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Modular library assembly via efficient proprietary technology
100% sequence-verified clones or high-diversity pooled libraries
Metabolic Pathway Engineering is a powerful approach to optimize production of desired biomolecules, but assembling and validating the DNA constructs encoding re-engineered metabolic pathways can be laborious. Our Metabolic Pathway Assembly service allows you to focus on design while we perform the molecular biology. Going beyond our expertise in gene synthesis, we offer customizable services to assemble genetic components into full-length constructs or modular libraries.
Our proprietary Oligo Linker Mediated Assembly (OLMA) technology was developed by, and is exclusively licensed through, our academic research partners Dr. Chunbo Lou and Dr. Yong Tao of The Institute of Microbiology of the Chinese Academy of Sciences (IMCAS). OLMA offer several advantages:
Our in-house scientists have performed numerous case studies with diverse assembly design strategies. We will discuss all parameters of the assembly project design with you to customize each experiment for your needs.
Metabolic Engineering is a powerful approach to optimize genetic circuits, such as biosynthetic pathways that drive "cellular factories" for efficient, industrial-scale production of natural products, such as:
In one example, Brazier-Hicks and Edwards developed a method for efficient production of C-glycosylated flavanoids for dietary studies by using gene synthesis to re-engineer a metabolic circuit in yeast. They designed synthetic variants of five genes that comprise the flavone-C-glycoside pathway in rice plants, which were subsequently codon-optimized for expression in yeast. These synthetic genes were used to construct a polyprotein cassette that expresses the entire metabolic circuit in a single step.
Metabolic Pathway Optimization approaches that can be used alone or in combination include:
Search or browse peer-reviewed publications on metabolic engineering that cite GenScript services & products.
The International Genetically Engineered Machine (iGEM) Competition showcases synthetic biology and metabolic engineering innovations that create new tools for research, healthcare, energy, or environmental applications. In the 2014 Jamboree, GenScript-sponsored teams won accolades for their work to develop novel genetic circuits that turned their genetically engineered bacteria into “cellular factories” to purify water of heavy metals or methane, to keep burn wound sites free of infection, to produce biodegradable elastin polymers that could replace plastics, and to detect pathogenic bacteria. Read more about the projects of GenScript-sponsored iGEM teams from 2009-2014.
To download a printable PDF of this case study, click here.
Introduction: Lycopene, a carotenoid phytochemical best known for its bright red color and anti-oxidant properties, has various biological functions and is widely used in pharmaceutical, food and cosmetic industries. Structurally, it consists of six isopentenyl diphosphate (IPP) and two dimethylallyl diphosphate (DMAPP) molecules. The precursors IPP and DMAPP can be converted to lycopene by co-expressing four exogenous genes in E.coli cells: isopentenyl-diphosphaste delta-isomerase (idi); geranylgeranyl disphosphate (GGPP) synthase (crtE); phytoene synthase (crtB); and phytoene desaturase (crtI).
In this case study, we applied a new technology to perform an all-in-one reaction to assemble multiple variants of each part of the lycopene biosynthetic pathway in many unique combinations. The resulting high-diversity pooled library was then transformed into E. coli hosts and colonies were screened to identify transformants with enhanced lycopene yield. The recombinant genetic circuits that gave rise to the best yield improvements could then be identified through restriction analysis and/or sequencing.
Experimental Design: Four homologs of crtE, crtB, and crtI from Pantoea ananatis, Pantoea agglomerans, Pantoea vagans, and Rhodobacter sphaeroides were cloned into a module plasmid with the same overhang for each gene. idi from E.coli K12 was cloned into the backbone plasmid, and fixed on the last position of the gene circuit. For each gene, 20 ribosome binding sites (RBS), including 10 reverse designed and 10 forward designed RBS, were applied to balance the expression of crtE, crtB, and crtI genes. Each RBS for crtE, crtB, and crtI was synthesized as an oligo linker containing one of three versions of overhangs, in order to enable assembly of the first three genes in any order. An all in-one-reaction was performed to generate a modular metabolic pathway assembly construct library.
Results: The plasmids mixture was transformed into E.coli PXIDF and the transformations were cultured for lycopene production. Lycopene was quantified by measuring OD472 absorption after extracting by ethanol-acetone (V4:1). 1080 red colonies were randomly picked for lycopene production measurement. A wide range of lycopene yield was observed. Simultaneous optimization of RBS, gene order, and homologs using oligo linker mediated assembly enabled the rapid identification of genetic circuits that drive vastly enhanced lycopene production in E. coli.
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