DNA Foundry

The DNA Foundry focuses on advancing strain development for biomanufacturing by integrating automation, modularization, and standardization. Key to our approach is the construction and testing of genetic components, coupled with precise genome engineering. Our methodology uses a diverse array of genetic parts such as gene variants, promoters, terminators, markers and plasmid replication elements to rapidly build and test complex designs to achieve desired phenotypes. Through the implementation of high-throughput plate-based workflows using automation, we streamline microbial genome engineering processes.

The two approaches towards strain improvement include (i) building and testing individual design in a single well and (ii) building and testing designs in a pool as libraries. In the first approach, we develop plate-based protocols for nucleic acid extraction, transformation, DNA assembly, editing, and colony verification as well as implement colorimetric assays for measuring target products for rapid phenotyping. In the second approach, we build libraries and apply selection pressure to enrich variants for desired phenotype. Typically, selection is based on either tolerance to products or increased substrate utilization. Usage of biosensors and Fluorescent Activated Cell Sorting (FACS) further facilitates screening of larger libraries. The variants from the library can be plated as single cells, analysed and sequenced to identify targets or the enriched pools can directly be sequenced to identify hits. The identified hits from single well or library testing is introduced into production strains either as single edits or as multiplexed edits for further testing for improved phenotype.

Our current microbial species of interest consists of Escherichia, Pseudomonas, Cupriavidus, Parageobacillus and Streptomycetes. The selection of these hosts is driven by their diverse capabilities such as thermophilic production, ability to utilize diverse substrates and secondary metabolite production. We work to develop methodologies to make platform strains robust for engineering. This involves expanding our repertoire of genetic parts and strains collection, as well as refining our techniques for transformation and genome editing. The commonly used editing methods include using integrative plasmids with homologous recombination and CRISPR based genome editing. Further, we use technologies developed by former biofoundry team led by Dr. Morten Nørholm including Standardized artificial genome architecture (SAGA) in vivo bacterial plasmid engineering. We combine our build capabilities with other Biofoundry teams to accelerate microbial strain improvement efforts for sustainable biobased production.

Technologies

• Nucleic acid extractions
• Microbial Transformations
• DNA assembly
• Expression parts libraries
• Standardized genome architecture (SEGA) bacterial genome engineering
• Standardized artificial genome architecture (SAGA) in vivo bacterial plasmid engineering
• Classical genome editing using integrative plasmids
• Lambda red recombineering
• CRISPR based genome engineering
• Flow cytometry and FACS
• Nanopore sequencing