Table 3.
Summary of bifidobacterial mutagenesis strategies.
| Concept | Benefits | Pitfalls | Example |
|---|---|---|---|
| SINGLE-CROSSOVER PLASMID INSERTION | |||
| • Non-replicative plasmid insertion | • Reproducible | • Prior knowledge of the strain is essential | • Disruption of galA and apuB genes in B. breve UCC2003 (O'Connell Motherway et al., 2009) |
| • Single cross-over homologous recombination | • Requires only a single transformation round | • Successful knock-outs left with antibiotic marker | |
| • Internal region of target gene and marker | • Requires high transformation efficiencies | ||
| • Unstable mutations | |||
| DOUBLE-CROSSOVER GENE DELETION | |||
| • Non-replicative plasmid insertion | • Stable mutation | • Successful knock-outs left with antibiotic marker | • Disruption of BL0033 in B. longum NCC2705 (Fukuda et al., 2011) |
| • Double crossover homologous recombination | • Gene target is deleted | • Time-consuming and laborious- multiple transformations and extensive screening of transformants | |
| • 5′ and 3′ regions of target gene separated by marker | • Requires high transformation efficiencies | ||
| DOUBLE CROSS-OVER MARKER-LESS GENE DELETION | |||
| • Non-replicative plasmid insertion | • Stable mutation | • Time-consuming and laborious- multiple transformations and extensive screening of transformants | • Disruption of aga in B. longum 105-A (Hirayama et al., 2012) |
| • Double cross-over homologous recombination | • Gene deletion | ||
| • 5′ and 3′ regions of target gene adjacent to marker | • Successful knock-outs left without antibiotic marker | ||
| • Marker-less gene disruption | |||
| TEMPERATURE SENSITIVE (TS) PLASMID FOR GENE DISRUPTION | |||
| • Ts plasmid unable to replicate at high temperatures | • Does not require high transformation efficiencies | • Time-consuming and laborious- multiple transformations and extensive screening of transformants | • Disruption of apuB in B. breve UCC2003 (O'Connell Motherway et al., 2008) |
| • Ts plasmid contains homologous sequence and marker | |||
| • Homologous recombination | |||
| TRANSPOSON MUTAGENESIS FOR GENE DISRUPTION | |||
| • Random mutagenesis with Transposome complex | • Generation of a large mutant bank | • Successful knock-outs left with antibiotic marker | • Creation of a mutant library in B. breve UCC2003 (Ruiz et al., 2013) |
| • Large number of gene disruptions | • High throughput screening of bank | • Requires high transformation efficiencies | |
| • Antibiotic marker | |||