Interlaboratory Reproducibility in Growth and Reporter Expression in the Cyanobacterium Synechocystis sp. PCC 6803

Abstract

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 CO2 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.

Cyanoremediation of heavy metals (As(v), Cd(ii), Cr(vi), Pb(ii)) by live cyanobacteria (Anabaena variabilis, and Synechocystis sp.): an eco-sustainable technology

“…Cyanobacterial growth was determined by measuring the optical density at 730 nm (ref. 25 ) (hereafter OD 730 ) and dry biomass (mg L −1 ) with and without HM contamination. The initial biomasses of both cyanobacteria based on OD 730 , dry biomass, chlorophyll a, and carotenoids were the same (OD 730 > 1.5) to ensure synchronized cultivation.…”