Fig. 2

Inactivation and overexpression of ntcA in S. PCC 6803 WT and ethylene-producing recombinant. a Schematic representation of the deletion of ntcA. The open reading frame (ORF) of ntcA (sll1423) was replaced by a kanamycin resistance (Km^r) cassette through homologous recombination with plasmid pHM003. DNA fragments were amplified by PCR and were analyzed by agarose gel electrophoresis, showing the partial segregation of ntcA in the mutant strains. PCR products from S. PCC 6803 were generated using the indicated primer pairs. Primer sequences are listed in Table 1. Lanes were loaded with PCR products that were generated with genomic DNA from the indicated strain as a template. The sizes of the PCR products are indicated on the right. b Schematic representation of the overexpression of ntcA. The Gm^r-P _cpcB -ntcA expression cassette was inserted into the phaAB loci through homologous recombination with plasmid pHM006. DNA fragments were amplified by PCR and were analyzed by agarose gel electrophoresis, which showed the complete replacement of phaAB by the Gm^r-P _cpcB -ntcA expression cassette. PCR products from S. PCC 6803 were generated using the indicated primer pairs. Primer sequences are listed in Table 1. Lanes were loaded with the PCR products that were generated with genomic DNA from the indicated strains as templates. The sizes of the PCR products are indicated on the right. c Quantitative PCR results of ntcA deletion and overexpression mutants. The copy numbers of ntcA were measured through qPCR. The reference genes for S. PCC 6803 were rnpB and the 16S rRNA gene. The relative ratios of gene copy numbers of ntcA were quantified in the WT strain, ethylene producer XX76, and ntcA mutant strains (MH015, MH021, MH017, and MH023). Data represent the means ± standard deviations from three independent experiments