Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering.
The BioBrick™ paradigm for the assembly of enzymatic pathways is being adopted and becoming a standard practice in microbial engineering. We present a strategy to adapt the BioBrick™ paradigm to allow the quick assembly of multi-gene pathways into a number of vectors as well as for the quick mobilization of any cloned gene into vectors with different features for gene expression and protein purification. A primary BioBrick™ (BB-eGFP) was developed where the promoter/RBS, multiple cloning sites, optional protein purification affinity tags and reporter gene were all separated into discrete regions by additional restriction enzymes. This primary BB-eGFP then served as the template for additional BioBrick™ vectors with different origins of replication, antibiotic resistances, inducible promoters (arabinose, IPTG or anhydrotetracycline), N- or C-terminal Histidine tags with thrombin cleavage, a LacZα reporter gene and an additional origin of mobility (oriT). All developed BioBricks™ and BioBrick™ compatible vectors were shown to be functional by measuring reporter gene expression. Lastly, a C(30) carotenoid pathway was assembled as a model enzymatic pathway to demonstrate in vivo functionality and compatibility of this engineered vector system.
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, 55108, USA.
Jacob E
JE
Vick
Ethan T
ET
Johnson
Swati
S
Choudhary
Sarah E
SE
Bloch
Fernando
F
Lopez-Gallego
Poonam
P
Srivastava
Ilya B
IB
Tikh
Grayson T
GT
Wawrzyn
Claudia
C
Schmidt-Dannert
D057005:Bioengineering / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic