Fig. 1
From: 6S RNA plays a role in recovery from nitrogen depletion in Synechocystis sp. PCC 6803
Strategy and confirmation of the Synechocystis 6803 ΔssaA mutant. a Strategy for the construction of the ΔssaA mutant. The ssaA gene was replaced by a kanamycin resistance cassette (KmR) and its promoter region (grey box) at the indicated genome position (ssaA locus: 1,886,879…1,887,066). The construction was realized by overlap extension PCR of three fragments using primer pairs P3 and P4 (red), P5 and 6 (green, P5: 15 bp 5′-end overlap with P4, 17 bp 3′-end overlap with KmR; P6: 14 bp 5’end overlap with P7, 24 bp 3’end overlap with KmR) and P7 and P8 (blue). The construct was further used to replace ssaA in the genome of Synechocystis 6803 by homologous recombination. P1 and P2 represent the location of primers used to verify complete segregation of the mutant strain. The location of genes slr1287, slr1288 and sll1166 are depicted upstream and downstream of the ssaA gene. b Complete segregation of the ΔssaA mutant gene copies and replacement with a kanamycin resistance cassette (KmR) and its flanking regions (998 nt) was tested by PCR analysis, using primers P1 and P2. c Deletion of 6S RNA in ΔssaA mutant was confirmed by Northern blot analysis using a radioactive [32P]-labelled oligonucleotide for 6S RNA. 5S rRNA was hybridized as a loading control