"If it ain't broke, don't fix it," goes the old adage, which Rice University professor James Chappell completely ignored in a recent Nature Communications publication . In the study, Chappell describes an innovation in plasmids, circular pieces of DNA that have been a workhorse of molecular biology research since the 1970s.
"For decades, we've been designing experiments around two major limitations of plasmids: fixed copy numbers and incompatibility," said Chappell, the corresponding author on the study. "While functional, such workarounds are clunky. We created a synthetic version of a part of the plasmid called the origin of replication that allows us to modify the plasmid instead of modifying the experiment."
Plasmids are typically put into bacterial cells, where they use the cell's machinery to build proteins and create copies of themselves. Each plasmid generates tiny pieces of a stop signal, called a negative regulator, which binds to the origin of replication (ORI). Once the ORI has bound enough of these stop signals, the plasmid will stop replicating. This allows plasmids to maintain a constant number of copies of themselves in the cell, called a copy number.
The exact number of negative regulators needed to create a full stop varies. Some plasmids need many stop signals and thus will create hundreds of copies of themselves, which results in more protein being made. Others need only a few pieces, creating just a few copies of themselves, which results in less protein being made. Since researchers will often want a specific amount of protein made, copy number is a crucial factor in experimental design.
Equally important is which ORI a plasmid has. There are about 27 different classes of ORIs, and putting two plasmids with the same or similar ORIs into one cell causes protein production to decrease, since both plasmids will create and bind the same negative regulators. This plasmid incompatibility limits researchers to putting only a few plasmids into a single cell at a time.
Chappell's team, working with collaborator Matt Lakin from the University of New Mexico, realized they could address both issues by creating a synthetic version of an ORI that gave the researcher control over copy number and stop signal. In their ORI, one module controls which stop signals the plasmid used and a second module controls how many signals are needed.
"Instead of using the natural stop signals, we used synthetically engineered RNA control elements," said Baiyang Liu '25, first author on this paper and former graduate student. "We have large libraries of unique RNA control elements that can be used in plasmids, meaning that we can potentially put large numbers of plasmids into a cell without incompatibility issues affecting plasmid replication."
Chappell's team tested this by placing six plasmids with synthetic ORIs in a cell. All six plasmids successfully replicated to the copy number set by the synthetic ORI; all successfully expressed their protein product. They also showed that the copy number can be programmed dynamically, with their synthetic ORIs allowing more or less plasmid replication in response to changing conditions in the cell.
"This represents a new, very fine level of control available in a critical molecular biology tool," Chappell said. "The modular design lets each researcher modify the plasmids to fit their experiments, simplifying their workflow and expanding experimental possibilities."
This work was supported by the National Science Foundation (2124306, 2237512, 2124308), the Welch Foundation (A24-0270-001) and the NSF/National Institute of General Medical Sciences Mathematical Biology Program (R01GM144959).
