Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering

The BioBrick (TM) 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 (TM) 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 (TM) (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 (TM) vectors with different origins of replication, antibiotic resistances, inducible promoters (arabinose, IPTG or anhydrotetracycline), N- or C-terminal Histidine tags with thrombin cleavage, a LacZ alpha reporter gene and an additional origin of mobility (oriT). All developed BioBricks (TM) and BioBrick (TM) 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.