Single Day Construction of Multigene Circuits with 3G Assembly.

Abstract:

:The ability to rapidly design, build, and test prototypes is of key importance to every engineering discipline. DNA assembly often serves as a rate limiting step of the prototyping cycle for synthetic biology. Recently developed DNA assembly methods such as isothermal assembly and type IIS restriction enzyme systems take different approaches to accelerate DNA construction. We introduce a hybrid method, Golden Gate-Gibson (3G), that takes advantage of modular part libraries introduced by type IIS restriction enzyme systems and isothermal assembly's ability to build large DNA constructs in single pot reactions. Our method is highly efficient and rapid, facilitating construction of entire multigene circuits in a single day. Additionally, 3G allows generation of variant libraries enabling efficient screening of different possible circuit constructions. We characterize the efficiency and accuracy of 3G assembly for various construct sizes, and demonstrate 3G by characterizing variants of an inducible cell-lysis circuit.

journal_name

ACS Synth Biol

journal_title

ACS synthetic biology

authors

Halleran AD,Swaminathan A,Murray RM

doi

10.1021/acssynbio.8b00060

subject

Has Abstract

pub_date

2018-05-18 00:00:00

pages

1477-1480

issue

5

issn

2161-5063

journal_volume

7

pub_type

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