PMID:22033566
| Citation |
Vick, JE, Johnson, ET, Choudhary, S, Bloch, SE, Lopez-Gallego, F, Srivastava, P, Tikh, IB, Wawrzyn, GT and Schmidt-Dannert, C (2011) Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering. Appl. Microbiol. Biotechnol. 92:1275-86 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1007/s00253-011-3633-4 |
| Keywords |
Bioengineering/instrumentation; Bioengineering/methods; Escherichia coli/genetics; Escherichia coli/metabolism; Genetic Vectors/genetics; Genetic Vectors/metabolism; Metabolic Networks and Pathways; Plasmids/genetics; Plasmids/metabolism; Promoter Regions, Genetic |
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