Figure 2.

Screen strategy and results for identification of mutant rox sites. (A) Strategy used for recombination test (for p4RKanP, p6RKanP, or p2NKanP libraries) and for library screening (p2NKanP library). For details, see also Material and Methods. (B) PvuI + PstI restriction digest analysis of pooled target vectors before (−) and after (+) exposure to Dre recombination. Linearized pGB2-Dre can be seen in the (+) lanes (5.3 kb). Target vectors show the unrecombined restriction pattern (1.4 kb + 1.8 kb) before pGB2-Dre cotransformation (− lanes). After cotransformation (+ lanes), various degrees of recombination (0.8 kb band) can be seen. (C) Densitometric analysis of recombination efficiency of libraries and wild type control. (D) Nucleotide distributions at positions 1–8 in 82 individual p2NKanP minipreps sequenced after cotransformation with pGB2-Dre, and Kan selection (top) and 24 control p2NKanP minipreps from the unrecombined library (bottom). Original roxP sequence marked as wild type (WT) is indicated at the top. Positions 1, 4, 5, and 8 were not mutated in the rox2N spacer library. Numbers in the colored bars represent number of spacer sequences having the indicated nucleotide at the respective position. Nucleotide distributions were not significantly different between Dre-treated and untreated libraries at positions 3 (χ² = 0.73, P = 0.87) and 6 (χ² = 3.84, P = 0.28). However, after Dre selection, position 2 shows a highly significant bias toward G and C (χ² = 18.44, P = 0.000357), and position 7 a significant bias toward G (χ² = 9.99, P = 0.0186). (E) Most frequent spacers recovered from the screen. Nucleotides differing from consensus are shown in black. Occ. Nr. represents the number of times that specific sequence was observed. Clone number is the representative clone used for further studies.