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 organismSynechocystissp. 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 PJ23100, PrhaBAD, and PpetEover 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 CO2supply. Despite the use of a regulatory system orthogonal toSynechocystissp. PCC 6803, PrhaBAD, 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.