Large Constructs and Genome Synthesis
DNA2.0 routinely uses our proprietary technologies to produce genes >50kb. The longest gene we have made thus far is >230kb.
- Rapid and cost effective
- Any sequence (specified by customer)
- Ideal for large constructs >10kb
- Convenient construction of expression units, multi-gene pathways, antibody variants, and more
- One step assembly of up to 20 “parts”
- Explore numerous combinations with a relatively small number of reusable parts
Modular part design facilitates combinatorial DNA assembly and allows you to easily explore:
- Gene variants (codon choices, species of origin, mutants, N- and C-terminal fusions)
- Promoters/RBS/Kozak sequences
- Chromosomal integration sites
- Expression plasmids
- Order of genes
DNA2.0′s modular assembly of Ab variants enables you to rapidly explore a large number of constructs with no restrictions on the final sequence.
The heavy chain and light chain are broken up into 3 segments each to isolate the regions for variant synthesis. Modular assembly enables exploration of several variables with a small number of compatible and reusable parts:
- Promoters, polycistrionic expression via 2A splice site or IRES
- Order of heavy and light chain genes
- HC and LC variants
- Vector backbones
- In vivo (recombination based) methods
- In vitro (ligation based) methods
These methods allow DNA2.0 to quickly produce a large number of variants using compatible and reusable parts, and to rapidly and affordably synthesize large constructs.
An antibody therapeutics researcher at a major biotechnology corporation wished to explore a number of Ab constructs with specific alterations. DNA2.0′s modular assembly of Ab variants enabled the researcher to rapidly and affordably test a large number of constructs. Here, DNA2.0 created 15 modular parts, allowing for a total of 72 possible constructs. The HC and LC were each split into two parts (HC.1, HC.2, LC.1, LC.2), in order to minimize the length of the sections which were varied. This same set of parts also allows for swapping of HC and LC regions relative to the IRES, which is not possible with standard synthesis where each variant is synthesized and priced separately. Furthermore, subsequent rounds of assembly have a significantly lower cost, as the individual parts are already synthesized. The customer found 12 first round constructs to be of immediate interest. All 11 desired constructs were synthesized by DNA2.0 in 2 weeks and are currently being functionally validated by the customer.
Twenty-one parts were synthesized to explore several versions of gene 1, gene 2, promoters driving expression, and different chromosomal integration sites. Using the 11 parts shown in the figure below, there are 24 different assemblies (promoter X gene X integration site combinations). Five assemblies were of immediate interest to the customer, a researcher in specialty chemicals. DNA2.0 made all 5 desired ∼15kb assemblies (8-11 parts each, pictured below) in 1 week. All final constructs were successfully integrated in the production host by DNA2.0 and verified by the customer to produce expected compound.
- Synthetic vaccine development
- Metabolic pathway engineering
- Organism engineering
- Full length Virus genome synthesis
J Ind Microbiol Biotechnol 2013. DNA assembly techniques for next‑generation combinatorial biosynthesis of natural products. Cobb et al.
Methods Mol Biol 2012. Building block synthesis using the polymerase chain assembly method. Marchand, Peccoud.
Nucleic Acids Res 2009. DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Shao et al.
Science 2008. Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Gibson et al.
Gene 2000. Chain reaction cloning: a one-step method for directional ligation of multiple DNA fragments. Pachuk et al.