Researchers created a new technology to gain knowledge of what is happening inside these complex biological systems.
Biotechnology is consistently experiencing newer innovations, and genetically encoded reporter proteins stay at the center of biotechnology research. These proteins allow scientists to track gene expression, understand intracellular processes, and debug engineered genetic circuits.
However, conventional reporting schemes rely on fluorescence and other optical approaches that come with their own limitations and hinders the progress of reporting technologies. Researchers from University of Washington and Microsoft have created a “nanopore-tal” to gain knowledge of what is happening inside these complex biological systems. According to the researchers, this technology allows them to see reporter proteins in a whole new light.
The team of researchers have reported a new kind of protein that can be directly read by a commercially available nanopore sensing device. The newly developed system called “Nanopore-addressable protein Tags Engineered as Reporters” or “NanoporeTERs” can detect multiple protein expression levels from bacterial and human cell cultures effectively as compared to existing techniques.
“NanoporeTERs offer a new and richer lexicon for engineered cells to express themselves and shed new light on the factors they are designed to track. They can tell us a lot more about what is happening in their environment all at once,” said co-lead author Nicolas Cardozo, a doctoral student with the UW Molecular Engineering and Sciences Institute. “We’re essentially making it possible for these cells to ‘talk’ to computers about what’s happening in their surroundings at a new level of detail, scale and efficiency that will enable deeper analysis than what we could do before.”
Reporter proteins are, however, difficult to distinguish between more than three different colors of fluorescent proteins at once. NanoporeTERs are designed to carry distinct protein “barcodes” composed of strings of amino acids that, when used in combination, allow at least ten times more multiplexing possibilities.
The NanoporeTER protein is designed with charged “tails” so that they can be pulled into the nanopore sensors by an electric field. Researchers then apply machine learning models to classify the electrical signals for each NanoporeTER barcode in order to determine each protein’s output levels.
“This is a fundamentally new interface between cells and computers,” said senior author Jeff Nivala, a UW research assistant professor in the Paul G. Allen School of Computer Science & Engineering. “One analogy I like to make is that fluorescent protein reporters are like lighthouses, and NanoporeTERs are like messages in a bottle.”
“Lighthouses are really useful for communicating a physical location, as you can literally see where the signal is coming from, but it’s hard to pack more information into that kind of signal. A message in a bottle, on the other hand, can pack a lot of information into a very small vessel, and you can send many of them off to another location to be read. You might lose sight of the precise physical location where the messages were sent, but for many applications that’s not going to be an issue.”
“We are currently working to scale up the number of NanoporeTERs to hundreds, thousands, maybe even millions more,” said Zhang, who graduated this year from the UW with bachelor’s degrees in both biochemistry and microbiology. “The more we have, the more things we can track.
“We’re particularly excited about the potential in single-cell proteomics, but this could also be a game-changer in terms of our ability to do multiplexed biosensing to diagnose disease and even target therapeutics to specific areas inside the body. And debugging complicated genetic circuit designs would become a whole lot easier and much less time-consuming if we could measure the performance of all the components in parallel instead of by trial and error.”
The synthetic proteins are secreted outside of a cell into the surrounding environment, where researchers can collect and analyze them using a commercially available nanopore array. Researchers use the MinION device here, which relies on barcodes comprising synthetic strands of DNA that could be decoded on demand using the portable reader.
Co-author Kathryn Doroschak, a computational biologist at Adaptive Biotechnologies who completed this work as a doctoral student at the Allen School, says, “This is exciting as a precursor for nanopore technology becoming more accessible and ubiquitous in the future. You can already plug a nanopore device into your cell phone. I could envision someday having a choice of ‘molecular apps’ that will be relatively inexpensive and widely available outside of traditional genomics.”
The research appeared in the journal Nature Biotechnology.