XL-1 Red E.coli大肠杆菌菌株/感受态细胞 BioVector NTCC质粒载体菌种细胞基因保藏中心

XL-1 Red E.coli大肠杆菌菌株/感受态细胞
A highly efficient, rapid and reproducible method for introducing random mutations in a cloned gene of interest was developed. This method involves propagating the cloned gene into an Escherichia coli strain, called XL1-Red, which is deficient in three of the primary DNA repair pathways. The mutS (error-prone mismatch repair),1 mutD (deficient in 3´- to 5´- exonuclease of DNA polymerase III)2 and mutT (unable to hydrolyze 8-oxodGTP)3 mutations were introduced into the XL1-Red strain using basic bacterial genetics. The random mutation rate in this triple mutant strain was measured to be ~5000-fold higher than that of wild-type. This strain is particularly suitable for generating random mutations within a gene that has no selectable or screenable phenotype, and the method does not require extensive genetic or biochemical manipulations. Because of the mutator genes engineered into the XL1-Red strain, this mutator strain possesses the following characteristics requiring special considerations: ♦ Although this mutator strain is recombination proficient (recA ), the strain grows extremely slowly in rich media (e.g., LB media), having a doubling time of ~90–120 minutes. ♦ The XL1-Red strain carries the Tn10 transposon that encodes the tetracycline-resistance gene. However, due to the rapid mutator phenotype, the cells will frequently give rise to tetracycline-sensitive (Tets) variants. Therefore, the resulting transformants may or may not be tetracycline resistant, and we do not recommend that tetracycline be added to the growth media. ♦ Following transformation into XL1-Red competent cells and subsequent plating on LB–ampicillin agar plates at 37°C, the transformant colonies become visible 24–30 hours later. If large-sized colonies are desired, even longer incubation times are often required. ♦ The high mutation rate of the XL1-Red mutator strain causes a wide range of colony sizes. ♦ Additionally, the XL1-Red mutator strain should not be propagated on plates for prolonged periods of time. The rapid mutation rate affects the chromosome, and after prolonged growth, the subsequent colonies are probably not genetically identical to the original strain. To avoid the potential problems outlined above, the XL1-Red mutator strain is available only as competent cells, since the mutator phenotype of the XL1-Red strain cannot be guaranteed if the strain is maintained by the researcher. Each lot of XL1-Red competent cells is strictly monitored to ensure that the competent cells retain their mutator phenotype. For the E. coli strain, the genes listed in the table below signify that the bacterium carries a mutant allele. The genes present on the F´ episome, however, represent the wild-type alleles unless indicated otherwise. Strains should be considered lambda– and F– unless designated otherwise. a:The XL1-Red strain carries the Tn10 transposon that encodes the tetracycline-resistance gene. However, due to the rapid mutator phenotype, the cells will frequently give rise to tetracycline-sensitive (Tets) variants. Therefore, the resulting transformants may or may not be tetracycline resistant.
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