Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Dec 15;12(12):3506-3513.
doi: 10.1021/acssynbio.3c00292. Epub 2023 Nov 10.

Automated Platform for the Plasmid Construction Process

Alberto A Nava  1   2   3 , Anna Lisa Fear  1   2 , Namil Lee  1   2 , Peter Mellinger  1   2 , Guangxu Lan  1   2 , Joshua McCauley  1   2   4 , Stephen Tan  1   2   4 , Nurgul Kaplan  1   2   4 , Garima Goyal  1   2   4 , R Cameron Coates  1   2   4 , Jacob Roberts  1   2   5 , Zahmiria Johnson  3 , Romina Hu  5 , Bryan Wu  5 , Jared Ahn  5 , Woojoo E Kim  1   2 , Yao Wan  1   2 , Kevin Yin  1   2   6 , Nathan Hillson  1   2   4 , Robert W Haushalter  1   2 , Jay D Keasling  1   2   3   5   7   8
Affiliations

Automated Platform for the Plasmid Construction Process

Alberto A Nava et al. ACS Synth Biol. .

Abstract

There is a growing need for applications capable of handling large synthesis biology experiments. At the core of synthetic biology is the process of cloning and manipulating DNA as plasmids. Here, we report the development of an application named DNAda capable of writing automation instructions for any given DNA construct design generated by the J5 DNA assembly program. We also describe the automation pipeline and several useful features. The pipeline is particularly useful for the construction of combinatorial DNA assemblies. Furthermore, we demonstrate the platform by constructing a library of polyketide synthase parts, which includes 120 plasmids ranging in size from 7 to 14 kb from 4 to 7 DNA fragments.

Keywords: automation; plasmid construction; polyketide synthases; synthetic biology.

PubMed Disclaimer

Conflict of interest statement

The authors declare the following competing financial interest(s): J.D.K. has financial interests in Amyris, Ansa Biotechnologies, Apertor Pharma, Berkeley Yeast, Demetrix, Lygos, Napigen, ResVita Bio, and Zero Acre Farms. N.H. has financial interests in TeselaGen Biotechnologies and Ansa Biotechnologies.

Figures

Figure 1
Figure 1
DNAda build workflow. DNA constructs are designed using the computer-aided design tool DeviceEditor, and build instructions are generated through the j5 DNA assembly algorithm. Those build instructions are then translated into step-by-step instructions for automated liquid handlers by DNAda.
Figure 2
Figure 2
DNAda core features. (A) DNAda contains numerous functions that automatically create customized liquid handler instructions given design files and result files. (B) An example intermediate workflow involves analyzing uploaded PCR result data from the ZAG. (C) DNAda can then use that uploaded PCR data in order to generate automated redo PCR instructions for the failed reactions.
Figure 3
Figure 3
PKS part library architecture. (A) There are two versions of yeast-assembly-modified Serine Recombinate-assisted Genome Engineering (SAGE) vectors in which PKS parts were assembled. The first drives PKS expression under a PlacUV5 promoter and is suited for inducible expression in a variety of hosts. The second drives PKS expression under a PT7 promoter and links each PKS part to an N-terminal 6X-His Tag and a TEV protease cleavage site. This second vector is suited for high expression in E. coli and could serve a variety of applications, including protein purification and rapid cell-free assays. (B) The architecture of PKS parts that were successfully assembled. In total, we assembled a library of 120 plasmids. Within those 120 plasmids, there are 9 unique loading modules linked to a variety of C-terminal docking domains, 25 unique extension modules flanked by a variety of docking domains, 11 unique extension modules with the KS domain swapped with that of another extension module, and 6 unique termination modules that consist of an N-terminal docking domain fused to a thioesterase.
See this image and copyright information in PMC

References

    1. Paddon C. J.; Westfall P. J.; Pitera D. J.; Benjamin K.; Fisher K.; McPhee D.; Leavell M. D.; Tai A.; Main A.; Eng D.; Polichuk D. R.; Teoh K. H.; Reed D. W.; Treynor T.; Lenihan J.; Jiang H.; Fleck M.; Bajad S.; Dang G.; Dengrove D.; Diola D.; Dorin G.; Ellens K. W.; Fickes S.; Galazzo J.; Gaucher S. P.; Geistlinger T.; Henry R.; Hepp M.; Horning T.; Iqbal T.; Kizer L.; Lieu B.; Melis D.; Moss N.; Regentin R.; Secrest S.; Tsuruta H.; Vazquez R.; Westblade L. F.; Xu L.; Yu M.; Zhang Y.; Zhao L.; Lievense J.; Covello P. S.; Keasling J. D.; Reiling K. K.; Renninger N. S.; Newman J. D. High-Level Semi-Synthetic Production of the Potent Antimalarial Artemisinin. Nature 2013, 496 (7446), 528–532. 10.1038/nature12051. - DOI - PubMed
    1. Martin V. J. J.; Pitera D. J.; Withers S. T.; Newman J. D.; Keasling J. D. Engineering a Mevalonate Pathway in Escherichia Coli for Production of Terpenoids. Nat. Biotechnol. 2003, 21 (7), 796–802. 10.1038/nbt833. - DOI - PubMed
    1. Yu V. Y; Chang M. C Y High-Yield Chemical Synthesis by Reprogramming Central Metabolism. Nat. Biotechnol. 2016, 34 (11), 1129–1129. 10.1038/nbt.3723. - DOI - PubMed
    1. Liao J. C.; Mi L.; Pontrelli S.; Luo S. Fuelling the Future: Microbial Engineering for the Production of Sustainable Biofuels. Nat. Rev. Microbiol. 2016, 14 (5), 288–304. 10.1038/nrmicro.2016.32. - DOI - PubMed
    1. Hillson N. J.; Rosengarten R. D.; Keasling J. D. j5 DNA Assembly Design Automation Software. ACS Synth. Biol. 2012, 1 (1), 14–21. 10.1021/sb2000116. - DOI - PubMed

Publication types