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PASIV: A Pooled Approach-Based Workflow to Overcome Toxicity-Induced Design of Experiments Failures and

Alexis Casas¹, Matthieu Bultelle¹, Charles Motraghi¹

¹Department of Bioengineering, Imperial College London, Exhibition Road, London SW7 2BX, United Kingdom.

ACS Synthetic Biology

|March 9, 2022

Summary

We developed a new workflow called PASIV (pooled approach, screening, identification, and visualization) to overcome challenges in experimental design. This method enables the identification of optimal biological conditions despite toxicity or burden issues.

Keywords:

construct matching design of experiments pooled approach scoping

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Area of Science:

Synthetic biology
Bioinformatics
Metabolic engineering

Background:

Design of experiments (DoE) methodology faces challenges in the scoping phase due to burden and toxicity issues.
These issues can make it impossible to proceed with screening, hindering the identification of optimal biological conditions.

Purpose of the Study:

To introduce PASIV (pooled approach, screening, identification, and visualization), a novel workflow addressing scoping phase limitations in DoE.
To enable the identification of viable regions of interest in biological systems despite toxicity or burden.

Main Methods:

PASIV integrates multiplex construction in a pooled, one-pot reaction approach.
It combines a viability assay with advanced bioinformatics tools, including a novel construct matching tool.
The workflow facilitates the interplay between biological experimentation and software analysis.

Main Results:

PASIV was successfully applied to the lycopene pathway under stressful constitutive expression.
The workflow identified a region of interest characterized by comparatively stronger producers.
This demonstrates the capability of PASIV to navigate complex biological constraints.

Conclusions:

PASIV offers a robust solution for overcoming toxicity and burden issues in the scoping phase of DoE.
The workflow enhances the efficiency and reliability of identifying optimal biological conditions.
PASIV represents a significant advancement in the application of DoE in synthetic biology and metabolic engineering.