Abstract
As an emerging discipline, synthetic biology is becoming increasingly important to design, construct, and optimize metabolic pathways leading to desired phenotypes such as overproduction of biofuels and pharmaceuticals in genetically tractable organisms. We have recently developed a versatile gene assembly platform ePathBricks supporting the modular assembly of multi-gene pathway components and combinatorial generation of pathway diversities. In this protocol, we will detail the process to assemble a seven gene flavonoid pathway (~9 kb) on one single ePathBrick vector. We will also demonstrate that a three-gene flavonoid pathway can be easily diversified to 54 pathway equivalents differing in pathway configuration and gene order; coupled with high-throughput screening techniques, we envision that this combinatorial strategy would greatly improve our ability to exploit the full potential of microbial cell factories for recombinant metabolite production.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cheng AA, Lu TK (2012) Synthetic biology: an emerging engineering discipline. Annu Rev Biomed Eng 14:155–178
Ellis T, Adie T, Baldwin GS (2011) DNA assembly for synthetic biology: from parts to pathways and beyond. Integr Biol (Camb) 3:109–118
Xu P, Koffas MAG (2010) Metabolic engineering of Escherichia coli for biofuel production. Biofuels 1:493–504
Westfall PJ et al (2012) Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc Natl Acad Sci USA 109:E111–E118
Ajikumar PK et al (2010) Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli. Science 330:70–74
Xu P, Ranganathan S et al (2011) Genome-scale metabolic network modeling results in minimal interventions that cooperatively force carbon flux towards malonyl-CoA. Metab Eng 13:578–587
Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4:251–256
Jeong J-Y et al (2012) One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. Appl Environ Microbiol 78:5440–5443
Gibson DG et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345
Schmid-Burgk JL et al (2012) Rapid hierarchical assembly of medium-size DNA cassettes. Nucleic Acids Res 40:e92
Wang R-Y, Shi Z-Y et al (2012) Cloning large gene clusters from E. coli using in vitro single-strand overlapping annealing. ACS Synth Biol 1:291–295
Shao Z, Zhao H (2009) DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res 37:e16
Wingler LM, Cornish VW (2011) Reiterative recombination for the in vivo assembly of libraries of multigene pathways. Proc Natl Acad Sci USA 108:15135–15140
Gibson DG et al (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329:52–56
Noskov VN et al (2012) Assembly of large, high G + C bacterial DNA fragments in yeast. ACS Synth Biol 1:267–273
Quan J, Tian J (2011) Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nat Protoc 6:242–251
Hillson NJ, Rosengarten RD, Keasling JD (2011) j5 DNA assembly design automation software. ACS Synth Biol 1:14–21
Xu P, Bhan N, Koffas MAG (2013) Engineering plant metabolism into microbes: from systems biology to synthetic biology. Curr Opin Biotechnol 24:291–299. doi:10.1016/j.copbio.2012.08.010
Xu P, Vansiri A et al (2012) ePathBrick: a synthetic biology platform for engineering metabolic pathways in E. coli. ACS Synth Biol 1:256–266
Vick JE et al (2011) Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering. Appl Microbiol Biotechnol 92:1275–1286
Du L, Villarreal S, Forster AC (2012) Multigene expression in vivo: supremacy of large versus small terminators for T7 RNA polymerase. Biotechnol Bioeng 109:1043–1050
Norville JE et al (2010) Introduction of customized inserts for s-treamlined assembly and optimization of BioBrick synthetic genetic circuits. J Biol Eng 4:17
Leonard E, Lim K et al (2007) Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli. Appl Environ Microbiol 73:3877–3886
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Xu, P., Koffas, M.A.G. (2013). Assembly of Multi-gene Pathways and Combinatorial Pathway Libraries Through ePathBrick Vectors. In: Polizzi, K., Kontoravdi, C. (eds) Synthetic Biology. Methods in Molecular Biology, vol 1073. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-625-2_10
Download citation
DOI: https://doi.org/10.1007/978-1-62703-625-2_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-624-5
Online ISBN: 978-1-62703-625-2
eBook Packages: Springer Protocols