Protein Engineering Protocols - Mycobacteriology research center

264 Campbell-Valois and Michnickvarious factors, such as the level of degeneracy of the libraries and the use of stringencymutants (see Subheading 3.1.2.).4. At any step, a 10 –4 to 10 –5 diluted aliquot of the saturated culture can be plated onsolid selective medium to qualitatively check the efficiency of the competition.The heterogeneity in colony size should decrease and the average size of coloniesshould increase with each successive competition step.5. At any passage, the clones represented in a pool mix can be recovered by inoculating2 mL LB (100 µg/mL ampicillin and 25 µg/mL kanamycin) with 10 µL of the poolmixture and incubated O/N. Then, DNA is prepared with QIAprep (see Note 14).3.6. Isolation of Clones and SequencingThis step consists of the isolation of the library-bearing plasmid from independentclones or from the mixed pools obtained by the manipulations describedin Subheadings 3.4. and 3.5.1. 300 ng of DNA from the pool of clones is digested with a mixture of restrictionenzymes that recognize sites present in the pREP4 and pQE-32 ras-F [3] plasmids,but absent in the library plasmid. For this purpose, we used HpaI, XmaI, EcoNI,and XbaI (see Note 15).2. One-tenth of the digested DNA is transformed in XL-1 Blue chemiocompetentcells, and 20 µL is plated on 24-well plates containing LB-agar with 100 µg/mLof ampicillin (see Note 15).3. Colonies are picked and incubated in LB, and supplemented with 100 µg/mL ofampicillin, in the appropriate culture vessels.4. High-quality DNA minipreps are prepared for sequencing. For processing 96 samplesin plates, we use the Montage kit. We have used QIAprep column kit forsmaller-scale preps.5. For sequencing, we have used a primer that anneals only to the library plasmid,i.e., inside F [1,2] (see Note 16).6. Sequencing.3.7. Protein Purification and CharacterizationAfter analysis of the obtained sequences, clones of interest are selected andrearrayed on the appropriate number of 96-well plates. At this moment, clonescan be retransformed in XL-1 Blue cells and frozen to serve as backup stock.1. The selected clones at this step are recloned to express them as fusions with the6xHis-tag only, i.e., without the DHFR fragment (see Note 17). The expression ofthe 6xHis clones is verified by an induction test (see Note 18).2. The preps of the clones that express correctly are performed with a Montage kitand rearrayed again.3. These clones are then transformed into BL21 pREP4 cells and plated on LB-agarmedium containing 100 µg/mL of ampicillin and 25 µg/mL kanamycin. The platesare incubated at 37°C O/N. When processing several clones in parallel, we use24-well plates. In this case, no more than 20 µL of competent cells should be used