{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,22]],"date-time":"2026-01-22T14:45:32Z","timestamp":1769093132851,"version":"3.49.0"},"reference-count":34,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2020,1,7]],"date-time":"2020-01-07T00:00:00Z","timestamp":1578355200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000774","name":"Defense Threat Reduction Agency","doi-asserted-by":"publisher","award":["CBT LF1BR CB380"],"award-info":[{"award-number":["CBT LF1BR CB380"]}],"id":[{"id":"10.13039\/100000774","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000006","name":"Office of Naval Research","doi-asserted-by":"publisher","award":["N0001414IP20004; N00014-12-1-0313"],"award-info":[{"award-number":["N0001414IP20004; N00014-12-1-0313"]}],"id":[{"id":"10.13039\/100000006","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>In vivo functionalization of diatom biosilica frustules by genetic manipulation requires careful consideration of the overall structure and function of complex fusion proteins. Although we previously had transformed Thalassiosira pseudonana with constructs containing a single domain antibody (sdAb) raised against the Bacillus anthracis Sterne strain, which detected an epitope of the surface layer protein EA1 accessible in lysed spores, we initially were unsuccessful with constructs encoding a similar sdAb that detected an epitope of EA1 accessible in intact spores and vegetative cells. This discrepancy limited the usefulness of the system as an environmental biosensor for B. anthracis. We surmised that to create functional biosilica-localized biosensors with certain constructs, the biosilica targeting and protein trafficking functions of the biosilica-targeting peptide Sil3T8 had to be uncoupled. We found that retaining the ER trafficking sequence at the N-terminus and relocating the Sil3T8 targeting peptide to the C-terminus of the fusion protein resulted in successful detection of EA1 with both sdAbs. Homology modeling of antigen binding by the two sdAbs supported the hypothesis that the rescue of antigen binding in the previously dysfunctional sdAb was due to removal of steric hindrances between the antigen binding loops and the diatom biosilica for that particular sdAb.<\/jats:p>","DOI":"10.3390\/biology9010014","type":"journal-article","created":{"date-parts":[[2020,1,8]],"date-time":"2020-01-08T03:59:57Z","timestamp":1578455997000},"page":"14","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for Bacillus anthracis Detection"],"prefix":"10.3390","volume":"9","author":[{"given":"Nicole R.","family":"Ford","sequence":"first","affiliation":[{"name":"Marine Biotechnology Group, Pacific Northwest National Laboratory, Sequim, WA 98382, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9832-1472","authenticated-orcid":false,"given":"Yijia","family":"Xiong","sequence":"additional","affiliation":[{"name":"Department of Basic Medical Sciences, Western University of Health Sciences, Lebanon, OR 97355, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Karen A.","family":"Hecht","sequence":"additional","affiliation":[{"name":"Marine Biotechnology Group, Pacific Northwest National Laboratory, Sequim, WA 98382, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thomas C.","family":"Squier","sequence":"additional","affiliation":[{"name":"Department of Basic Medical Sciences, Western University of Health Sciences, Lebanon, OR 97355, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gregory L.","family":"Rorrer","sequence":"additional","affiliation":[{"name":"School of Chemical Biological and Environmental Engineering, Oregon State University, Corvallis, OR 97331, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guritno","family":"Roesijadi","sequence":"additional","affiliation":[{"name":"Marine Biotechnology Group, Pacific Northwest National Laboratory, Sequim, WA 98382, USA"},{"name":"School of Chemical Biological and Environmental Engineering, Oregon State University, Corvallis, OR 97331, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,1,7]]},"reference":[{"key":"ref_1","unstructured":"Round, F.E., Crawford, R.M., and Mann, D.G. (1990). Diatoms: Biology and Morphology of the Genera, Cambridge University Press."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1016\/j.tibtech.2008.11.003","article-title":"The Glass Menagerie: Diatoms for novel applications in nanotechnology","volume":"27","author":"Gordon","year":"2009","journal-title":"Trends Biotechnol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Russell, J.J., Theriot, J.A., Sood, P., Marshall, W.F., Landweber, L.F., Fritz-Laylin, L., Polka, J.K., Oliferenko, S., Gerbich, T., and Gladfelter, A. (2017). Non-model model organisms. BMC Biol., 15.","DOI":"10.1186\/s12915-017-0391-5"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"659","DOI":"10.1002\/jbio.201400070","article-title":"Ultra-sensitive immunoassay biosensors using hybrid plasmonic-biosilica nanostructured materials","volume":"8","author":"Yang","year":"2015","journal-title":"J. Biophotonics"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Cicco, S.R., Vona, D., Gristina, R., Sardella, E., Ragni, R., Lo Presti, M., and Farinola, G.M. (2016). Biosilica from Living Diatoms: Investigations on Biocompatibility of Bare and Chemically Modified Thalassiosira weissflogii Silica Shells. Bioengineering, 3.","DOI":"10.3390\/bioengineering3040035"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1843","DOI":"10.1002\/anie.200603928","article-title":"Silica immobilization of an enzyme through genetic engineering of the diatom Thalassiosira pseudonana","volume":"46","author":"Poulsen","year":"2007","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Narayan, R., Nayak, U.Y., Raichur, A.M., and Garg, S. (2018). Mesoporous Silica Nanoparticles: A Comprehensive Review on Synthesis and Recent Advances. Pharmaceutics, 10.","DOI":"10.3390\/pharmaceutics10030118"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ccr.2019.02.024","article-title":"Chemical, physical, and biological coordination: An interplay between materials and enzymes as potential platforms for immobilization","volume":"388","author":"Bilal","year":"2019","journal-title":"Coord. Chem. Rev."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"17496","DOI":"10.1021\/acsomega.8b02784","article-title":"A Toolbox for the Synthesis of Multifunctionalized Mesoporous Silica Nanoparticles for Biomedical Applications","volume":"3","author":"Renfer","year":"2018","journal-title":"ACS Omega"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1016\/j.msec.2015.05.027","article-title":"Bone tissue engineering using silica-based mesoporous nanobiomaterials: Recent progress","volume":"55","author":"Shadjou","year":"2015","journal-title":"Mater. Sci. Eng. C Mater. Biol. Appl."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Gotz, W., Tobiasch, E., Witzleben, S., and Schulze, M. (2019). Effects of Silicon Compounds on Biomineralization, Osteogenesis, and Hard Tissue Formation. Pharmaceutics, 11.","DOI":"10.3390\/pharmaceutics11030117"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"8791","DOI":"10.1038\/ncomms9791","article-title":"Targeted drug delivery using genetically engineered diatom biosilica","volume":"6","author":"Delalat","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Terracciano, M., De Stefano, L., and Rea, I. (2018). Diatoms Green Nanotechnology for Biosilica-Based Drug Delivery Systems. Pharmaceutics, 10.","DOI":"10.20944\/preprints201810.0512.v1"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Manayil, J.C., Lee, A.F., and Wilson, K. (2019). Functionalized Periodic Mesoporous Organosilicas: Tunable Hydrophobic Solid Acids for Biomass Conversion. Molecules, 24.","DOI":"10.3390\/molecules24020239"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1128\/AEM.06698-11","article-title":"Live diatom silica immobilization of multimeric and redox-active enzymes","volume":"78","author":"Sheppard","year":"2012","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"20100","DOI":"10.1074\/jbc.M113.469379","article-title":"Pentalysine clusters mediate silica targeting of silaffins in Thalassiosira pseudonana","volume":"288","author":"Poulsen","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Marshall, K.E., Robinson, E.W., Hengel, S.M., Pasa-Tolic, L., and Roesijadi, G. (2012). FRET imaging of diatoms expressing a biosilica-localized ribose sensor. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0033771"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1021\/acssynbio.5b00191","article-title":"Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms","volume":"5","author":"Ford","year":"2016","journal-title":"ACS Synth. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1205","DOI":"10.1021\/acs.bioconjchem.6b00165","article-title":"Dynamic Stabilization of Expressed Proteins in Engineered Diatom Biosilica Matrices","volume":"27","author":"Xiong","year":"2016","journal-title":"Bioconjug. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Walper, S.A., Anderson, G.P., Brozozog Lee, P.A., Glaven, R.H., Liu, J.L., Bernstein, R.D., Zabetakis, D., Johnson, L., Czarnecki, J.M., and Goldman, E.R. (2012). Rugged single domain antibody detection elements for Bacillus anthracis spores and vegetative cells. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0032801"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1146\/annurev-biochem-063011-092449","article-title":"Nanobodies: Natural single-domain antibodies","volume":"82","author":"Muyldermans","year":"2013","journal-title":"Annu. Rev. Biochem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1111\/j.1529-8817.2006.00269.x","article-title":"Molecular genetic manipulation of the diatom thalassiosira pseudonana (bacillariophyceae)1","volume":"42","author":"Poulsen","year":"2006","journal-title":"J. Phycol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"420","DOI":"10.1038\/s41587-019-0036-z","article-title":"SignalP 5.0 improves signal peptide predictions using deep neural networks","volume":"37","author":"Tsirigos","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1093\/protein\/10.1.1","article-title":"Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites","volume":"10","author":"Nielsen","year":"1997","journal-title":"Protein Eng."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"W296","DOI":"10.1093\/nar\/gky427","article-title":"SWISS-MODEL: Homology modelling of protein structures and complexes","volume":"46","author":"Waterhouse","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"D313","DOI":"10.1093\/nar\/gkw1132","article-title":"The SWISS-MODEL Repository-new features and functionality","volume":"45","author":"Bienert","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1093\/bioinformatics\/btq662","article-title":"Toward the estimation of the absolute quality of individual protein structure models","volume":"27","author":"Benkert","year":"2011","journal-title":"Bioinformatics"},{"key":"ref_28","unstructured":"Schrodinger, L.L.C. (2019, November 30). The PyMOL Molecular Graphics System, Version 1.7. Available online: https:\/\/pymol.org\/2\/support.html?."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2984","DOI":"10.1073\/pnas.1400546111","article-title":"Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11","volume":"111","author":"Pathare","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"25374","DOI":"10.1074\/jbc.M114.566257","article-title":"The molecular mechanism of Shiga toxin Stx2e neutralization by a single-domain antibody targeting the cell receptor-binding domain","volume":"289","author":"Lo","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"859","DOI":"10.1128\/JB.182.4.859-868.2000","article-title":"S-Layer proteins","volume":"182","author":"Sleytr","year":"2000","journal-title":"J. Bacteriol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"36824","DOI":"10.1038\/srep36824","article-title":"Establishing super-resolution imaging for proteins in diatom biosilica","volume":"6","author":"Groger","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1016\/j.jim.2012.11.014","article-title":"Comparison of single domain antibody immobilization strategies evaluated by surface plasmon resonance","volume":"388","author":"Walper","year":"2013","journal-title":"J. Immunol. Methods"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Anderson, G.P., Shriver-Lake, L.C., Walper, S.A., Ashford, L., Zabetakis, D., Liu, J.L., Breger, J.C., Brozozog Lee, P.A., and Goldman, E.R. (2018). Genetic Fusion of an Anti-BclA Single-Domain Antibody with Beta Galactosidase. Antibodies, 7.","DOI":"10.3390\/antib7040036"}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/9\/1\/14\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T13:28:58Z","timestamp":1760362138000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/9\/1\/14"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,1,7]]},"references-count":34,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2020,1]]}},"alternative-id":["biology9010014"],"URL":"https:\/\/doi.org\/10.3390\/biology9010014","relation":{},"ISSN":["2079-7737"],"issn-type":[{"value":"2079-7737","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,1,7]]}}}