Digital-to-biological converter for on-demand production of biologics

ABSTRACT Manufacturing 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. Access through your

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APPLICATIONS IN INFORMATION TECHNOLOGY Article Open access 17 January 2022 DNA-BASED PROGRAMMABLE GATE ARRAYS FOR GENERAL-PURPOSE DNA COMPUTING Article 13 September 2023 PLUG-AND-PLAY

PROTEIN BIOSENSORS USING APTAMER-REGULATED IN VITRO TRANSCRIPTION Article Open access 12 September 2024 ACCESSION CODES ACCESSIONS NCBI REFERENCE SEQUENCE * KY199424 * KY199425 * KY199426 *

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potential expression host. _Nucleic Acids Res._ 33, W526–W5331 (2005). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS The authors thank C. Beard, S. Farah, O. Fetzer, K.

Han, C. Hutchison, A. Lee, D. Lomelin, A. Nandi, T. Newman-Lehman, T. Peterson, S. Riedmuller, J. Robinson, H. Smith, J. Strauss, J. Thielmier, L. Warden, M. Winstead, and A. 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 INFORMATION

Author notes * Kent S Boles and Krishna Kannan: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Synthetic Genomics, Inc., La Jolla, California, USA Kent S Boles, 

Krishna Kannan, John Gill, Martina Felderman, Heather Gouvis, Bolyn Hubby, Kurt I Kamrud, J Craig Venter & Daniel G Gibson Authors * Kent S Boles View author publications You can also

search for this author inPubMed Google Scholar * Krishna Kannan View author publications You can also search for this author inPubMed Google Scholar * John Gill View author publications You

can also search for this author inPubMed Google Scholar * Martina Felderman View author publications You can also search for this author inPubMed Google Scholar * Heather Gouvis View author

publications You can also search for this author inPubMed Google Scholar * Bolyn Hubby View author publications You can also search for this author inPubMed Google Scholar * Kurt I Kamrud

View author publications You can also search for this author inPubMed Google Scholar * J Craig Venter View author publications You can also search for this author inPubMed Google Scholar *

Daniel G Gibson View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS J.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 AUTHOR

Correspondence 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 INFORMATION

SUPPLEMENTARY 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 ΦX174, 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˚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˚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 μl of unpurified T7-GFP template was used in 25 μl 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 μl 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˚C or 4˚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 μg) 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™ 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 FIGURES Supplementary

Figures 1–10 (PDF 1236 kb) SUPPLEMENTARY TABLES Supplementary Tables 1–12 (PDF 241 kb) SUPPLEMENTARY CODES Oligonucleotide Designer Script (PDF 226 kb) SUPPLEMENTARY DATA Amplicon 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

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Boles, K., Kannan, K., Gill, J. _et al._ Digital-to-biological converter for on-demand production of biologics. _Nat

Biotechnol_ 35, 672–675 (2017). https://doi.org/10.1038/nbt.3859 Download citation * Received: 05 August 2016 * Accepted: 24 March 2017 * Published: 29 May 2017 * Issue Date: July 2017 *

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