BASIC: a new Biopart Assembly Standard for Idempotent Cloning provides accurate, single-tier DNA assembly for synthetic biology
File(s)Storch et al BASIC.pdf (678.01 KB)
Accepted version
Author(s)
Type
Journal Article
Abstract
The ability to quickly and reliably assemble DNA constructs is one of the key enabling technologies for synthetic biology. Here we define a new Biopart Assembly Standard for Idempotent Cloning (BASIC), which exploits the principle of orthogonal linker based DNA assembly to define a new physical standard for DNA parts. Further, we demonstrate a new robust method for assembly, based on type IIs restriction cleavage and ligation of oligonucleotides with single stranded overhangs that determine the assembly order. It allows for efficient, parallel assembly with great accuracy: 4 part assemblies achieved 93% accuracy with single antibiotic selection and 99.7% accuracy with double antibiotic selection, while 7 part assemblies achieved 90% accuracy with double antibiotic selection. The linkers themselves may also be used as composable parts for RBS tuning or the creation of fusion proteins. The standard has one forbidden restriction site and provides for an idempotent, single tier organisation, allowing all parts and composite constructs to be maintained in the same format. This makes the BASIC standard conceptually simple at both the design and experimental levels.
Date Issued
2015-07-17
Date Acceptance
2015-03-01
Citation
ACS Synthetic Biology, 2015, 4 (7), pp.781-787
ISSN
2161-5063
Publisher
American Chemical Society
Start Page
781
End Page
787
Journal / Book Title
ACS Synthetic Biology
Volume
4
Issue
7
Copyright Statement
© 2015 American Chemical Society
Identifier
http://dx.doi.org/10.1021/sb500356d
Notes
doi: 10.1021/sb500356d The ability to quickly and reliably assemble DNA constructs is one of the key enabling technologies for synthetic biology. Here we define a new Biopart Assembly Standard for Idempotent Cloning (BASIC), which exploits the principle of orthogonal linker based DNA assembly to define a new physical standard for DNA parts. Further, we demonstrate a new robust method for assembly, based on type IIs restriction cleavage and ligation of oligonucleotides with single stranded overhangs that determine the assembly order. It allows for efficient, parallel assembly with great accuracy: 4 part assemblies achieved 93% accuracy with single antibiotic selection and 99.7% accuracy with double antibiotic selection, while 7 part assemblies achieved 90% accuracy with double antibiotic selection. The linkers themselves may also be used as composable parts for RBS tuning or the creation of fusion proteins. The standard has one forbidden restriction site and provides for an idempotent, single tier organisation, allowing all parts and composite constructs to be maintained in the same format. This makes the BASIC standard conceptually simple at both the design and experimental levels.
Publication Status
Published
Date Publish Online
2015-03-08