Plasmid design and construction are important for quickly narrowing plasmid candidates to achieve a master cell bank (MCB). To ensure quality, plasmids must go through initial screens before scaling up production. After plasmids are built, they are tested, screened and selected based on functional and product quality parameters.

Constructing diverse plasmid libraries is challenging, and evaluating them introduces new complexities. The selection of analytical methods at this stage of post-transformation into microbial cells and later transfection into mammalian or insect cell lines is crucial. Analytical method selection at this step lays the groundwork for routine quality control procedures later in drug development.

• Plasmid selection - Determining which plasmids to add to the cell bank often involves identifying the best plasmid out of thousands.
• Validation methods - Robust and high-quality methods are essential for defining and determining the best candidates.
• Initial screening - Researchers usually focus on initial screenings, such as verifying plasmid sequence with NGS and measuring product sizes, to evaluate many products quickly.

A key consideration for researchers is evaluating the maximum number of candidates without compromising precision and quality. Time-saving measures can include using tools capable of rapidly validating products in a high-throughput fashion. Using an automated liquid handler like the Beckman Coulter Life Sciences Biomek i-Series Workstation to perform plasmid extraction  with Beckman Coulter Life Sciences CosMCPrep plasmid DNA extraction kits can significantly increase throughput and improve the consistency of NGS library preparation.

Beyond assessing the plasmid sequences, it's crucial to evaluate plasmid components and how the microbial host maintains the plasmid in culture. If E. coli strains do not grow robustly, or appropriate fermentation conditions cannot be quickly found, strains may be cut from the master cell bank. This may be because of poor plasmid component selection or an inducible plasmid toxic to the host cell. Carrying out fermentation trials using traditional bioreactors is low-throughput and resource-intensive, making strain optimization challenging and expensive.

Using a microbioreactor for early fermentation trials and experiments can aid in evaluating plasmid-carrying strains and identify optimal culture scale-up conditions. The Beckman Coulter Life Sciences BioLector XT Microbioreactor is a small-scale, automated microbioreactor system that enables:

• High-throughput and real-time screening of strains
• Monitoring of cultivation parameters
• Feeding strategy optimization

The BioLector XT presents opportunities for efficient mass spectrometry analysis of culture media to analyze metabolic conditions. Recent work using the SCEX Echo MS System analyzed 60 metabolites from microbial strains, finding:

• Analysis time was 5.6x quicker than conventional LC-MS methods
• No compromise in reproducibility or sensitivity¹

Tools like the Echo MS and liquid handling systems make assessing large plasmid libraries easier and faster than conventional methods. In a separate study, the SCIEX Echo MS system:

• Quantified 10 unique metabolites directly from fermentation broth
• Completed the experiment in 20 minutes, compared to ~ 32 hours with a conventional LC-MS/MS method²

Identifying top plasmid candidates from large libraries can be challenging. In addition to analyzing the plasmid itself using methods like NGS, strain viability, plasmid component viability and ideal culturing conditions need to be determined. The Life Sciences companies of Danaher offer advanced tools to streamline and improve this assessment, accelerating early-stage research with greater precision and quality.

Contact our experts  today to learn more about testing plasmid designs or explore ways of partnering with us.

References

1. High-throughput metabolite quantification for synthetic biology. https://sciex.com/tech-notes/life-science-research/metabolomics/high-throughput-metabolite-quantification-for-synthetic-biology ?sfdcname=Global_EchoMS_ProductPage_TechNotes_2021&utm_term=G-Echo%20MS%20Product%20Page%20-%20Metabolite%20Synthetic%20Biology
2. Accelerating synthetic biology with very fast screening of metabolites in fermentation broth. https://sciex.com/tech-notes/life-science-research/metabolomics/accelerating-synthetic-biology-with-very-fast-screening-of-metab