Table 2. Functional and genotype analysis of recombinant pools.
| Round-1 | Round-2 | |||||||
|---|---|---|---|---|---|---|---|---|
| Phenotype (fluorescence) | GFP | + | - | + | + | - | + | |
| RFP | + | - | + | - | ||||
| genotype 1 (PCR) | NT 2 | + | - | NT 2 | + | + | NT 2 | |
| HK cassette | 85(90%) | 1(1%) | 8(9%) | 0(0%) | 0(0%) | 0(0%) | 94(100%) | |
| HKH cassette | 94(100%) | 0(0%) | 0(0%) | 90(96%) | 2(2%) | 2(2%) | 0(0%) | |
After each round of recombineering depicted in Fig. 4B, 94 clones were randomly picked, grown, and analyzed with regard to both phenotype (fluorescence) and in genotype (genomic PCR).
1. Number of clones with PCR bands corresponding to ΔlacZ:: p L-gfp mut3.1 (for round-1) or ΔlacZ::p L-gfp mut3.1 and ΔproV::p T5-mrfp (for round-2). For the sequences of primers used for genotype analysis (P34 and P42 for p L-gfp mut3.1, P35 and P39 for p T5-mrfp), see S1 Table.
2. NT; not tested.