Protein Engineering Protocols - Mycobacteriology research center

262 Campbell-Valois and MichnickFig. 4. Schematic representation of the strategy for the synthesis of degeneratedlibraries (see Subheading 3.2. for detail). The strategy is divided into three distinctsteps: first, the template is obtained by replacing the wild-type sequence of the regionto be varied by an in-frame stop codon, a frame shift (FS), and a unique restriction site(RS) to allow for identification of the mutant (the solid line and dashed line correspond,respectively, to the DNA of the gene of interest and of the vector). Next, two PCR reactionsare performed on this template. PCR 1 product corresponds to the 5′-end of thegene, whereas PCR 2 corresponds to the 3′-end of the gene. In the latter case, theSTOP-FS-RS sequence in the template is replaced by the appropriate number of NNKdegenerated codons. Note that the products from the first round of PCR partiallyhybridize through their 3′- and 5′-ends. Thus, in PCR 3, the products from the firstround PCRs act as the template and primers for the reaction. After a low number ofcycles, usually 10, the full-length degenerated gene is recovered by digestion with therestriction enzymes Sph I and XhoI. The library is then ready to be cloned.3.3. Library Cloning and Recovery1. The ideal insert vs vector ratio for ligation is 2:1 to 3:1. We try to limit the concentrationof DNA ligated to 10 ng/µL and we use 1 mM adenosine triphosphate.We allow the ligation to proceed O/N at 16°C (see Note 8).2. The enzyme is heat inactivated at 65°C, chloroform extracted, and precipitatedwith ethanol. The DNA pellet is then air-dried for several minutes, and resuspendedin 30 µL of deionized water before electroporation.3. SS320 E. coli strain (see Note 9) electrocompetent cells are prepared the same day(see Subheading 3.8.). The ligation reaction from step 2 is mixed in 300 µL ofresuspended SS320 cells. The mix is transferred to 2-mm-wide electroporatingcuvets and electroporated on the Genepulser II. The apparatus parameters are