Transformation is an essential first step for metabolic engineering, and we have expertise in developing transformation methods for bacterial strains. We can evade bacterial host defenses against foreign DNA via targeted DNA methylation to protect DNA from native restriction systems. We have extensive collections of plasmids that replicate in phylogenetically diverse hosts, which can be introduced into organisms via electroporation or conjugation.  

Effective rational engineering requires an expanded set of parts to control gene expression. We have large collections of genetic parts for phylogenetically diverse bacteria. This includes selectable and counter-selectable markers, plasmid origins of replication, constitutive and regulated promoters, terminators, riboregulators, biosensors, and aerobic and anaerobic reporter genes. These parts can be rapidly prototyped and optimized in diverse new organisms.

We have demonstrated and optimized a number of tools required for efficient genome engineering to enable production and rapid optimization of bioproducts in bacteria. This includes methodologies for targeted and random introduction of heterologous DNA and targeted genome editing using tools such as homologous recombination, recombineering, and CRISPR that enable gene deletion, decreased expression, and overexpression of native and heterologous genes. These tools are connected with other Agile BioFoundry capabilities for analysis of transcripts, proteins, and metabolites which are used to verify the genetic modifications function as desired. We have also developed orthogonal DNA integration tools that use site-specific recombinases, which can enable multiplexed DNA insertion into the chromosome for combinatorial pathway evaluation in the organism of choice.

We develop and adapt high throughput methods for bacterial strain engineering and evaluation. Automation can be applied to bacterial genetic transformation to increase throughput. Transposon mutagenesis and CRISPRi can be applied for genome-scale analysis of genotype-phenotype relationships involved in substrate utilization, product formation, and robustness. Site-specific recombination enables simple, rapid, and high throughput insertion of DNA into the chromosome for rapid evaluation of heterologous DNA at single copy instead of from plasmids. Biosensors combined with fluorescence-activated cell sorting (FACS), especially in combination with other genome-scale methods, allows rapid screening of large libraries of strains and constructs.

SAGE (serine recombinase-assisted genome engineering) is an easy-to-use, highly efficient, and extensible technology that enables selection marker-free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. Learn more about SAGE. 

Randomly barcoded transposon insertion sequencing (RB-TnSeq) is a tool for assigning gene function. Transposons, each containing a random 20 nt barcode sequence, are randomly inserted throughout the genome of a bacterial strain of interest. Next-generation sequencing then identifies the genomic location of each barcode. The library can then be grown under any selection condition of interest, and the abundance of barcodes in the selection condition (e.g., a single carbon source) is compared to the abundance of barcodes in the baseline condition (e.g., rich medium) to assign a “fitness” value to each gene. Genes with large fitness magnitudes play a key role in growth under the selection condition. RB-TnSeq is commonly used for metabolic pathway identification. By identifying genes with strong fitness scores under a certain condition, a user can assign metabolic function to those genes. Learn more about RB-TnSeq and watch a video about this capability.

Transformation is an essential part of biomanufacturing and we have expertise in development and application of electroporation, chemical and Agrobacterium-based transformation methods. Development may also include preparation and stabilization of protoplasts for filamentous fungi and optimizing the various parameters for each transformation method for optimal efficiency. 

Effective rational engineering requires an expanded set of parts to control gene expression. We have developed methods for omics-enabled identification and validation of promoters and terminators to control expression of native and heterologous genes and is developing a set of universal fungal promoters to expedite engineering of non-model fungi. We also have an extensive library of selectable and counter-selectable markers (antibiotic resistance and auxotrophies) to enable marker and marker-free engineering.

We have demonstrated and optimized a number of tools required for efficient genome engineering to enable rapid optimization of fungal strains for producing bioproducts and biofuel precursors. This includes methodologies for targeted and random introduction of heterologous DNA and targeted genome editing using tools such as CRISPR that enable reduction and overexpression of native and heterologous genes. These tools are connected with the Agile BioFoundry Test capabilities for analysis of transcripts, proteins, and metabolites which are used to verify the genetic modifications function as desired and identify additional gene targets for improving the efficiency and productivity of the desired product in the host.

We have expertise in modifying bench-scale protocols for transformation and strain screening to a plate-based format that is compatible with automation platforms to reduce the time and resources required for strain building and functional validation. Examples include, plate-based chemical transformation of yeast, PCR-based methods for rapid validation of genetic modifications and assessment of heterologous gene copy number, plate-based HPLC analysis of products and metabolites, etc.