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 (KmR)-, chloramphenicol (CmR)-, or spectinomycin-resistance (SpR) 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.

ORFOligonucleotidesInsertion SiteInserted Cartridge
sll1349Fw1 5′-TTG GGC GGA ACG GGC CG-3′KmR
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
slr0458Fw1 5′-TGA CCT CGA TGG AGT AT-3′CmR
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
sll0404Fw 5′-CTC GAG ATG GCC ATT TTC TCC-3′EcoRVKmR
Rev 5′-GAA TTC TCA ATA AAT TTC CTC-3′
sll1559Fw 5′-CTC GAG ATG GAT AAT AAG CAA-3′StuISpR
Rev 5′-GAA TTC TTA ACC TTT AGC CAA-3′
sll0171Fw 5′-AGA CCT GAA GGA AGC TGT AG-3′MscI, DSpR
Rev 5′-GAG GAA GTG GTG CAC AGG TT-3′
sll1931Fw 5′-GCT ATT ACG GCG GCT GTG AA-3′SmaIKmR
Rev 5′-CCA TGA CGG CCA CAA CTG AA-3′
sll1981Fw 5′-CCG GAT TCG TTA GGC TAG-3′AfeIKmR
Rev 5′-AGT TAG CGT CGA TTT GGT-3′
slr2088Fw 5′-CGA TTG AGT TAA AAT TAG-3′SnaBI, DKmR
Rev 5′-GGT CAA GAA AAA CCG AGG-3′
slr0229Fw 5′-ATA AGT CAG AGA AGT GAA-3′HpaIKmR
Rev 5′-CCA TGT TTA CTC CAG TAA-3′