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

bioRxiv

Department of Molecular Biosciences, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas 78712, U.S.A.

Published: September 2024

Foundational techniques in molecular biology-such as cloning genes, tagging biomolecules for purification or identification, and overexpressing recombinant proteins-rely on introducing non-native or synthetic DNA sequences into organisms. These sequences may be recognized by the transcription and translation machinery in their new context in unintended ways. The cryptic gene expression that sometimes results has been shown to produce genetic instability and mask experimental signals. Computational tools have been developed to predict individual types of gene expression elements, but it can be difficult for researchers to contextualize their collective output. Here, we introduce CryptKeeper, a software pipeline that visualizes predictions of bacterial gene expression signals and estimates the translational burden possible from a DNA sequence. We investigate several published examples where cryptic gene expression in interfered with experiments. CryptKeeper accurately postdicts unwanted gene expression from both eukaryotic virus infectious clones and individual proteins that led to genetic instability. It also identifies off-target gene expression elements that resulted in truncations that confounded protein purification. Incorporating negative design using CryptKeeper into reverse genetics and synthetic biology workflows can help to mitigate cloning challenges and avoid unexplained failures and complications that arise from unintentional gene expression.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11398486PMC
http://dx.doi.org/10.1101/2024.09.05.611466DOI Listing

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