Project description:Cyanobacteria are valuable organisms for studying the physiology of photosynthesis and carbon fixation as well as metabolic engineering for the production of fuels and chemicals. This work describes a novel counter selection method for the cyanobacterium Synechococcus sp. PCC 7002 based on organic acid toxicity. The organic acids acrylate, 3-hydroxypropionate, and propionate were shown to be inhibitory towards PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA. Loss of AcsA function was used as a basis for an acrylate counter selection method. DNA fragments of interest were inserted into the acsA locus and strains harboring the insertion were isolated on selective medium containing acrylate. This methodology was also used to introduce DNA fragments into a pseudogene, glpK. Application of this method will allow for more advanced genetics and engineering studies in PCC 7002 including the construction of markerless gene deletions and insertions. The acrylate counter-selection could be applied to other cyanobacterial species where AcsA activity confers acrylate sensitivity (e.g. Synechocystis sp. PCC 6803).
Project description:Application of genome-scale 'omics approaches to dissect subcellular pathways and regulatory networks governing the fast-growing response of Synechococcus sp. PCC 7002 response to variable irradience levels. We employed controlled cultivation and next-generation sequencing technology to identify transcriptional responses of euryhaline unicellular cyanobacterium Synechococcus sp. PCC 7002 grown under steady state conditions at six irradiance levels ranging from 33 to 760 µmol photons m-2 sec-1.
Project description:Cyanobacteria are valuable organisms for studying the physiology of photosynthesis and carbon fixation as well as metabolic engineering for the production of fuels and chemicals. This work describes a novel counter selection method for the cyanobacterium Synechococcus sp. PCC 7002 based on organic acid toxicity. The organic acids acrylate, 3-hydroxypropionate, and propionate were shown to be inhibitory towards PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA. Loss of AcsA function was used as a basis for an acrylate counter selection method. DNA fragments of interest were inserted into the acsA locus and strains harboring the insertion were isolated on selective medium containing acrylate. This methodology was also used to introduce DNA fragments into a pseudogene, glpK. Application of this method will allow for more advanced genetics and engineering studies in PCC 7002 including the construction of markerless gene deletions and insertions. The acrylate counter-selection could be applied to other cyanobacterial species where AcsA activity confers acrylate sensitivity (e.g. Synechocystis sp. PCC 6803). Cultures were grown in medium modified with 5mM acrylic acid at pH 8 and compared to cultures grown in unmodified medium. Samples were processed in duplicate.
Project description:In this study, we characterized the homeostasis of the marine cyanobacteria Synechococcus sp. PCC7002 (BMB04) growing in chemically characterized synthetic seawater with three different levels of iron limitation representative of the modern ocean. Using transcriptomic approach, we identified the sequence of physiological responses to increasing Fe limitation. Our results showed an increase in the number of dysregulated genes and in the complexity of the response to increasing Fe limitation. Genes involved in photosynthesis were strongly down-regulated under MiFeL, while membrane transporters were up-regulated. Genes involved in regulation of energy metabolism responded under strong Fe limitation, while fine metabolic regulation of co-factors expression and activation of specific cellular mechanisms to minimize oxidative stress were only observed under severe Fe limitation. Additionally, our results demonstrate the limitations in the construct of the bioreporter BMB04 that hamper its application in areas of the ocean strongly Fe limited.