.1.For transformation of phage DNA, after step 3, add 1.5 mL of mid-log SS-320 cells, then transfer the suspension to prewarmed 120 mL of 2xYT-Tc. Titerthe phages as described in Subheading 3.1.2. Incubate for 4 h at 37°C. Preparephages as described in Subheading 3.1.1.3.1.7. Construction of a Yeast LibraryWe construct vector libraries for yeast two-hybrid screening first in E. coli,as described in Subheading 3.1. and then introduce the libraries in yeast.Transformation of yeast is based on the method of Gietz (18). We typicallyobtain approx 10 6 independent clones using this method.1. Grow yeast EGY48 in 20 mL YPD with shaking at 30°C overnight. Add the cultureto prewarmed 300 mL YPD in such a way that the OD 600nmis 0.25. Grow thecells until OD 600nmis approx 1.2. Centrifuge at 3600g (4.5 krpm, JA-10 rotor) for 5 min at room temperature. Discardsupernatant, and suspend cells in 300 mL sterile water. Repeat centrifugation and