Liquid Handling Automation Enabling Computer-Aided Genetic Design

Written by Bioscribe on October 04, 2018

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Developing scalable new biotechnology applications requires a close integration between software development, liquid handling robotics, and protocol development.

At the London DNA Foundry, scientists are keen to optimize the construction of genetic constructs using robotic automation and computer-aided design.

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The Foundry, which is affiliated with Imperial College London, enables synthetic biologists and metabolic engineers from academia and industry to easily build designed DNA constructs from standardized constituent parts. Its processes were first optimized to work with Escherichia coli, but this can also be used as a workhorse to produce larger pieces of DNA for engineering Saccharomyces cerevisiae.

In a paper published recently in SLAS Technology, David McClymont, Priscilla Rajakumar and colleagues describe a methodology they developed to abstract and automate the construction of yeast-compatible designs.

The Labcyte Echo 550 and Echo 525 Liquid Handlers were used alongside an in-house software tool, AMOS, and design software, JMP, to successfully manage the construction of a library of yeast expression plasmids. The design-build-test platform allows rapid iteration through design cycles using the leading JMP design and statistical analysis tools, making complex genetic engineering applications quick and easy for scientists to understand and execute.

“The automated design, execution and analysis platform of complex experiments also builds a framework for future ‘closed loop’ applications,” McClymont said.

As the authors explain, the yeast assembly process using kits like the MoClo (Golden Gate)—Yeast Toolkit (YTK), with its set of standardized and well-characterized promoters, terminators, coding sequences, assembly connectors, E. coli markers, yeast markers and origins of replication, involves two tiers of DNA assembly, with different type II restriction enzymes and two rounds of amplification in E. coli prior to transformation of the DNA constructs into S. cerevisiae.

The first-tier assembly is made up of single-transcription unit constructs containing a promoter, coding sequence, and terminator, called “clips.” These are then amplified in E. coli and subjected to a restriction digest-based quality control (QC) screen. These clips are combined into the desired combinations into a yeast expression plasmid to form a “stitch” that, after amplification in E. coli, is subjected to further QC. Finally, these stitches are transformed into yeast and characterized.

Such a process is difficult to scale by hand.

“Producing just one 384-well plate of DNA assemblies might require multiple thousands of sub-microlitre liquid transfers, with each well possibly requiring different combinations of parts,” McClymont said.

One mistake can also lead to large numbers of downstream failures, limiting scale.

So the team developed the AMOS Laboratory Information Management System platform to translate a design into a set of physical parts and picklists with well locations and barcodes, while scaling correctly for larger processes within the precise capabilities of the lab’s robotic systems, including the Labcyte Access.

"The platform was 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," the authors write. "By bringing together assay development, automation platforms, and software expertise around a specific problem, we can build new capabilities such as more rapid design–build–test cycles or larger, more high-throughput applications using YTK-formatted DNA parts."

For a more detailed description of the protocol, read the full paper.

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