Rapid Prototyping Platform for Saccharomyces cerevisiae Using Computer-Aided Genetic Design Enabled by Parallel Software and Workcell Platform Development.

Biofoundries have enabled the ability to automate the construction of genetic constructs using computer-aided design. In this study, we have developed the methodology required to abstract and automate the construction of yeast-compatible designs. We demonstrate the use of our in-house software tool, AMOS, to coordinate with design software, JMP, and robotic liquid handling platforms to successfully manage the construction of a library of 88 yeast expression plasmids. In this proof-of-principle study, we used three fluorescent genes as proxy for three enzyme coding sequences. Our platform has been designed to quickly iterate around a design cycle of four protein coding sequences per plasmid, with larger numbers possible with multiplexed genome integrations in Saccharomyces cerevisiae. This work highlights how developing scalable new biotechnology applications requires a close integration between software development, liquid handling robotics, and protocol development.

Delivery of Formulated Industrial Enzymes with Acoustic Technology.

Industrial enzymes are instrumental in many applications, including carbohydrate processing, fabric and household care, biofuels, food, and animal nutrition, among others. Enzymes have to be active and stable not only in harsh application conditions, but also during shipment and storage. In protein stability studies, formulated concentrated enzyme solutions are frequently diluted gravimetrically prior to enzyme activity measurements, making it challenging to move toward more high-throughput techniques using conventional robotic equipment. Current assay methods pose difficulties when measuring highly concentrated proteins. For example, plastic pipette tips can introduce error because proteins adsorb to the tip surface, despite the presence of detergents, decreasing precision and overall efficiency of protein activity assays. Acoustic liquid handling technology, frequently used for various dilute small-molecule assays, may overcome such problems. Originally shown to effectively deliver dilute solutions of small molecules, this technology is used here as an effective alternative to the aforementioned challenge with viscous concentrated protein solutions. Because the acoustic liquid handler transfers nanoliter quantities of liquids without using pipette tips and without sample loss, it rapidly and uniformly prepares assay plates for enzyme activity measurements within minutes. This increased efficiency transforms the nature of enzyme stability studies toward high precision and throughput.

Acoustic Liquid Handling for Rapid siRNA Transfection Optimization.

Gene knockdown by small interfering RNA (siRNA) has been used extensively to investigate the function of genes in targeted and genome-wide studies. One of the primary challenges of siRNA studies of any scale is to achieve sufficient gene knockdown to produce the biological changes that lead to measurable phenotypes. Reverse, lipid-based transfection efficiency minimally requires the optimization of the following parameters: cell number, knockdown duration, siRNA oligonucleotide concentration, type/brand of transfection lipid, and transfection lipid concentration. In this study, we describe a methodology to utilize the flexibility and low-volume range of the Echo acoustic liquid handler to rapidly screen a matrix of transfection conditions. The matrix includes six different transfection lipids from three separate vendors across a broad range of concentrations. Our results validate acoustic liquid transfer for the delivery of siRNAs and transfection reagents. Finally, this methodology is applied to rapidly optimize transfection conditions across many tissue culture cell lines derived from various originating tissues.