Journal: mBio

Article Title: Investigation of the Cyanothece nitrogenase cluster in Synechocystis : a blueprint for engineering nitrogen-fixing photoautotrophs

doi: 10.1128/mbio.04052-24

Figure Lengend Snippet: Expression levels of nitrogenase structural genes affect nitrogen fixation activities in engineered Synechocystis 6803. ( A ) Schematic map of changes on the 5′-UTR sequence before the nifH gene. Three promoters were tested, P ssl0452 , P sll1626 , and P trc1O . ( B ) The strengths of the tested promoters were compared to the native promoter P nifH . The strengths were tested from the expression levels of the reporter protein EYFP. ( C ) Nitrogenase activity under different strength promoters preceding the nifH gene. Samples were collected from cultures under 12 h light/12 h dark conditions in BG11 0 medium. Nitrogen fixation activity was assayed by acetylene reduction, and error bars represent the standard deviations observed from at least three independent experiments.

Article Snippet: In a first-of-its kind effort in this direction, the unicellular non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) was engineered to fix nitrogen by introduction of the nitrogenase gene cluster from Cyanothece sp. ATCC 51142 ( ).

Techniques: Expressing, Sequencing, Activity Assay

Journal: mBio

Article Title: Investigation of the Cyanothece nitrogenase cluster in Synechocystis : a blueprint for engineering nitrogen-fixing photoautotrophs

doi: 10.1128/mbio.04052-24

Figure Lengend Snippet: Rebuilding the functional nitrogenase enzyme in Synechocystis 6803. ( A ) A schematic map of reconstruction of nif genes using a bottom–up strategy. Five operons were added sequentially into the plasmid pCB-SC101, which were transferred into Synechocystis 6803 as the CK strain. For each operon, promoters, RBS, and terminators were organized with ORFs to form an independent expression cassette. Shown are promoters (red), transcription terminators (black), and the genes for the three structural proteins, nifHDK (green), necessary cofactors (blue), accessory proteins (orange). ( B ) Nitrogenase activities of engineered strains containing sets of reconstructed nif operons. Samples were collected from cultures under 12 h light/12 h dark conditions in BG11 0 medium. Nitrogen fixation activity was assayed by acetylene reduction, and error bars represent the standard deviations observed from at least three independent experiments.

Article Snippet: In a first-of-its kind effort in this direction, the unicellular non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) was engineered to fix nitrogen by introduction of the nitrogenase gene cluster from Cyanothece sp. ATCC 51142 ( ).

Techniques: Functional Assay, Plasmid Preparation, Expressing, Activity Assay

Journal: mBio

Article Title: Investigation of the Cyanothece nitrogenase cluster in Synechocystis : a blueprint for engineering nitrogen-fixing photoautotrophs

doi: 10.1128/mbio.04052-24

Figure Lengend Snippet: Ferredoxins affect nitrogenase activity in engineered Synechocystis 6803. ( A ) Scheme showing the combination of promoters and ferredoxin genes. The expression cassettes were integrated into the plasmid pRSF1010. ( B ) Nitrogenase activities are affected by type and expression level of ferredoxins in engineered Synechocystis 6803. Samples were collected from cultures under 12 h light/12 h dark conditions in BG11 0 medium. Nitrogen fixation activity was assayed by acetylene reduction, and error bars represent the standard deviations observed from at least three independent experiments.

Article Snippet: In a first-of-its kind effort in this direction, the unicellular non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) was engineered to fix nitrogen by introduction of the nitrogenase gene cluster from Cyanothece sp. ATCC 51142 ( ).

Techniques: Activity Assay, Expressing, Plasmid Preparation

Journal: mBio

Article Title: Investigation of the Cyanothece nitrogenase cluster in Synechocystis : a blueprint for engineering nitrogen-fixing photoautotrophs

doi: 10.1128/mbio.04052-24

Figure Lengend Snippet: Deletion analysis of the genes shown in the re-constructed nif operons. ( A ) Scheme showing the deletion of specific genes in the operons using the CRISPR/cpf1 system. The hollow rectangles represent the deleted genes, and the colored rectangles represent the remaining genes. ( B ) Nitrogenase activity of the deletion strains in engineered Synechocystis 6803. Samples were collected from cultures under 12 h light/12 h dark conditions in a BG11 0 medium. Nitrogen fixation activity was assayed by acetylene reduction, and error bars represent the standard deviations observed from at least three independent experiments.

Article Snippet: In a first-of-its kind effort in this direction, the unicellular non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) was engineered to fix nitrogen by introduction of the nitrogenase gene cluster from Cyanothece sp. ATCC 51142 ( ).

Techniques: Construct, CRISPR, Activity Assay