title: Enhanced error-prone RCA mutagenesis by concatemer resolution.
abstract: Error-prone rolling circle amplification (RCA) is a promising alternative to error-prone PCR for random mutagenesis. The main disadvantage of error-prone RCA is the low transformation efficiency of the DNA concatemer produced in the amplification reaction. We improved the method by introducing loxP recombination site of bacteriophage P1 Cre recombinase into the target plasmid and reducing the concatemer by Cre recombinase to plasmid-sized units, increasing the number of transformants 50-fold in non-error-prone and 13-fold in error-prone conditions. The efficiency improvement was verified by obtaining 115 ± 57 ceftazidime resistant colonies per recombined RCA reaction from randomly mutated TEM-1 β-lactamase gene library whereas only 9 ± 11 colonies were gained without recombination. Supplementation of the error-prone RCA with Cre/loxP recombination is a simple and useful tool to increase the transformable library size.
authors: Department of Biochemistry and Food Chemistry, University of Turku, Tykistökatu 6A, 6th floor, 20520 Turku, Finland. Tuomas.Huovinen@utu.fi
authors: Tuomas
authors: T
authors: Huovinen
authors: Marja
authors: M
authors: Julin
authors: Hanna
authors: H
authors: Sanmark
authors: Urpo
authors: U
authors: Lamminmäki
mesh_terms: D019943:Amino Acid Substitution; D002442:Ceftazidime / Q000378:metabolism; D003001:Cloning, Molecular; D004270:DNA, Circular / Q000235:genetics / Q000378:metabolism*; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D005822:Genetic Vectors; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D004270:DNA, Circular; D002442:Ceftazidime; C045073:Cre recombinase; D019426:Integrases; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1