Interlaboratory reproducibility in growth and reporter expression in the cyanobacterium Synechocystis sp. PCC 6803

Maurice Mager, Hugo Pineda Hernandez, Fabian Brandenburg, Luis López-Maury, Alistair J McCormick, Dennis J Nürnberg, Tim Orthwein, David A Russo, Angelo Joshua Victoria, Xiaoran Wang, Julie A Z Zedler, Filipe Branco dos Santos, Nicolas M. Schmelling

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

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 J23100 , 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.

Original languageEnglish
Pages (from-to)1823-1835
Number of pages13
JournalACS Synthetic Biology
Volume12
Issue number6
Early online date29 May 2023
DOIs
Publication statusPublished - 16 Jun 2023

Keywords / Materials (for Non-textual outputs)

  • promoter
  • cyanobacteria
  • reproducibility
  • interlab

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