PT  - JOURNAL ARTICLE
AU  - Andrew D. Halleran
AU  - Anandh Swaminathan
AU  - Richard M. Murray
TI  - Single day construction of multi-gene circuits with 3G assembly
AID  - 10.1101/260851
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 260851
4099  - http://biorxiv.org/content/early/2018/02/10/260851.short
4100  - http://biorxiv.org/content/early/2018/02/10/260851.full
AB  - 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 multi-gene 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.