Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci

Table 1 Direct comparison of integration using linearised plasmid and pcr products

Locus	Strain	Template	Colonies	WT locus	Single X	Double X	Background
arsB	W	Plasmid	38	33	2	3	92%
	W	PCR	2	0	2	0	100%
	MG1655	Plasmid	38	4	15	19	50%
	MG1655	PCR	14	0	2	12	14%
lacZ	W	Plasmid	8	5	1	2	75%
	W	PCR	6	0	1	5	17%
	MG1655	Plasmid	32	25	6	1	97%
	MG1655	PCR	39	5	12	22	44%
rbsAR	W	Plasmid	22	0	16	6	73%
	W	PCR	7	0	5	2	71%
	MG1655	Plasmid	8	0	8	0	100%
	MG1655	PCR	15	0	14	1	93%

Templates for each transformation (linearised plasmid or PCR) are noted, as well as the number of colonies obtained from each transformation, the number of colonies having a wild type locus (no integration), the number of colonies having a single cross-over (Single X; integration of entire plasmid) and the number of colonies having a double cross-over (Double X; integration of only the region contained between the homologous arms). Background % is calculated based on the number of WT locus + Single X colonies.

ISSN: 1475-2859