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CryptKeeper: a negative design tool for reducing unintentional gene expression in bacteria.

Cameron T Roots¹, Jeffrey E Barrick¹

¹Department of Molecular Biosciences, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX 78712, USA.

Synthetic Biology (Oxford, England)

|December 26, 2024

Summary

CryptKeeper software predicts unintended gene expression from synthetic DNA sequences in Escherichia coli. This tool helps researchers avoid genetic instability and experimental failures in molecular biology and synthetic biology applications.

Keywords:

computational DNA sequence design design-build-test cycle plasmid instability recombinant protein overexpression reliability and reproducibility

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Area of Science:

Molecular Biology
Synthetic Biology
Bioinformatics

Background:

Introducing synthetic DNA is crucial for molecular biology techniques like gene cloning and protein expression.
Unintended recognition of synthetic DNA by host machinery can lead to cryptic gene expression.
Cryptic gene expression causes genetic instability and masks experimental results, complicating research.

Purpose of the Study:

To introduce CryptKeeper, a computational pipeline for visualizing and analyzing cryptic gene expression signals in *Escherichia coli*.
To estimate the translational burden imposed by synthetic DNA sequences.
To provide a tool for negative design in synthetic biology and reverse genetics workflows.

Main Methods:

Development of the CryptKeeper software pipeline.
Visualization of predicted *Escherichia coli* gene expression signals.
Estimation of translational burden from DNA sequences.
Analysis of published experimental data where cryptic expression interfered.

Main Results:

CryptKeeper accurately predicts unintended gene expression from eukaryotic virus clones and individual proteins, correlating with observed genetic instability.
The software identifies off-target expression elements causing protein truncations that hindered purification.
CryptKeeper successfully postdicts cryptic expression events in previously published studies.

Conclusions:

CryptKeeper aids researchers in identifying and mitigating challenges arising from unintentional gene expression in synthetic DNA constructs.
Integrating CryptKeeper into experimental design can prevent cloning difficulties and experimental failures.
The tool enhances the reliability of reverse genetics and synthetic biology endeavors by enabling negative design.