Interlaboratory Reproducibility in Growth and Reporter Expression in the Cyanobacterium Synechocystis sp. PCC 6803
Open accessAbstract
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.
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Various inducible promoter systems have been adapted for cyanobacteria, including metal-responsive promoters such as PpetE, which reacts to copper levels, and sugar-responsive systems like PrhaBAD, activated by rhamnose presence.
“Various inducible promoter systems have been adapted for cyanobacteria, including metal-responsive promoters such as PpetE, which reacts to copper levels, and sugar-responsive systems like PrhaBAD, activated by rhamnose presence [104,105].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [105]
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Cites
Wickham, H., Averick, M., Bryan, J., Chang, W., McGowan, L., François, R., Grolemund, G., Hayes, A., Henry, L., Hester, J., et al. (2019) · Journal of Open Source Software · doi:10.21105/joss.01686 · Cited by 22,138 · see citing works
1,500 scientists lift the lid on reproducibility
Baker, M. (2016) · Nature · doi:10.1038/533452a · Cited by 4,209 · see citing works
A Modular Cloning System for Standardized Assembly of Multigene Constructs
Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011) · PLoS ONE · doi:10.1371/journal.pone.0016765 · Cited by 1,385 · see citing works
Precise and reliable gene expression via standard transcription and translation initiation elements
Mutalik, V. K., Guimaraes, J. C., Cambray, G., Lam, C. S. C., Christoffersen, M. J., Mai, Q.-A., Tran, A. B., Paull, M. L., Keasling, J. D., Arkin, A. P., et al. (2013) · Nature Methods · doi:10.1038/nmeth.2404 · Cited by 748 · see citing works
Ritchie, R. J. (2008) · Photosynthetica · doi:10.1007/s11099-008-0019-7 · Cited by 684 · see citing works
General calibration of microbial growth in microplate readers
Stevenson, K., McVey, A. F., Clark, I. B. N., Swain, P. S., & Pilizota, T. (2016) · Scientific Reports · doi:10.1038/srep38828 · Cited by 407 · see citing works
Improving accuracy of cell and chromophore concentration measurements using optical density
Myers, J. A., Curtis, B. S., & Curtis, W. R. (2013) · BMC Biophysics · doi:10.1186/2046-1682-6-4 · Cited by 391 · see citing works
Cpf1 Is a Versatile Tool for CRISPR Genome Editing Across Diverse Species of Cyanobacteria
Ungerer, J., & Pakrasi, H. B. (2016) · Scientific Reports · doi:10.1038/srep39681 · Cited by 308 · see citing works
Robust estimation of bacterial cell count from optical density
Beal, J., Farny, N. G., Haddock-Angelli, T., Selvarajah, V., Baldwin, G. S., Buckley-Taylor, R., Gershater, M., Kiga, D., Marken, J., Sanchania, V., et al. (2020) · Communications Biology · doi:10.1038/s42003-020-01127-5 · Cited by 290 · see citing works
Self-citationCyanogate: a modular cloning suite for engineering cyanobacteria based on the plant moclo syntax
Vasudevan, R., Gale, G. A. R., Schiavon, A. A., Puzorjov, A., Malin, J., Gillespie, M. D., Vavitsas, K., Zulkower, V., Wang, B., Howe, C. J., et al. (2019) · Plant Physiology · doi:10.1104/pp.18.01401 · Cited by 189 · see citing works
Self-citationLuimstra, V. M., Schuurmans, J. M., Verschoor, A. M., Hellingwerf, K. J., Huisman, J., & Matthijs, H. C. P. (2018) · Photosynthesis Research · doi:10.1007/s11120-018-0561-5 · Cited by 173 · see citing works
Englund, E., Liang, F., & Lindberg, P. (2016) · Scientific Reports · doi:10.1038/srep36640 · Cited by 166 · see citing works
Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction
Hays, S. G., Yan, L. L. W., Silver, P. A., & Ducat, D. C. (2017) · Journal of Biological Engineering · doi:10.1186/s13036-017-0048-5 · Cited by 147 · see citing works
Microevolution in Cyanobacteria: Re-sequencing a Motile Substrain of Synechocystis sp. PCC 6803
Trautmann, D., Voß, B., Wilde, A., Al‐Babili, S., & Hess, W. R. (2012) · DNA Research · doi:10.1093/dnares/dss024 · Cited by 144 · see citing works
A Regulatory Cascade in the Induction of rhaBAD
Egan, S. M., & Schleif, R. F. (1993) · Journal of Molecular Biology · doi:10.1006/jmbi.1993.1565 · Cited by 126 · see citing works
Production of Squalene in Synechocystis sp. PCC 6803
Englund, E., Pattanaik, B., Ubhayasekera, S. J. K. A., Stensjö, K., Bergquist, J., & Lindberg, P. (2014) · PLoS ONE · doi:10.1371/journal.pone.0090270 · Cited by 103 · see citing works
A Rhamnose-Inducible System for Precise and Temporal Control of Gene Expression in Cyanobacteria
Kelly, C. L., Taylor, G. M., Hitchcock, A., Torres-Méndez, A., & Heap, J. T. (2018) · ACS Synthetic Biology · doi:10.1021/acssynbio.7b00435 · Cited by 80 · see citing works
Zavřel, T., Očenášová, P., & Červený, J. (2017) · PLoS ONE · doi:10.1371/journal.pone.0189130 · Cited by 76 · see citing works
Quantification of bacterial fluorescence using independent calibrants
Beal, J., Haddock, T. L., Baldwin, G. S., Gershater, M. C., Dwijayanti, A., Storch, M., de Mora, K., Lizarazo, M., Rettberg, R., & Contributors, W. T. I. I. S. (2018) · PLoS ONE · doi:10.1371/journal.pone.0199432 · Cited by 76 · see citing works
Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli
Beal, J., Haddock, T. L., Gershater, M. C., de Mora, K., Lizarazo, M., Hollenhorst, J., Rettberg, R., & Contributors, I. I. S. (2016) · PLoS ONE · doi:10.1371/journal.pone.0150182 · Cited by 68 · see citing works
Comparative Dose–Response Analysis of Inducible Promoters in Cyanobacteria
Behle, A., Saake, P., Germann, A. T., Dienst, D., & Axmann, I. M. (2020) · ACS Synthetic Biology · doi:10.1021/acssynbio.9b00505 · Cited by 67 · see citing works
Sode, K., Tatara, M., Takeyama, H., Burgess, J. G., & Matsunaga, T. (1992) · Applied Microbiology and Biotechnology · doi:10.1007/bf00210994 · Cited by 65 · see citing works
Giner‐Lamia, J., López‐Maury, L., & Florencio, F. J. (2014) · PLoS ONE · doi:10.1371/journal.pone.0108912 · Cited by 63 · see citing works
Self-citationGeerts, D., Bovy, A. G., de Vrieze, G., Borrias, M., & Weisbeek, P. J. (1995) · Microbiology · doi:10.1099/13500872-141-4-831 · Cited by 62 · see citing works
Ferreira, E. A., Pacheco, C. C., Pinto, F., Pereira, J. A., Lamosa, P., Oliveira, P., Kirov, B., Jaramillo, A., & Tamagnini, P. (2018) · Synthetic Biology · doi:10.1093/synbio/ysy014 · Cited by 59 · see citing works
Live-cell imaging of cyanobacteria
Yokoo, R., Hood, R. D., & Savage, D. F. (2014) · Photosynthesis Research · doi:10.1007/s11120-014-0049-x · Cited by 58 · see citing works
van Alphen, P., Najafabadi, H. A., dos Santos, F. B., & Hellingwerf, K. J. (2018) · Biotechnology Journal · doi:10.1002/biot.201700764 · Cited by 56 · see citing works
Bland, E., & Angenent, L. T. (2016) · Bioresource Technology · doi:10.1016/j.biortech.2016.05.116 · Cited by 46 · see citing works
Liu, D., Johnson, V. M., & Pakrasi, H. B. (2020) · ACS Synthetic Biology · doi:10.1021/acssynbio.0c00106 · Cited by 37 · see citing works
García-Cañas, R., Giner‐Lamia, J., Florencio, F. J., & López‐Maury, L. (2021) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2017898118 · Cited by 37 · see citing works
Self-citationElomaa, H., Seisko, S., Junnila, T., et al. (2017) · Recycling · doi:10.3390/recycling2030014 · Cited by 29 · see citing works
Price, C. E., dos Santos, F. B., Hesseling, A., Uusitalo, J. J., Bachmann, H., Benavente, V., Goel, A., Berkhout, J., Bruggeman, F. J., Marrink, . J., et al. (2019) · BMC Evolutionary Biology · doi:10.1186/s12862-018-1331-x · Cited by 29 · see citing works
Gale, G. A. R., Wang, B., & McCormick, A. J. (2021) · Frontiers in Microbiology · doi:10.3389/fmicb.2020.624011 · Cited by 20 · see citing works
Self-citationTietze, L., Mangold, A., Hoff, M. W., et al. (2022) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2022.826142 · Cited by 17 · see citing works
Beal, J., Baldwin, G. S., Farny, N. G., Gershater, M. C., Haddock, T. L., Buckley-Taylor, R., Dwijayanti, A., Kiga, D., Lizarazo, M., Marken, J. P., et al. (2021) · PLoS ONE · doi:10.1371/journal.pone.0252263 · Cited by 14 · see citing works
Author Correction: Robust estimation of bacterial cell count from optical density
Beal, J., Farny, N. G., Haddock-Angelli, T., Selvarajah, V., Baldwin, G. S., Buckley-Taylor, R., Gershater, M., Kiga, D., Marken, J., Sanchania, V., et al. (2020) · Communications Biology · doi:10.1038/s42003-020-01371-9 · Cited by 12 · see citing works
Self-citationMoCloFlex: A Modular Yet Flexible Cloning System
Klein, C., Emde, L., Kuijpers, A., & Sobetzko, P. (2019) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2019.00271 · Cited by 9 · see citing works
El‐Shehawy, R. M., & Kleiner, D. (2003) · Physiologia Plantarum · doi:10.1034/j.1399-3054.2003.00108.x · Cited by 3 · see citing works
Triparental mating of Synechocystis v1Preprint
Behle, A. (2016) · doi:10.17504/protocols.io.ftpbnmn · Cited by 1 · see citing works
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Walshe, J., & Saha, S. K. (2026) · Current Issues in Molecular Biology · doi:10.3390/cimb48060638 · Cited by 1 · see citing works
“Various inducible promoter systems have been adapted for cyanobacteria, including metal-responsive promoters such as P petE , which reacts to copper levels, and sugar-responsive systems like P rhaBAD , activated by rhamnose presence [104,105].”
- Authors
- Mager, M., Pineda Hernandez, H., Brandenburg, F., López-Maury, L., McCormick, A. J., Nürnberg, D. J., Orthwein, T., Russo, D. A., Victoria, A. J., Wang, X., Zedler, J. A. Z., Branco dos Santos, F., & Schmelling, N. M.
- Affiliations
- Heinrich Heine University Düsseldorf (DE)
- Molecular Microbial Physiology Group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Science Park 904, Amsterdam 1098 XH, The Netherlands
- Helmholtz Centre for Environmental Research (DE)
- Instituto de Bioquímica Vegetal y Fotosíntesis, University of Seville − CSIC, Américo Vespucio 49, 41092 Sevilla, Spain
- University of Edinburgh (GB)
- Freie Universität Berlin (DE)
- University of Tübingen (DE)
- Friedrich Schiller University Jena (DE)
- Corresponding author
- Schmelling, Nicolas M.
- ORCID
- Venue
- ACS Synthetic Biology
- Publisher
- American Chemical Society (ACS)
- Published
- 2023-05-29
- Volume
- 12
- Issue
- 6
- Pages
- 1823-1835
- ISSN
- 2161-5063 (ISSN-L)
- PMC
- PMC10278186
- OpenAlex
- W4378639830
- Cited by
- 11
- References
- 39
- Citations by year
- 2026: 4 · 2025: 4 · 2024: 3
- FWCI
- 0.98
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- ACS Synthetic Biology (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- UvA-DARE (University of Amsterdam) (submittedVersion, cc-by)
- DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion, cc-by)
- Deposito de Investigacion Universidad de Sevilla (University of Seville) (submittedVersion, cc-by)
- Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, cc-by)
- Refubium (Universitätsbibliothek der Freien Universität Berlin) (submittedVersion, cc-by)
- Universitätsbibliothek der FU Berlin Hochschulschriftenstelle u. Dokumentenserver (cc-by)
- Funders
- Darwin Trust of Edinburgh
- European Regional Development Fund (100361842)
- Deutsche Forschungsgemeinschaft (CRC 1127/2 239748522, ID 390713860, NU 421/1)
- H2020 LEIT Biotechnology (760994)
- Alexander von Humboldt-Stiftung
- Biotechnology and Biological Sciences Research Council (BB/S020128/1)
- Agencia Estatal de Investigación,Ministerio de Ciencia, Innovación y Universidades (PID2020-112645GB-I00)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Keywords
- interlaboratory reproducibility, Synechocystis sp. PCC 6803, cyanobacteria, promoter activity, growth rate variability, optical density measurement, cell counting, biomass measurement, synthetic biology tools
- MeSH terms
- Promoter Regions, Genetic, Reproducibility of Results, Genes, Reporter, Biomass, Synechocystis
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