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Combinatorial DNA libraries are powerful, high-throughput tools used in a variety of applications, including metabolic pathway and microbial strain engineering for optimizing biological systems and producing high-value chemicals, biofuels, pharmaceuticals, and sustainable food. Combinatorial DNA libraries, or combinatorial assembly libraries, are made as a set of predefined DNA parts strategically assembled in a specific arrangement.
Built on our advanced, high-throughput platforms and world-renowned expertise in gene synthesis, GenScript offers comprehensive combinatorial DNA library services to accelerate the build phase of your “design, build, exam & learn” development cycle so you can efficiently move through the process of optimizing or developing bio-based products.
You can use our custom-built combinatorial DNA libraries in a variety of applications across many disciplines of life sciences, from plant biology to drug development. The following lists three major areas of combinatorial DNA library applications:
Over the last several years, GenScript has partnered with researchers in both industry and academia to build sophisticated combinatorial DNA libraries. These highly-customized libraries were used to develop new drugs, enzymes, or to discover novel gene functions and networks. The following is one example of such successful partnerships:
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.
To download a printable PDF of this case study, click here.
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