Journal
ACS SYNTHETIC BIOLOGY
Volume 6, Issue 7, Pages 1380-1392Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.7b00016
Keywords
mammalian synthetic biology; mammalian expression vectors; DNA assembly; combinatorial assembly
Categories
Funding
- University of Edinburgh
- SULSA
- Biotechnology and Biological Sciences Research Council (BBSRC) [BB/M025640/1]
- Engineering and Physical Sciences Research Council (EPSRC) [IAA J273I2]
- BBSRC UK mammalian Synthetic biology centre [BB/M018040/1]
- BBSRC [BB/M005690/2, BB/M018040/1, BB/M025640/1, BB/M005690/1] Funding Source: UKRI
- EPSRC [EP/P017401/1] Funding Source: UKRI
- MRC [MR/K017047/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/M005690/1, BBS/OS/GC/000001, BB/M025640/1, BB/M005690/2, BB/M018040/1] Funding Source: researchfish
- Cancer Research UK [17368] Funding Source: researchfish
- Engineering and Physical Sciences Research Council [EP/P017401/1] Funding Source: researchfish
- Medical Research Council [MR/K017047/1] Funding Source: researchfish
- The Brain Tumour Charity [8/105] Funding Source: researchfish
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Mammalian plasmid expression vectors are critical reagents underpinning many facets of research across biology, biomedical research, and the biotechnology industry. Traditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive, and error-prone. New tools and strategies that facilitate the efficient design and production of bespoke vectors would help relieve a current bottleneck for researchers. To address this, we have developed an extensible mammalian modular assembly kit (EMMA). This enables rapid and efficient modular assembly of mammalian expression vectors in a one-tube, one-step golden-gate cloning reaction, using a standardized library of compatible genetic parts. The high modularity, flexibility, and extensibility of EMMA provide a simple method for the production of functionally diverse mammalian expression vectors. We demonstrate the value of this toolkit by constructing and validating a range of representative vectors, such as transient and stable expression vectors (transposon based vectors), targeting vectors, inducible systems, polycistronic expression cassettes, fusion proteins, and fluorescent reporters. The method also supports simple assembly combinatorial libraries and hierarchical assembly for production of larger multigenetic cargos. In summary, EMMA is compatible with automated production, and novel genetic parts can be easily incorporated, providing new opportunities for mammalian synthetic biology.
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