HOW TO BIOSYNTHESIZE ALMOST ANYTHING

Lecture 2: Gene Synthesis

MIT MAS.S65

9.22.26

J. Jacobson

Next Generation Gene Synthesis

HOW TO BIOMANUFACTURE ALMOST ANYTHING

MIT MAS.S65

September, 2026

Metabolic Circuit

to Program Cells to

Become Cellular Factories,

Sensors …

​

to Make High Value Products

(Biofuels, Chemicals,

Pharmaceuticals)

Phosphoramidite DNA Synthesis Cycle

http://www.nature.com/nmeth/journal/v11/n5/images/nmeth.2918-F2.jpg

Acid Based Deprotection

Light Based Deprotection

5 Minute Cycles

Machine ~ .5 Mbp / yr

DNA Synthesis

template dependant 5'-3'

primer extension

5'-3' error-correcting

exonuclease

3'-5' proofreading

exonuclease

Error Rate: 1:10⁶

Throughput: 10 mS per

Base Addition

Beese et al. (1993), Science, 260, 352-355. http://www.biochem.ucl.ac.uk/bsm/xtal/teach/repl/klenow.html

​

Next Generation Sequencing by Synthesis

https://raw.githubusercontent.com/lmmx/shots/master/2015/May/Illinois-BioE416-Illumina-polonies.jpg

1M

10G

1T

100T

Base Pairs

100M

~ 200 Mbp

~ 500 Mbp

~ 1.5 Gbp

~ 1.6 Gbp

~ 3.2 Gbp

235 Gbp

~ 250 Gbp

10000000000 G Transistors

97 Mbp

~1-13 Mb

Scale Factors in DNA Synthesis & Applications

Next Generation (Chip Based) DNA Synthesis

12.2 Mbp

Up to ~ 1M Oligos/Chip

Next Generation (Chip Based) DNA Synthesis

MutS Repair System

Lamers et al. Nature 407:711 (2000)

Error Correction

Error Correcting Gene Synthesis

Error Rate 1:10⁴

Nucleic Acids Research 2004 32(20):e162

http://www.pnas.org/content/100/26/15440/F1.large.jpg

Generating a synthetic genome by whole genome assembly: φX174 bacteriophage from synthetic oligonucleotides – 5,386 bp

AGCTAGCTAGTTTTTTTTTT

GGGGGGGGGGAAGGCCTTAA

AAAAAAAAAACCCCCCCCCC

2003

https://www.neb.com/~/media/Catalog/All-Products/0AA961B294E444AFBEDD5C4A904C76E6/Long%20Description/E2611a.jpg

Gibson Assembly (2009)

Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009). "Enzymatic assembly of DNA molecules up to several hundred kilobases". Nature Methods. 6 (5): 343–345. doi:10.1038/nmeth.1318. PMID 19363495.

Multiplex Synthesis (2010)

Kosuri, Sriram, et al. "Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips." Nature biotechnology 28.12 (2010): 1295-1299.

Parallel gene synthesis in a microfluidic device

D.S. Kong, P.A. Carr, L. Chen, S. Zhang, J.M. Jacobson Nucleic Acid Research , 2007, Vol. 35, No. 8 e61

�Accurate multiplex gene synthesis from programmable DNA microchips

Jingdong Tian, Hui Gong, Nijing Sheng, Xiaochuan Zhou, Erdogan Gulari, Xiaolian Gao and George Church

Nature 432, 1050-1054(23 December 2004)

doi:10.1038/nature03151

Chip Based Gene Synthesis

http://www.synthesis.cc/assets_c/2014/02/Carlson_Price_Seq_Synth_Feb2014.html

1M

10G

1T

100T

Base Pairs

100M

~ 200 Mbp

~ 500 Mbp

~ 1.5 Gbp

~ 1.6 Gbp

~ 3.2 Gbp

235 Gbp

~ 250 Gbp

10000000000 G Transistors

97 Mbp

~1-13 Mb

Scale Factors in DNA Synthesis & Applications

Next Generation (Chip Based) DNA Synthesis

12.2 Mbp

(a) Scheme for two-step oligonucleotide extension using TdT–dNTP conjugates consisting of a TdT molecule site-specifically labeled with a dNTP via a cleavable linker. In the extension step, a DNA primer is exposed to an excess of TdT–dNTP conjugate. Upon incorporation of the tethered nucleotide into the 3′ end of the primer, the conjugate becomes covalently attached and prevents further extensions by other TdT–dNTP molecules. In the deprotection step, the remaining TdT–dNTP conjugates are inactivated (or removed) and the linkage between the incorporated nucleotide and TdT is cleaved by addition of the cleavage reagent (e.g., DTT, 365 nm light, peptidase), thereby releasing the primer for subsequent extension. The cycle can be iterated to extend a primer by a defined sequence. (b) Chemical structures of two types of TdT–linker-dNTP conjugates used in this study, based on the amine-to-thiol crosslinkers PEG₄-SPDP (upper, “TdT–PEG₄-dTTP”) and BP-23354 (lower, “TdT–dTTP”) and the dTTP analogs 5-aminoallyl dUTP (aa-dUTP) and 5-propargylamino dUTP (pa-dUTP), respectively. Upon cleavage of the linker, the atoms indicated in red remain attached to the nucleobase and are referred to as a scar. The cleavable bond is indicated with a black dotted line.

97.7% yield

20 S reaction time

Enzymatic Synthesis

Terminal Deoxynucleotidyl

Transferase(TdT)

Synthesizing DNA

Enzymatically

Applications

Cells as Chemical Factories

1,3 Propanediol

http://3rdpartylogistics.blogspot.com/2011/10/genetic-bacteria-genetic-modification.html

http://www.latonkorea.com/Plant.html

http://3rdpartylogistics.blogspot.com/2011/10/genetic-bacteria-genetic-modification.html

http://www.latonkorea.com/Plant.html

Cells as Chemical Factories

http://3rdpartylogistics.blogspot.com/2011/10/genetic-bacteria-genetic-modification.html

http://www.latonkorea.com/Plant.html

http://www.latonkorea.com/Plant.html

~6 Gb Synthesis For top 1000 small molecules drug intermediates

Paddon, Christopher J., et al. "High-level semi-synthetic production of the potent antimalarial artemisinin." Nature 496.7446 (2013): 528-532.

Artemisinic Acid (Antimalarial)

http://neweuropeans.org/eurowine-3-yeast-europe-and-economics/

Paclitaxel (Taxol) 

Therapeutics from Natural Product Pathways

Pacific Yew

~2/3 of Small Molecule Pharmaceuticals are Natural Products

1000’s of Annotated PKS’s

~5 Gb Synthesis for Known PKS Pathway Modules

The Genetic Code

Life’s Operating System

Recoding

Conferring New Function

Phage Resistance

Building:

​

• Therapeutics
• Nanostructures
• Nanomachines
• Data Storage

​

With Nucleotides

​

BIOMANUFACTURING ARCHITECTURE

Moderna mRNA-4157 (V940)

Needle-to-Needle Circular Flow

CRITICAL WINDOW

Post-surgical adjuvant therapy must begin before micrometastatic recurrence, making the 45-day cycle clinically vital.

TARGET TURNAROUND TIME

~45 Days

Days 1 – 12

Genomics & AI

Biopsy, WES/RNA-Seq, HLA-epitope ranking

Days 13 – 24

DNA & Transcription

Enzymatic gene block & cell-free IVT (m1Ψ)

Days 25 – 36

LNP & Purification

Microfluidic impingement assembly & TFF

Days 37 – 45

QC & Clinical Cryo

Assays, isolator vial fill & -80°C delivery

ssrna/dna-sensors via embedded strand-displacement programs in crispr/cas9 guides Jakimo, N., Chatterjee, P. and Jacobson, J.M., 2018.. bioRxiv, p.264424.

Nano Letters, 1 (1), 22 -26, 2001. 10.1021/nl000182v S1530-6984(00)00182-X

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Holliday Junctions

http://seemanlab4.chem.nyu.edu/HJ.arrays.html

Holliday Junctions

Science 15 April 2011: �Vol. 332 no. 6027 pp. 342-346 �DOI: 10.1126/science.1202998

3D DNA Origami

https://www.researchgate.net/figure/Summary-of-DNA-tile-and-DNA-origami-a-Multistranded-DNA-tile-Each-tile-is-composed-of_fig1_321675577

https://www.pnas.org/content/112/3/713

DNA NANO- MACHINES

Programmable motion of DNA origami mechanisms

1. Alexander E. Marras, Lifeng Zhou, Hai-Jun Su, and Carlos E. CastroDepartment of Mechanical and Aerospace Engineering, The Ohio State University, Columbus, OH 43210

DNA Data Storage

980,000 Sq. ft

=

~ 10 Petabits

Flash: 64GB = $10

DNA Chip: 12 MB

~ $20

10⁴x COST

$12,400 / MB