Large Constructs and Genome Synthesis

Longest Gene is 230KbDNA2.0 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
Large Construct Assembly 1000 Image

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.

Large Construct Assembly_15000 Constructs Image

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

DNA2.0 offers multiple methods for DNA assembly to maximize success with ANY sequence.
  • 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.

Large Construct Assembly Overview Image
Modular Assembly of Antibody Variants for an Antibody Therapeutics Researcher

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.

Antibody Variant Case Study Image
Chromosomal integration of 2 genes for chemical product biosynthesis.

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.

Large Construct Assembly 24 Combinations Image

GenomeGPS™ Applications:

  • Synthetic vaccine development
  • Metabolic pathway engineering
  • Organism engineering
  • Full length Virus genome synthesis
The rapid expansion of synthetic biology demands increased efficiency and effectiveness in gene synthesis. In particular, pathway and organism engineering require gene synthesis lengths of 100kb or more. An excellent example is the publication of the first synthetic life produced with whole genome synthesis of Mycoplasma capricolum (106 bp).

Figure 1: Completely synthetic 143,659 bp construct digested with BstBI after successful assembly.
Ladders are NEB Lambda DNA-Mono Cut mix and NEB 1kb DNA ladder. Expected sizes are 28015, 30484, 5323, 27476, 28127, 14632, 9602 bp.

Figure 2: Completely synthetic 83,263 bp gene digested with SwaI after successful assembly.
Ladders are NEB Lambda DNA-Mono Cut mix and NEB 1kb DNA ladder. Expected sizes are 4769, 9754, 13413, 18091, 3745, 5031, 26874bp.

The GenomeGPS bioengineering technology developed by DNA2.0 can be used to develop completely novel genomes or to optimize pathways. GenomeGPS builds on DNA2.0’s other GPS systems to explore higher order combinations of multiple genes into functionally improved metabolic pathways. Our capability for high-capacity gene synthesis enables synthesis of multi-component, multi-gene pathways up to several hundred kilobases in size.

Learn More

Consult with a DNA Assembly/GenomeGPS Specialist today at +1 877 362 8646 or info@DNA20.com to achieve your research goals quickly and affordably.