Large Constructs and Genome Synthesis

ATUM routinely uses our proprietary technologies to produce genes >50kb. The longest gene we have made thus far is >230kb.

 

Advantages

  • 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 Constructs 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


Application: Antibody Fragment (Fab) Variants

ATUM’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


Methodology

ATUM 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 ATUM to quickly produce a large number of variants using compatible and reusable parts, and to rapidly and affordably synthesize large constructs.

Large Constructs Assembly Overview Image

Case Study: Modular Assembly of Antibody Variants

An antibody therapeutics researcher at a major biotechnology corporation wished to explore a number of Ab constructs with specific alterations. ATUM’s modular assembly of Ab variants enabled the researcher to rapidly and affordably test a large number of constructs. Here, ATUM 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 ATUM in 2 weeks and are currently being functionally validated by the customer.

Antibody Variant Case Study Image

Case Study: Chromosomal Integration

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. ATUM 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 ATUM and verified by the customer to produce expected compound.

Large Constructs Assembly 24 Combinations Image

Learn More

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