Putting the pieces together: building and validating plasmid libraries

Plasmids are the “building blocks” of biomanufacturing. To express a product, a high-quality plasmid vector must be created. Plasmid DNA is required from initial research and discovery expression trials through continuous, cGMP-grade manufacturing. “Synthetic Biology” is a commonly used term used to refer to rapid engineering of plasmids under a “design, build, test, learn” cycle. These describe how plasmid components are created, built into a library, tested for expression and efficiency with analytical methods, and learnings are then analyzed to go back to the design phase to create better plasmid candidates for bioproduction. Rapid iteration on libraries of putative candidate plasmids allows for quick optimization and identification of the best plasmids to bring forward to development.

While plasmid library development seems straightforward, the sheer volumes of plasmid candidates may present difficulty. Individual plasmids may be created from a set of components, with different promoters to test for optimizing expression, for example. Libraries may extend to thousands of candidates. Distribution of plasmid fragments and ligation reaction reagents can be automated and miniaturized with a Beckman Coulter Life Sciences Echo 525 Acoustic Liquid Handler. Adopting the Beckman Coulter Life Sciences Echo 525 for plasmid library arraying allows for performing gene assemblies up to 93% faster and 80% more efficiently.

When an initial workflow bottleneck is removed, it may lead to bottlenecks downstream. Plasmids are transformed into cell lines, typically Escherichia coli for microbial expression, and screened for critical quality attributes such as sequence and microbial strain viability in culture. After transformation, colonies are plated on selection media that eliminates colonies that did not have plasmid DNA introduced. However, there is a degree of subjectivity involved in colony picking by hand– for example, when using a fluorescent marker, some colonies may fluoresce brighter than others, indicating lower copy number of the plasmid of interest or suboptimal plasmid arraying. Large plasmid libraries require a prohibitive amount of colony picking by hand to screen candidates and continue to evaluation of CQAs. Bringing aMolecular Devices QPix XE Microbial Colony Picker automates this step, removing subjectivity and working with multiple selection markers. Automating this step with a Molecular Devices QPix XE allows researchers to increase efficiency compared to manual methods by 4.75x.

Large plasmid libraries are within reach. Development does not have to be difficult or laborious. Optimizing expression for bio-based manufacturing is key for disrupting traditional chemical synthesis markets, from small molecule medicinal chemistry to making polymers and plastics more sustainable. Contact an expert at the Life Sciences companies of Danaher Corporation to learn more about plasmid library development and scaling up to your throughput needs. Our holistic workflow approach mitigates bottlenecks before they happen and designs solutions to meet your problems head-on.