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产品产品实验室服务应用资源网络研讨会新闻与活动贸易展览新闻发布公司关于我们联系方式提交样品产品文献产品Rainbow AgarMitoPlateSherlock SoftwareFF（丝状真菌）软件解决方案表型微阵列定制表型微阵列EcoPlate微生物鉴定微孔板实验室服务YT MicroPlateMicroStationOdin应用教育发酵生物工艺可持续农业环境监测基础研究资源类型宣传册应用说明使用说明测试面板用户指南数据库分析证书Safe ty 数据表一般过滤器：数据库清除所有数据库FF（丝状真菌）表型微阵列定制表型微阵列EcoPlate微生物鉴定微孔板YT MicroPlateMicroStationOdin数据库00A 005 Rev G – MI Biolog 系统数据库下载版权所有 2023 Biolog，版权所有rved用户隐私政策Site by Metropolis
产品产品实验室服务应用资源网络研讨会新闻与活动贸易展览新闻发布公司关于我们联系方式提交样品Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. AbstractManufacturing processes for biological molecules in the research laboratory have failed to keep pace with the rapid advances in automization and parellelization1,2,3. We report the development of a digital-to-biological converter for fully automated, versatile and demand-based production of functional biologics starting from DNA sequence information. Specifically, DNA templates, RNA molecules, proteins and viral particles were produced in an automated fashion from digitally transmitted DNA sequences without human intervention. Subscription info for Chinese customersWe have a dedicated website for our Chinese customers. Please go to naturechina.com to subscribe to this journal.Go to naturechina.comRent or Buy articleGet time limited or full article access on ReadCube.from$8.99Rent or BuyAll prices are NET prices. References1Rauch, S., Dallinger, M., Dallasega, P. Matt, D.T. Sustainability in manufacturing through distributed manufacturing systems (DMS). Procedia CIRP 29, 544鈥?49 (2015).Article聽 Google Scholar聽 2Srai, J.S. et al. Distributed manufacturing: scope, challenges and opportunity. Int. J. Prod. Res. 54, 6917鈥?935 (2016).Article聽 Google Scholar聽 3Pearce, J.M. Materials science. Building research equipment with free, open-source hardware. Science 337, 1303鈥?304 (2012).CAS聽 Article聽 Google Scholar聽 4Jones, R. et al. RepRap鈥攖he replicating rapid prototyper. Robotica 29, 177鈥?91 (2011).Article聽 Google Scholar聽 5Pearce, J.M., Anzalone, N.C. Heldt, C.L. 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Witschi for their contributions to this work, and the US Defense Advanced Research Projects Agency (contract HR0011-13-C-0073 for D.G.G. and J.C.V.) for funding aspects of this work.Author informationAuthor notesKent S Boles and Krishna Kannan: These authors contributed equally to this work.AffiliationsSynthetic Genomics, Inc., La Jolla, California, USAKent S Boles,聽Krishna Kannan,聽John Gill,聽Martina Felderman,聽Heather Gouvis,聽Bolyn Hubby,聽Kurt I Kamrud,聽J Craig Venter聽 聽Daniel G GibsonAuthorsKent S BolesView author publicationsYou can also search for this author in PubMed聽Google ScholarKrishna KannanView author publicationsYou can also search for this author in PubMed聽Google ScholarJohn GillView author publicationsYou can also search for this author in PubMed聽Google ScholarMartina FeldermanView author publicationsYou can also search for this author in PubMed聽Google ScholarHeather GouvisView author publicationsYou can also search for this author in PubMed聽Google ScholarBolyn HubbyView author publicationsYou can also search for this author in PubMed聽Google ScholarKurt I KamrudView author publicationsYou can also search for this author in PubMed聽Google ScholarJ Craig VenterView author publicationsYou can also search for this author in PubMed聽Google ScholarDaniel G GibsonView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsJ.C.V. and D.G.G. conceived the study; K.S.B., K.K., J.G., H.G., B.H., K.I.K. and D.G.G. designed experiments and analyzed data; K.S.B., K.K., M.F. and D.G.G. performed experiments; and K.S.B., K.K., J.C.V. and D.G.G. wrote the paper.Corresponding authorCorrespondence to Daniel G Gibson.Ethics declarations Competing interests The authors are or have been employed by Synthetic Genomics, Inc. (SGI), a privately held company, and may hold stock or stock options. SGI has filed provisional applications with the US Patent and Trademark Office on aspects of this research (PCT/US2013/055454). Integrated supplementary informationSupplementary Figure 1 Prototype of the digital-to-biological converter (DBC).Components that constitute the DBC are marked as indicated in the following equipment list.Supplementary Figure 2 Route map of synthesis of a biological in DBC.Schematic of the sequence of processes that are automated to occur on the DBC subsequent to recognizing the oligonucleotide synthesis file and the name of the instruments used in these processes are shown.Supplementary Figure 3 Importance of error-correction reaction to produce functional DNA amplicons.(a) Genome of the phage 桅X174, was synthesized in vitro from oligonucleotides while including or not including an error-correction step. Error-corrected genome produced many more functional viral particles (as demonstrated by plaque formation on susceptible E. coli strain HF4704, Supplementary Methods), than the genome that was not subjected to an error-correction step during synthesis. (b) In vitro coupled transcription-translation reaction (PURExpress, New England Biolabs) was used to produce green fluorescent protein (GFP) from DNA templates that were error-corrected or not subjected to the error-correction reaction. A no template control was included. Relative fluorescence units (RFU) (Ex: 480nm; Em: 510nm) was measured every 20min after the first 60min of incubation at 37藲C. A consistent lag in the GFP yield was observed when a non-error corrected template was used (green curve vs orange curve).Supplementary Figure 4 Optimizing protein production conditions for the DBC.T7- or tac- promoter driven- green fluorescent protein (GFP) encoding template with or without the T7 terminator was transcribed and translated using PURExpress in vitro Protein Synthesis Kit (New England Biolabs) or E. coli S30 Extract System for Linear Templates (Promega). Transcription-translation reactions were incubated for two hours at 37藲C before measuring relative fluorescence units (RFU) (Ex: 480nm; Em: 510nm). (a) To avoid DNA purification on the DBC, unpurified DNA amplicon directly from a PCR was tested for the production of protein, when incubated with in vitro coupled transcription-translation systems. 3-6 渭l of unpurified T7-GFP template was used in 25 渭l of PURExpress and the RFUs were recorded. (b) Two in vitro transcription-translation systems were compared, PURExpress in vitro Protein Synthesis Kit (New England Biolabs) or E. coli S30 Extract System for Linear Templates (Promega), for the production of GFP using none or 3 渭l of unpurified PCR product carrying T7 or the tac promoter. (c) Robustness of the PURExpress system was tested by pre-incubating the reaction mix under sub-optimal conditions such as -20藲C or 4藲C for 16 hours prior to the coupled transcription-translation reaction for the ease-of-use in the DBC. Temperature conditions were tested with T7-promoter driven GFP templates with or without the T7-terminator to assess the importance of the T7-terminator regulatory element.Supplementary Figure 5 Visualizing GFP produced by DBC.DNA template carrying encoding GFP with T7 -promoter and -terminator as regulatory elements was transcribed and translated using the PURExpress system. A negative control for the in vitro coupled transcription-translation reaction was included with no DNA template added to the reaction. These two reactions were tested for protein production on the Typhoon Imager (Amersham Biosciences) using the Green laser setting along with a GFP standard (10 渭g) purchased from Vector Laboratories. GFP fluorescence from the DBC sample is indicative of the production of properly-folded and functional GFP.Supplementary Figure 6 Production of polypeptides constituting antibodies on the DBC.Lucentis (a) and Herceptin (b) were synthesized by assembling a tac-promoter driven bi-cistronic light and heavy chain ORFs and translating this construct using the E. coli S30 Extract System for Linear Templates (Promega). Translation was done in the presence of FluoroTect鈩?GreenLys in vitro Translation Labeling System (Promega), which enabled fluorescence-based visualization of the protein. Lucentis (a) is a combination of two polypeptides of very similar molecular weight (24 kDa and 25 kDa), which could not be successfully resolved with our system. In both (a) and (b), the control and the antibody lanes were contrasted more than the protein marker lane to visualize the proteins produces.Supplementary Figure 7 Verifying H7 assembly into an RNA replicon backbone using RT-PCR.Hemagglutinin antigen H7 was assembled from oligo nucleotides in the DBC and ligated into a self-amplifying RNA replicon based on the genome of the Venezuelan equine encephalitis virus before transcription. To verify the presence of H7 coding region within the RNA replicon mRNA, an oligo dT primer was used in RT-PCR (NEB ProtoScript, with or without RT, +/-) to convert the RNA to cDNA. cDNA, thus generated, was subsequently used as a PCR template for PCR with primers flanking the H7 coding region (1790bp).Supplementary Figure 8 Immunofluorescence analysis (IFA) of Vero cells transfected with H7 replicon RNA synthesized on the DBC.Cells were fixed and immuno-stained with an anti-H7 primary antibody and Alexa Flour 488 secondary antibody. (a) Bright-field image of H7 Replicon transfected Vero cells. (b) IFA of H7 Replicon transfected cells showing H7 specific expression. (c) Overlay of H7 and DAPI channels. (d) Overlay of H7, DAPI, and bright-field channels.Supplementary Figure 9 GC flux of the various DNA templates synthesized on the DBC.Each of the nine DNA templates (listed on Supplementary Table 4) assembled on the DBC, was subjected to GC-flux analysis using GPMiner (http://gpminer.mbc.nctu.edu.tw/) across the entire length of the template using a 60-nucleotide window. The number of windows (X-axis) was plotted against the GC% of the windows (Y-axis).Supplementary Figure 10 Biological material synthesized on the DBC.Full-length DNA and protein gels corresponding to those in Figure 2 are shown. The expected DNA and protein products are indicated by arrows.Supplementary information Supplementary Text and FiguresSupplementary Figures 1鈥?0 (PDF 1236 kb) Supplementary TablesSupplementary Tables 1鈥?2 (PDF 241 kb) Supplementary CodesOligonucleotide Designer Script (PDF 226 kb) Supplementary DataAmplicon Sequencing (PDF 23955 kb)Oligonucleotide Synthesis. (MOV 10927 kb)Oligonucleotide Deprotection. (MOV 26162 kb)Oligonucleotide Pooling. (MOV 14451 kb)DNA Assembly. (MOV 26774 kb)Rights and permissionsReprints and PermissionsAbout this articleCite this articleBoles, K., Kannan, K., Gill, J. et al. Digital-to-biological converter for on-demand production of biologics. Nat Biotechnol 35, 672鈥?75 (2017). https://doi.org/10.1038/nbt.3859Download citationReceived: 05 August 2016Accepted: 24 March 2017Published: 29 May 2017Issue Date: July 2017DOI: https://doi.org/10.1038/nbt.3859 Leonard Katz, Yvonne Y Chen, Ramon Gonzalez, Todd C Peterson, Huimin Zhao Richard H Baltz Journal of Industrial Microbiology and Biotechnology (2018) Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.