Available methods for assembling expression cassettes for synthetic biology.
Studies in the structural biology of the multicomponent protein complex, metabolic engineering, and synthetic biology frequently rely on the efficient over-expression of these subunits or enzymes in the same cell. As a first step, constructing the multiple expression cassettes will be a complicated and time-consuming job if the classic and conventional digestion and ligation based cloning method is used. Some more efficient methods have been developed, including (1) the employment of a multiple compatible plasmid expression system, (2) the rare-cutter-based design of vectors, (3) in vitro recombination (sequence and ligation independent cloning, the isothermally enzymatic assembly of DNA molecules in a single reaction), and (4) in vivo recombination using recombination-efficient yeast (in vivo assembly of overlapping fragments, reiterative recombination for the chromosome integration of foreign expression cassettes). In this review, we systematically introduce these available methods.
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
Tianwen
T
Wang
Xingyuan
X
Ma
Hu
H
Zhu
Aitao
A
Li
Guocheng
G
Du
Jian
J
Chen
D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011995:Recombination, Genetic; D058615:Synthetic Biology / Q000379:methods*; D015003:Yeasts / Q000235:genetics