Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering

Jacob E. Vick, Ethan T. Johnson, Swati Choudhary, Sarah E. Bloch, Fernando Lopez-Gallego, Poonam Srivastava, Ilya B. Tikh, Grayson T. Wawrzyn, Claudia Schmidt-Dannert

Research output: Contribution to journalArticlepeer-review

45 Scopus citations

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 C30 carotenoid pathway was assembled as a model enzymatic pathway to demonstrate in vivo functionality and compatibility of this engineered vector system.

Original languageEnglish (US)
Pages (from-to)1275-1286
Number of pages12
JournalApplied Microbiology and Biotechnology
Volume92
Issue number6
DOIs
StatePublished - Dec 1 2011

Keywords

  • BioBrick™
  • Carotenoid
  • Escherichia coli
  • Metabolic engineering
  • Pathway engineering
  • Synthetic biology
  • Vector system

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