(1163-D) Automating end-to-end plasmid preparation and functional assays on the Beckman Coulter Biomek i7 Hybrid Workstation

Abstract: Synthetic Biology is a rapidly growing multidisciplinary field of biotechnology which utilizes principles of genetic engineering to modify or create genetic material in different organisms to address challenges in areas of agriculture, medicine, and conservation of the environment. Liquid handling systems minimize human errors, reduce hands-on time, and increase throughput and reproducibility. Molecular cloning is one of the many genetic engineering techniques that greatly benefit from these platforms, which are widely used for high-throughput gene assembly for applications such as genetically modified crops, antibody discovery, proteomics, and oligo/gene synthesis.

In this poster we describe an end-to-end automated workflow; starting with cloning using In-Fusion Snap Assembly Kit from Takara, followed by plasmid purification using the CosMCprep and EMnetik PLP Beads from Beckman Coulter Life Sciences, and finally transfection of plasmids into cells for functional assays on the Biomek i7 Dual Hybrid Workstation.

In-Fusion cloning is simple, scalable, directional, and highly efficient (>95%), making it ideal for high-throughput workflows. Furthermore, this automated In-Fusion Snap Assembly protocol showcases the ability of the Biomek i7 Dual Hybrid Workstation to perform 1 μL transfers using the 1,200 μL Multichannel head. To demonstrate a 96-sample plate setup, we assembled a 3.7 kb insert into a 2.6 kb plasmid and tested two reaction volumes: 10 μL and 5 μL for the Takara In-Fusion Snap Assembly Kit. CosMCPrep kit utilizes bead-based purification which makes it flexible and easy scalable for high-throughput screenings. Here we demonstrate data from a 96-sample automated plasmid purification starting with 2mL of culture. The average yield of the 96 samples was 151 ng/µL with A260/280 of 1.87 and A230/280 of 2.38.

For the automated dual luciferase functional assay, the cell plating, transfection, and luminescence measurements were carried out on the Biomek i7 workstation with a HEPA filter and an integrated SpectraMax i3 X (Molecular Devices) plate reader to measure luminescence. The DLR setup demonstrated here used a firefly luciferase (FLuc) reporter to detect Nuclear Factor Kappa B (NF-κB) signaling in response to Tumor Necrosis Factor α (TNFα). The control luciferase with constant expression used was NanoLuc (Promega). The automated method performed excellently in both 96-well and 384-well format as compared to manual, allowing miniaturization to increase throughput and reduce reagent cost. TNFα EC50 values were 0.4 and 0.3 ng/mL for the automated and manual workflows, respectively. Finally, we show that the automated method developed here was ready to be adapted for high-throughput screening, as Z’ values ranging from 0.70 to 0.80 were observed.

In summary, we show the ability of the Biomek i7 workstation to perform important workflows in synthetic biology along with downstream application for high-throughput screening, thus increasing reproducibility of the assays and reducing user hands-on time and handling errors.