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Appl Microbiol Biotechnol. 2011 Dec;92(6):1275-86. doi: 10.1007/s00253-011-3633-4. Epub 2011 Oct 28.

Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering.

Author information

1
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, 55108, USA.

Abstract

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.

PMID:
22033566
DOI:
10.1007/s00253-011-3633-4
[Indexed for MEDLINE]

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