Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells

BMC Molecular Biology

Table 1 Evaluation of host context and dependence on phage protein expression in ssOR.

Strain + plasmid	Genotype + expressed proteins	Normalised ssOR
MM294	wild-type	0
+ pBADET	+ RecE, RecT	50
+ pBADETγ	+ RecE, RecT, Redγ	46
+ pBADαβ	+ Redα, Redβ	55
+ pBADαβγ	+ Redα, Redβ, Redγ	52
JC8679	SbcA, recBC	15
JC5519	RecBC	0
+ pBADrecET	+RecE, RecT	49
+ pBADredαβ	+ Redα, Redβ	52
+ pBADerf	+ Erf	35
JC9366	recA	0
+ pBADrecET	+RecE, RecT	45
+ pBADrecETγ	+RecE, RecT, Redγ	50
+ pBADredαβ	+ Redα, Redβ	54
+ pBADredαβγ	+ Redα, Redβ, Redγ	51
+pBADerf	+ Erf	32
JM103	sbcBC	0.1
JC9387	sbcBC, rac, recBC	0.3
JC15329	sbcBC, recA, recAB, rac	0
JC8111	sbcBC, rac, recBC, recF	0.1

Various E. coli hosts were transformed with pBAD24 expression plasmids or not as indicated before co-electroporation with a 48 mer oligonucleotide and pGKneoΔ to test for ssOR. The strains used were MM294, JC8679 [44], JC5519 [45], JC9366 [45], JM103 [46], JC 9387 [45], JC15329 [47] and JC8111 [48]. Relevant aspects of genotype and expressed proteins, after arabinose induction, are listed in the central column. Numbers refer to the ratio of kanamycin to ampicillin resistant colonies and were generated from the same master mixture of oligonucleotide and pGKneoΔ.

ISSN: 1471-2199