Interlaboratory Reproducibility in Growth and Reporter Expression in the Cyanobacterium Synechocystis sp. PCC 6803
Year: 2023
Type: article
Abstract: High Resolution Image Download MS PowerPoint Slide In recent years, a plethora of new synthetic biology tools for use in cyanobacteria have been published; however, their reported characterizations often cannot be reproduced, greatly limiting the comparability of results and hindering their applicability. In this interlaboratory study, the reproducibility of a standard microbiological experiment for the cyanobacterial model organism Synechocystis sp. PCC 6803 was assessed. Participants from eight different laboratories quantified the fluorescence intensity of mVENUS as a proxy for the transcription activity of the three promoters P J 23100, P rhaBAD, and P petE over time. In addition, growth rates were measured to compare growth conditions between laboratories. By establishing strict and standardized laboratory protocols, reflecting frequently reported methods, we aimed to identify issues with state-of-the-art procedures and assess their effect on reproducibility. Significant differences in spectrophotometer measurements across laboratories from identical samples were found, suggesting that commonly used reporting practices of optical density values need to be supplemented by cell count or biomass measurements. Further, despite standardized light intensity in the incubators, significantly different growth rates between incubators used in this study were observed, highlighting the need for additional reporting requirements of growth conditions for phototrophic organisms beyond the light intensity and CO 2 supply. Despite the use of a regulatory system orthogonal to Synechocystis sp. PCC 6803, P rhaBAD, and a high level of protocol standardization, ∼32% variation in promoter activity under induced conditions was found across laboratories, suggesting that the reproducibility of other data in the field of cyanobacteria might be affected similarly.
Source: ACS Synthetic Biology
Institutions: Heinrich Heine University Düsseldorf, University of Amsterdam, Helmholtz Centre for Environmental Research, Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla, University of Edinburgh, Freie Universität Berlin, University of Tübingen, Friedrich Schiller University Jena
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $5,000
Awards: G3143732916, G3606927638, EXC 2051: Balance of the Microverse, SFB 1127: Chemische Mediatoren in komplexen Biosystemen (ChemBioSys), CyanoSource: A foundry generated barcoded mutant library resource for the model cyanobacterium Synechocystis sp. PCC 6803, Engineered microbial factories for CO2 exploitation in an integrated waste treatment platform
OpenAlex ID: W4378639830