Synthetic genes designed into a cell’s genetic material that can be induced or suppressed at will have had one major problem: precise control. Where even trace amounts of a protein can mean the difference between life and death, strict, precise, and binary genetic control, just like switching a light on or off, is crucial. There is no room for leaks in the system.

Sustainable and clean production of fuels, chemicals and medicines is increasingly being accomplished through engineering synthetic genes and genetic circuits into microorganisms. This requires precise control of sets of genes.

Bioengineers at the University of Bristol have found a simple solution to the problem of precisely regulating the expression of synthetic inducible genes through harnessing the central dogma at the levels of both RNA and protein synthesis.

These findings are published in the article “Harnessing the central dogma for stringent multi-level control of gene expression” in the journal Nature Communications. The study was funded by the Royal Society, Max Planck Society, European Molecular Biology Organization (EMBO), BBSRC and EPSRC with support from the Bristol BioDesign Institute (BBI).

“Although turning ‘on’ or ‘off’ a gene sounds simple, getting a living cell to do it on command is a real challenge. Every cell is slightly different, and the processes involved are not 100 percent reliable,” says Veronica Greco, PhD student at Bristol’s School of Biological Sciences and lead author on the study.

Nature generally builds in redundancy into all critical bioprocesses. This natural design is messy but it allows biological systems to override errors and survive. The team took inspiration from this basic understanding of nature and systematically studied the combined use of transcriptional (RNA synthesis) and translational (protein synthesis) regulators on protein expression.

“If you look at a Venus flytrap you find that a trap will only close when multiple hairs are triggered together. This helps reduce the chance of a trap closing by accident. We wanted to do something similar when controlling the expression of a gene inside a cell, adding multiple-levels of regulation to ensure it only comes on precisely when we want it to,” says Greco.

“What was wonderful about this project was how well it worked to harness two of the core processes present in every cell and underpinning all of life—transcription and translation,” says Claire Grierson, PhD, professor, and head of the School of Biological Sciences at Bristol, and one of the senior authors on the study.

The evidence reported in this study shows that by multi-level regulation, one could create some of the most precisely regulated switches for gene expression built to date.

In collaboration with Amir Pandi, PhD, and Tobias Erb, PhD, from Bristol’s Max Planck Institute for Terrestrial Microbiology, the team demonstrated that even when used outside living cells (in vitro), these multi-level systems offered stringent control over gene expression.

“When we engineer microbes, we often try to simplify our systems as much as possible, thinking we’ll have better control over what is happening. But what we’ve shown is that embracing some of the inherent complexity of biology might be the key to fully unlocking its potential for the high-precision biotechnologies of tomorrow,” says Thomas Gorochowski, PhD, senior corresponding author on the study and a Royal Society University Research Fellow at Bristol.

Since the first inducible genetic systems designed in the 80’s, control of gene expression in biotechnology has been revolutionized using small molecules (chemogenetics), light (optogenetics) and other signals. Recently, synthetic biologists have developed more advanced methods to control gene expression involving regulators based on DNA binding proteins, such as zinc fingers, TALENs, CRISPRi, RNA–RNA interactions, post-transcriptional/translational processes such as RNA and protein degradation and directed evolution.

Instead of using just one of the available choices for controlling gene expression, this study shows that to strictly regulate gene expression one must couple multiple forms of regulation to reduce unwanted expression and improve the robustness of a system. Few multi-level regulatory systems have been implemented to date. Through embracing this inherent redundancy of nature may be a messy design but it can clarify the nuts and bolts of how stringent multi-level control can be achieved, and the trade-offs that exist between performance, regulatory complexity, and cellular burden.

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