Table I.

Strategies applied to inactivate selected ORFs in Synechocystis

The oligonucleotides were used to amplify the sequences encoding the candidate genes together with flanking regions. The insertion sites for the kanamycin (Km^R)-, chloramphenicol (Cm^R)-, or spectinomycin-resistance (Sp^R) cartridges are given. ORFs sll1349 and slr0458 were obtained as two PCR fragments. The resistance genes were inserted in between added restriction sites leading to deletions (D) of 267 bp in sll1349 and 59 bp in slr0458, respectively. ORF sll0171 was cut with MscI, leading to deletion of 1.0 kb. In the case of ORF slr2088, restriction with SnaBI led to a deletion of 0.24 kb. All other insertion sites were unique. Additionally introduced restriction sites are underlined in the oligonucleotide sequences.

ORF	Oligonucleotides	Insertion Site	Inserted Cartridge
sll1349	Fw1 5′-TTG GGC GGA ACG GGC CG-3′		Km^R
	Rev1 5′-GGG GGA TCC AGA ATG CGG TAA TCC CG-3′	BamHI
	Fw2 5′-TAA CTC TGC TCA ACC AA-3′	D
	Rev2 5′-GGG CTG CAG ACT AGT AAC CAG GGC ATG-3′	PstI
slr0458	Fw1 5′-TGA CCT CGA TGG AGT AT-3′		Cm^R
	Rev1 5′-GGG GGA TCC AGA ATG CGG TAA TCC CG-3′	BamHI
	Fw2 5′-GAG TGG GTT TAT GGT CG-3′	D
	Rev2 5′-GGG GCA TGC CTC CTC ACT AAA GCT CC-3′	SphI
sll0404	Fw 5′-CTC GAG ATG GCC ATT TTC TCC-3′	EcoRV	Km^R
	Rev 5′-GAA TTC TCA ATA AAT TTC CTC-3′
sll1559	Fw 5′-CTC GAG ATG GAT AAT AAG CAA-3′	StuI	Sp^R
	Rev 5′-GAA TTC TTA ACC TTT AGC CAA-3′
sll0171	Fw 5′-AGA CCT GAA GGA AGC TGT AG-3′	MscI, D	Sp^R
	Rev 5′-GAG GAA GTG GTG CAC AGG TT-3′
sll1931	Fw 5′-GCT ATT ACG GCG GCT GTG AA-3′	SmaI	Km^R
	Rev 5′-CCA TGA CGG CCA CAA CTG AA-3′
sll1981	Fw 5′-CCG GAT TCG TTA GGC TAG-3′	AfeI	Km^R
	Rev 5′-AGT TAG CGT CGA TTT GGT-3′
slr2088	Fw 5′-CGA TTG AGT TAA AAT TAG-3′	SnaBI, D	Km^R
	Rev 5′-GGT CAA GAA AAA CCG AGG-3′
slr0229	Fw 5′-ATA AGT CAG AGA AGT GAA-3′	HpaI	Km^R
	Rev 5′-CCA TGT TTA CTC CAG TAA-3′