A novel biosensor for ferrous iron developed via CoBiSe: A computational method for rapid biosensor design
Open accessPreprint — published as 10.1021/acssensors.5c02481 · Paper page
Abstract
Genetically encoded biosensors enable monitoring of metabolite dynamics in living organisms. We present CoBiSe, a computational approach using Constraint Network Analysis to identify optimal insertion sites for reporter modules in molecular recognition elements (MREs). Applied to the iron-binding protein DtxR from Corynebacterium glutamicum , CoBiSe identified a flexible connective loop (residues 138-150) for inserting the reporter module, resulting in IronSenseR, a novel ratiometric biosensor for ferrous iron (Fe²⁺). IronSenseR demonstrates high specificity for Fe²⁺ with dissociation constants of 1.55 ± 0.08 µM (FeSO 4 ) and 2.44 ± 0.28 µM (FeCl 2 ), while showing no binding to Fe³⁺ and other divalent cations. In vivo assessment in Escherichia coli , Pseudomonas putida and Corynebacterium glutamicum confirmed IronSenseR’s capability to detect changes in the intracellular iron pool. The creation of IronSenseR underlines that, by reducing search space and eliminating labor-intensive screening, CoBiSe streamlines biosensor development and enables precise creation of next-generation biosensors for diverse metabolites.
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Fiji: an open-source platform for biological-image analysis
Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., et al. (2012) · Nature Methods · doi:10.1038/nmeth.2019 · Cited by 66,890 · see citing works
Highly accurate protein structure prediction with AlphaFold
Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., et al. (2021) · Nature · doi:10.1038/s41586-021-03819-2 · Cited by 43,764 · see citing works
Accurate structure prediction of biomolecular interactions with AlphaFold 3
Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., Ronneberger, O., Willmore, L., Ballard, A. J., Bambrick, J., et al. (2024) · Nature · doi:10.1038/s41586-024-07487-w · Cited by 15,861 · see citing works
Enzymatic assembly of DNA molecules up to several hundred kilobases
Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009) · Nature Methods · doi:10.1038/nmeth.1318 · Cited by 9,742 · see citing works
UniProt: a worldwide hub of protein knowledge
The UniProt Consortium (2019) · Nucleic Acids Research · doi:10.1093/nar/gky1049 · Cited by 7,698 · see citing works
Svergun, D. I. (1992) · Journal of Applied Crystallography · doi:10.1107/s0021889892001663 · Cited by 3,709 · see citing works
Svergun, D. I., Barberato, C., & Koch, M. (1995) · Journal of Applied Crystallography · doi:10.1107/s0021889895007047 · Cited by 3,388 · see citing works
PRIMUS: a Windows PC-based system for small-angle scattering data analysis
Konarev, P. V., Волков, В. В., Sokolova, A. V., Koch, M. H. J., & Svergun, D. I. (2003) · Journal of Applied Crystallography · doi:10.1107/s0021889803012779 · Cited by 2,850 · see citing works
Andrews, S. C., Robinson, A. K., & Rodríguez-Quiñones, F. (2003) · FEMS Microbiology Reviews · doi:10.1016/s0168-6445(03)00055-x · Cited by 2,702 · see citing works
Engineering and characterization of a superfolder green fluorescent protein
Pédelacq, J. D., Cabantous, S., Tran, T. H., Terwilliger, T. C., & Waldo, G. S. (2005) · Nature Biotechnology · doi:10.1038/nbt1172 · Cited by 2,295 · see citing works
Iron Metabolism in Pathogenic Bacteria
Ratledge, C., & Dover, L. G. (2000) · Annual Review of Microbiology · doi:10.1146/annurev.micro.54.1.881 · Cited by 1,539 · see citing works
Cellpose 2.0: how to train your own model
Pachitariu, M., & Stringer, C. (2022) · Nature Methods · doi:10.1038/s41592-022-01663-4 · Cited by 1,381 · see citing works
Guinier, A. (1939) · Annales de Physique · doi:10.1051/anphys/193911120161 · Cited by 1,213 · see citing works
ATSAS 3.0 : expanded functionality and new tools for small-angle scattering data analysis
Manalastas-Cantos, K., Konarev, P. V., Hajizadeh, N. R., Kikhney, A., Petoukhov, M. V., Molodenskiy, D. S., Panjkovich, A., Mertens, H. D. T., Gruzinov, A. Y., Borges, C., et al. (2020) · Journal of Applied Crystallography · doi:10.1107/s1600576720013412 · Cited by 942 · see citing works
mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa
Choi, K., & Schweizer, H. P. (2006) · Nature Protocols · doi:10.1038/nprot.2006.24 · Cited by 882 · see citing works
Protein flexibility predictions using graph theory
Jacobs, D. J., Rader, A. J., Kuhn, L. A., & Thorpe, M. F. (2001) · Proteins Structure Function and Bioinformatics · doi:10.1002/prot.1081 · Cited by 729 · see citing works
Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon
Keilhauer, C., Eggeling, L., & Sahm, H. (1993) · Journal of Bacteriology · doi:10.1128/jb.175.17.5595-5603.1993 · Cited by 584 · see citing works
Greenwald, E. C., Mehta, S., & Zhang, J. (2018) · Chemical Reviews · doi:10.1021/acs.chemrev.8b00333 · Cited by 488 · see citing works
Tantama, M., Martínez‐François, J. R., Mongeon, R., & Yellen, G. (2013) · Nature Communications · doi:10.1038/ncomms3550 · Cited by 486 · see citing works
van der Rest, M. E., Lange, C. M., & Douwe Molenaar, B. (1999) · Applied Microbiology and Biotechnology · doi:10.1007/s002530051557 · Cited by 467 · see citing works
Graphical analysis of pH-dependent properties of proteins predicted using PROPKA
Rostkowski, M., Olsson, M. H. M., Søndergaard, C. R., & Jensen, J. H. (2011) · BMC Structural Biology · doi:10.1186/1472-6807-11-6 · Cited by 434 · see citing works
Panjkovich, A., & Svergun, D. I. (2017) · Bioinformatics · doi:10.1093/bioinformatics/btx846 · Cited by 332 · see citing works
Akerboom, J., Rivera, J. D. V., Rodríguez‐Guilbe, M. M., Malavé, E. C. A., Hernández, H. H., Tian, L., Hires, S. A., Marvin, J. S., Looger, L. L., & Schreiter, E. R. (2008) · Journal of Biological Chemistry · doi:10.1074/jbc.m807657200 · Cited by 286 · see citing works
Bilan, D. S., Pase, L. B., Joosen, L., Gorokhovatsky, A. Y., Ermakova, Y. G., Gadella, T. W. J., Grabher, C., Schultz, C., Lukyanov, S., & Belousov, V. V. (2012) · ACS Chemical Biology · doi:10.1021/cb300625g · Cited by 280 · see citing works
Structure- and mechanism-guided design of single fluorescent protein-based biosensors
Nasu, Y., Shen, Y., Kramer, L., & Campbell, R. E. (2021) · Nature Chemical Biology · doi:10.1038/s41589-020-00718-x · Cited by 231 · see citing works
The ins and outs of bacterial iron metabolism
Frawley, E. R., & Fang, F. C. (2014) · Molecular Microbiology · doi:10.1111/mmi.12709 · Cited by 222 · see citing works
The biosynthesis of pyoverdines
Ringel, M. T., & Brüser, T. (2018) · Microbial Cell · doi:10.15698/mic2018.10.649 · Cited by 176 · see citing works
Fluorescent proteins and genetically encoded biosensors
Wang, M., Da, Y., & Tian, Y. (2023) · Chemical Society Reviews · doi:10.1039/d2cs00419d · Cited by 156 · see citing works
Engineering allosteric protein switches by domain insertion
Ostermeier, M. (2005) · Protein Engineering Design and Selection · doi:10.1093/protein/gzi048 · Cited by 149 · see citing works
Bacterial iron detoxification at the molecular level
Bradley, J. M., Svistunenko, D. A., Wilson, M. T., Hemmings, A. M., Moore, G. R., & Le Brun, N. E. (2020) · Journal of Biological Chemistry · doi:10.1074/jbc.rev120.007746 · Cited by 140 · see citing works
A Natural Coarse Graining for Simulating Large Biomolecular Motion
Gohlke, H., & Thorpe, M. F. (2006) · Biophysical Journal · doi:10.1529/biophysj.106.083568 · Cited by 136 · see citing works
Self-citationBrune, I., Werner, H., Hüser, A. T., Kalinowski, J., Pühler, A., & Tauch, A. (2006) · BMC Genomics · doi:10.1186/1471-2164-7-21 · Cited by 128 · see citing works
Yagi, H., Kajiwara, N., Tanaka, H., Tomitake Tsukihara, A., Kato‐Yamada, Y., Yoshida, M., & Akutsu, H. (2007) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0701045104 · Cited by 125 · see citing works
Rapid construction of metabolite biosensors using domain-insertion profiling
Nadler, D. C., Morgan, S.-A., Flamholz, A. I., et al. (2016) · Nature Communications · doi:10.1038/ncomms12266 · Cited by 124 · see citing works
Bilan, D. S., Matlashov, M. E., Gorokhovatsky, A. Y., Schultz, C., Enikolopov, G., & Belousov, V. V. (2013) · Biochimica et Biophysica Acta (BBA) - General Subjects · doi:10.1016/j.bbagen.2013.11.018 · Cited by 121 · see citing works
Ast, C., Foret, J., Oltrogge, L. M., Michele, R. D., Kleist, T. J., Ho, C.-H., & Frommer, W. B. (2017) · Nature Communications · doi:10.1038/s41467-017-00400-2 · Cited by 118 · see citing works
The DtxR Regulon ofCorynebacterium glutamicum
Frunzke, J., & Bott, M. H. (2006) · Journal of Bacteriology · doi:10.1128/jb.188.8.2907-2918.2006 · Cited by 115 · see citing works
Self-citationKoveal, D., Rosen, P. C., Meyer, D. J., Díaz‐García, C. M., Wang, Y., Cai, L., Chou, P. J., Weitz, D. A., & Yellen, G. (2022) · Nature Communications · doi:10.1038/s41467-022-30685-x · Cited by 105 · see citing works
Hensen, U., Meyer, T., Haas, J., Rex, R., Vriend, G., & Grubmüller, H. (2012) · PLoS ONE · doi:10.1371/journal.pone.0033931 · Cited by 103 · see citing works
Diez, J., Diederichs, K., Greller, G., et al. (2001) · Journal of Molecular Biology · doi:10.1006/jmbi.2000.4203 · Cited by 102 · see citing works
Volke, D. C., Friis, L., Wirth, N. T., Turlin, J., & Nikel, P. I. (2020) · Metabolic Engineering Communications · doi:10.1016/j.mec.2020.e00126 · Cited by 98 · see citing works
Wynands, B., Lenzen, C., Otto, M., et al. (2018) · Metabolic Engineering · doi:10.1016/j.ymben.2018.03.011 · Cited by 96 · see citing works
Pseudomonas putida as a synthetic biology chassis and a metabolic engineering platform
Martínez‐García, E., & de Lorenzo, V. (2023) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2023.103025 · Cited by 93 · see citing works
Pfleger, C., Rathi, P. C., Klein, D. L., et al. (2013) · Journal of Chemical Information and Modeling · doi:10.1021/ci400044m · Cited by 90 · see citing works
Self-citationLinking the functions of unrelated proteins using a novel directed evolution domain insertion method
Edwards, W. R., Busse, K., Allemann, R. K., et al. (2008) · Nucleic Acids Research · doi:10.1093/nar/gkn363 · Cited by 88 · see citing works
Design Strategies of Fluorescent Biosensors Based on Biological Macromolecular Receptors
Tainaka, K., Sakaguchi, R., Hayashi, H., et al. (2010) · Sensors · doi:10.3390/s100201355 · Cited by 77 · see citing works
Sadoine, M., Ishikawa, Y., Kleist, T. J., et al. (2021) · PLANT PHYSIOLOGY · doi:10.1093/plphys/kiab353 · Cited by 69 · see citing works
Tully, M. D., Kieffer, J., Brennich, M., Aberdam, R. C., Florial, J.-B., Hutin, S., Oscarsson, M., Beteva, A., Popov, A. M., Moussaoui, D., et al. (2022) · Journal of Synchrotron Radiation · doi:10.1107/s1600577522011286 · Cited by 65 · see citing works
Genetically Encoded Biosensors Based on Fluorescent Proteins
Kim, H., Ju, J.-M., Lee, H. N., Chun, H., & Seong, J. (2021) · Sensors · doi:10.3390/s21030795 · Cited by 64 · see citing works
Next-Generation Genetically Encoded Fluorescent Biosensors Illuminate Cell Signaling and Metabolism
Frei, M. S., Mehta, S., & Zhang, J. (2024) · Annual Review of Biophysics · doi:10.1146/annurev-biophys-030722-021359 · Cited by 64 · see citing works
Claassens, N. J., Siliakus, M. F., Spaans, S. K., Creutzburg, S. C. A., Nijsse, B., Schaap, P. J., Quax, T. E. F., & John van der, O. O. S. T. (2017) · PLoS ONE · doi:10.1371/journal.pone.0184355 · Cited by 61 · see citing works
Creating a bifunctional protein by insertion of β-lactamase into the maltodextrin-binding protein
Betton, J., Jacob, J. P., Hofnung, M. J., et al. (1997) · Nature Biotechnology · doi:10.1038/nbt1197-1276 · Cited by 60 · see citing works
Functionally Accepted Insertions of Proteins within Protein Domains
Collinet, B., Hervé, M., Pecorari, F., et al. (2000) · Journal of Biological Chemistry · doi:10.1074/jbc.m000666200 · Cited by 55 · see citing works
Toxicity of Dipyridyl Compounds and Related Compounds
Li, S., Crooks, P. A., Wei, X., et al. (2004) · Critical Reviews in Toxicology · doi:10.1080/10408440490503143 · Cited by 51 · see citing works
Generic Rigidity of Network Glasses
Thorpe, M. F., Jacobs, D. J., Chubynsky, N. V., et al. (2005) · Kluwer Academic Publishers eBooks · doi:10.1007/0-306-47089-6_14 · Cited by 41 · see citing works
Bakkes, P. J., Ramp, P., Bida, A., et al. (2020) · Plasmid · doi:10.1016/j.plasmid.2020.102540 · Cited by 37 · see citing works
Self-citationBecker, F., Wienand, K., Lechner, M., Frey, E., & Jung, H. (2018) · Scientific Reports · doi:10.1038/s41598-018-22306-9 · Cited by 30 · see citing works
Kaczmarski, J. A., Mitchell, J. A., Spence, M. A., Vongsouthi, V., & Jackson, C. J. (2019) · Current Opinion in Structural Biology · doi:10.1016/j.sbi.2019.01.013 · Cited by 29 · see citing works
Pfleger, C., & Gohlke, H. (2013) · Structure · doi:10.1016/j.str.2013.07.012 · Cited by 23 · see citing works
Self-citationStructure Prediction of Domain Insertion Proteins from Structures of Individual Domains
Berrondo, M., Ostermeier, M., & Gray, J. J. (2008) · Structure · doi:10.1016/j.str.2008.01.012 · Cited by 22 · see citing works
Proteomic response of Streptococcus pneumoniae to iron limitation
Hoyer, J., Bartel, J., Gómez-Mejía, A., Rohde, M., Hirschfeld, C., Heß, N., Sura, T., Maaß, S., Hammerschmidt, S., & Becher, D. (2018) · International Journal of Medical Microbiology · doi:10.1016/j.ijmm.2018.02.001 · Cited by 20 · see citing works
Nakamura, A., Sosa, A., Komori, H., et al. (2006) · Proteins Structure Function and Bioinformatics · doi:10.1002/prot.21222 · Cited by 18 · see citing works
Rathi, P. C., Mulnaes, D., & Gohlke, H. (2015) · Bioinformatics · doi:10.1093/bioinformatics/btv139 · Cited by 17 · see citing works
Self-citationLiang, L., Lin, D., Chen, Y., Li, J., Liang, W., Zhao, H., Luo, W., Tian, G., & Feng, S. (2022) · mSystems · doi:10.1128/msystems.00209-22 · Cited by 14 · see citing works
Ejike, J. O., Sadoine, M., Shen, Y., et al. (2023) · Biochemistry · doi:10.1021/acs.biochem.3c00451 · Cited by 13 · see citing works
Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors
Sevimli, G., Alston, A., Funk, F., Flühmann, B., Malli, R., Graier, W. F., & Eroğlu, E. (2022) · Biosensors · doi:10.3390/bios12100903 · Cited by 10 · see citing works
A genetically encoded probe for monitoring and detection of iron in real-time
Soleja, N., & Mohsin, M. (2024) · Sensors & Diagnostics · doi:10.1039/d4sd00091a · Cited by 7 · see citing works
Sadoine, M., Castro‐Rodríguez, V., Poloczek, T., et al. (2020) · BIO-PROTOCOL · doi:10.21769/bioprotoc.3773 · Cited by 2 · see citing works
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- Authors
- Papadopoulos, A., Anlauf, M. T., Reiners, J., Paik, S.-H., Krüger, A., Lückel, B., Bott, M., Drepper, T., Frunzke, J., Gohlke, H., Weidtkamp-Peters, S., Smits, S. H. J., & Gertzen, C. G. W.
- Affiliations
- Heinrich Heine University Düsseldorf (DE)
- Forschungszentrum Jülich (DE)
- Corresponding author
- Smits, Sander H.J.
- ORCID
- Venue
- bioRxiv (Cold Spring Harbor Laboratory)
- Publisher
- openRxiv
- Published
- 2025-07-21
- OpenAlex
- W4412539260
- Cited by
- 0
- References
- 68
- Type
- posted content
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- JuSER (Forschungszentrum Jülich) (publishedVersion, other-oa)
- DESY Publication Database (PUBDB) (Deutsches Elektronen-Synchrotron) (submittedVersion, other-oa)
- Funders
- Deutsche Forschungsgemeinschaft (SFB1535)
- Forschungszentrum Jülich
- Heinrich-Heine-Universität Düsseldorf
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › bioluminescence and chemiluminescence research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Keywords
- genetically encoded biosensors, DtxR, iron-binding proteins, Corynebacterium glutamicum, ratiometric biosensor, molecular recognition elements, biosensor development
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