Uniformed gene expression is a critical function for accurate biological studies and imperative for biomanufacturing workflows. And while this process may sound straightforward, it is anything but and has vexed scientists for decades. Yet now, a team of investigators at Baylor College of Medicine and Rice University may have found a solution to this confounding problem through the creation of new genetic circuits, they dubbed Equalizers.
The findings—published recently in Nature Communications through an article titled, “A synthetic circuit for buffering gene dosage variation between individual mammalian cells”—show how researchers engineered these genetic circuits to buffer protein output from variations in the number of copies of the gene inside the cell, thereby helping to create consistent protein expression. A property called gene dosage compensation.
The research team used the analogy of heating a house to help explain how Equalizers work. Imagine using randomly placed space heaters to heat your home. To ensure each room gets a heater, you might purchase some extra ones, but that would mean some rooms could have additional heaters. Those rooms might become too hot, so a solution would be to have thermostats on each heater to downregulate the heat when a space becomes too hot. Those thermostats act as the Equalizer.
Researchers typically encode genes to be expressed on circular pieces of DNA called plasmids. Excess plasmids are often used to ensure that most cells get one, but some cells will get several. The Equalizer is composed of transcriptionally negative feedback and post-transcriptional incoherent feedforward loops. These loops counteract the presence of extra plasmids: they sense the outputs, in this case, the proteins and mRNAs that the plasmids produce, and tune down their expression if they rise too high.
“We didn’t invent the parts, but rather we invented a new way to connect them together into a circuit,” said co-lead study investigator Jin Yang, a doctoral candidate at the Massachusetts Institute of Technology. “In natural systems, some gene networks must control gene dosage variation to remain functional and conserve their properties. We repurposed and combined two types of gene dosage compensation circuits to create a version that enables the uniform expression of any protein scientists to want to produce in the lab.”
Negative feedback and incoherent feedforward circuit subcircuits can each help compensate for gene dosage, but the researchers found that coupling the two improved overall performance. This is because each circuit is not perfect. For example, the incoherent feedforward loop can saturate because it requires other proteins that are present in limited quantities in the cell. The negative feedback loop is limited in its inhibitory capacity, similar to a leaky sink faucet that cannot be fully closed. But combining these two imperfect circuits produced robust performance, with each circuit helping mitigate the limitation of the other.
“The process we used for these findings was a collaborative effort bringing together computer simulations and biology. This is similar to how engineers work—they draw up their plans, create a model, and then build their structure,” explained co-senior study investigator Oleg Igoshin, PhD, a professor of bioengineering, biosciences, and chemistry at Rice University. “In this case, we were able to create our model and show the effectiveness through computational models before it was then synthetically engineered in the lab.”
But why is minimizing expression variation important when it comes to biological research?
“The effect of a protein can depend on its abundance in a cell. If you’re studying a new protein and its concentration is too low, you may not be able to observe its function in the cell,” concluded co-senior study investigator François St-Pierre, PhD, assistant professor of neuroscience and McNair Scholar at Baylor. “If its concentration is too high, the protein may mislocalize, aggregate, produce cytotoxicity, or otherwise produce responses that are not physiological. It is, therefore, important that a protein is expressed at the desired level in every cell under investigation. We believe Equalizers will be of high value both for basic research and for industry.”
