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

doi:10.1007/s00253-011-3633-4

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

Biochemistry, Molecular Biology, and Biophysics (CBS)

Research output: Contribution to journal › Article › peer-review

51 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 language	English (US)
Pages (from-to)	1275-1286
Number of pages	12
Journal	Applied Microbiology and Biotechnology
Volume	92
Issue number	6
DOIs	https://doi.org/10.1007/s00253-011-3633-4
State	Published - Dec 2011

Bibliographical note

Funding Information:
Acknowledgements This manuscript is dedicated to the memory of Dr. Ethan Thoreau Johnson, our dear friend, colleague and manuscript co-author, who died due to injuries suffered in a hit-and-run car accident on September 21st of 2010. The authors acknowledge support from the National Science Foundation (Grant CBET-0756296), the National Institute of Health (Grant GM080299), the Office of Naval Research (Grant N00014-10-1-0157) and the Institute for Renewable Energy and Environment (University of Minnesota). We thank Ana Correa, Heather Kokesh, Andrew Le for assistance in plasmid construction and testing.

Keywords

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

Access

10.1007/s00253-011-3633-4

OpenUrl availability

Full text

Cite this

@article{8b3f7f1502434e41a9e6feefc08c7955,

title = "Optimized compatible set of BioBrick{\texttrademark} vectors for metabolic pathway engineering",

abstract = "The BioBrick{\texttrademark} 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{\texttrademark} 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{\texttrademark} (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{\texttrademark} 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{\texttrademark} and BioBrick{\texttrademark} 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.",

keywords = "BioBrick{\texttrademark}, Carotenoid, Escherichia coli, Metabolic engineering, Pathway engineering, Synthetic biology, Vector system",

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

note = "Funding Information: Acknowledgements This manuscript is dedicated to the memory of Dr. Ethan Thoreau Johnson, our dear friend, colleague and manuscript co-author, who died due to injuries suffered in a hit-and-run car accident on September 21st of 2010. The authors acknowledge support from the National Science Foundation (Grant CBET-0756296), the National Institute of Health (Grant GM080299), the Office of Naval Research (Grant N00014-10-1-0157) and the Institute for Renewable Energy and Environment (University of Minnesota). We thank Ana Correa, Heather Kokesh, Andrew Le for assistance in plasmid construction and testing.",

year = "2011",

month = dec,

doi = "10.1007/s00253-011-3633-4",

language = "English (US)",

volume = "92",

pages = "1275--1286",

journal = "Applied Microbiology and Biotechnology",

issn = "0175-7598",

publisher = "Springer Verlag",

number = "6",

}

TY - JOUR

T1 - Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering

AU - Vick, Jacob E.

AU - Johnson, Ethan T.

AU - Choudhary, Swati

AU - Bloch, Sarah E.

AU - Lopez-Gallego, Fernando

AU - Srivastava, Poonam

AU - Tikh, Ilya B.

AU - Wawrzyn, Grayson T.

AU - Schmidt-Dannert, Claudia

N1 - Funding Information: Acknowledgements This manuscript is dedicated to the memory of Dr. Ethan Thoreau Johnson, our dear friend, colleague and manuscript co-author, who died due to injuries suffered in a hit-and-run car accident on September 21st of 2010. The authors acknowledge support from the National Science Foundation (Grant CBET-0756296), the National Institute of Health (Grant GM080299), the Office of Naval Research (Grant N00014-10-1-0157) and the Institute for Renewable Energy and Environment (University of Minnesota). We thank Ana Correa, Heather Kokesh, Andrew Le for assistance in plasmid construction and testing.

PY - 2011/12

Y1 - 2011/12

N2 - 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.

AB - 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.

KW - BioBrick™

KW - Carotenoid

KW - Escherichia coli

KW - Metabolic engineering

KW - Pathway engineering

KW - Synthetic biology

KW - Vector system

UR - http://www.scopus.com/inward/record.url?scp=82455208881&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=82455208881&partnerID=8YFLogxK

U2 - 10.1007/s00253-011-3633-4

DO - 10.1007/s00253-011-3633-4

M3 - Article

C2 - 22033566

AN - SCOPUS:82455208881

SN - 0175-7598

VL - 92

SP - 1275

EP - 1286

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

IS - 6

ER -

Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering

Abstract

Bibliographical note

Keywords

Access

OpenUrl availability

Other files and links

Fingerprint

Cite this