Rychli, K,Guinane, CM,Daly, K,Hill, C,Cotter, PD,Jordan, K,Fox, EM,Wagner, M

2014

November

Methods in molecular biology (Clifton, N.J.)

Generation of Nonpolar Deletion Mutants in Listeria monocytogenes Using the "SOEing" Method

Validated

()

Nonpolar deletion mutants Listeria monocytogenes SOEing method pKSV7 vector pORI280/pVE6007 system GRAM-POSITIVE BACTERIA VECTOR GENE

1157

187

200

The ability to manipulate chromosomally encoded genes is a fundamental biological tool for the analysis of gene function. Here, we provide in greater depth protocols for the creation of nonpolar unlabeled gene deletions in Listeria (L.) monocytogenes that are facilitated by the splicing overlap extension PCR technique. For mutagenesis in L. monocytogenes, we describe two different plasmid-based approaches, which facilitate the introduction of this spliced amplicon in place of the corresponding segment of chromosomal DNA: the pKSV7 system and the pORI280/pVE6007 system.

10.1007/978-1-4939-0703-8_16