Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes

BMC Molecular Biology

Table 2 Summary of the diverse conditions used and final protocol

Step^a	Condition	Obtention of recombinant colonies (CFU/ml)^b	Final established condition
Construction of the amplimer	Short homologous arms	No	Long primers
	Long homologous arms	Yes	Single PCR reaction for tet
	Single PCR reaction	21 for Tc	Three PCR reaction for cat
	Three PCR reactions	19 for Cm
Amount of amplified DNA to be transformed	0.1 μg	0	0.5 μg
	0.25 μg	0
	0.5 μg	21 Tc, 19 Cm
Culture volume used for preparation of electrocompetent cells	5 ml (2 × 10⁹ CFU/ml)	0	50 ml (5.10¹⁰ CFU/ml)
	10 ml (10¹⁰ CFU/ml)	0
	25 ml (2.5 × 10¹⁰ CFU/ml)	1 Tc, 0 Cm
	50 ml (5 × 10¹⁰ CFU/ml)	9 Tc, 8 Cm
Temperature after electroporation	30°C	2 Tc, 1 Cm	37°C
	37°C	15 Tc, 9 Cm
Arabinose concentration media	1 mM	18 Tc, 9 Cm	0.1 M
	10 mM	10 Tc, 8 Cm
	0.1 M	20 Tc, 11 Cm
Concentration of antibiotic in plating media	Tc 20 μg/ml, Cm 20 μg/ml	1 Tc, 0 Cm	Tc 5 μg/ml, Cm 5 μg/ml
	Tc 5 μg/ml, Cm 5 μg/ml	21 Tc, 19 Cm

^a Each set of experiments was performed independently of the other tests with one phage for each antibiotic.
^b Phage used for tet was ØVTB55; Phage used for cat was ØA9.

ISSN: 1471-2199