Professor, University of Minnesota
First developed in the 1960s to understand the process of protein synthesis in living organisms, DNA-dependent cell-free expression became a research tool to analyze gene products and to unravel the regulation of natural genetic elements. Cell-free transcription-translation (TXTL) systems, optimized for large-scale protein synthesis as an alternative to the recombinant protein technology, are now used in an increasing number of applications in biotechnology, industry and proteomics.
With the emergence of synthetic biology, a new generation of cell-free systems has been engineered, and University of Minnesota Professor Vincent Noireaux has been at the forefront. While a postdoc at Rockefeller University, Noireaux demonstrated that elementary gene networks could be executed in commercially available TXTL systems. At the University of Minnesota, the Noireaux Lab developed a novel all E. coli TXTL platform for cell-free synthetic biology applications. It has since been used to study biological network prototyping, biosynthesis, and functional membrane protein production; elementary gene circuits, pattern formation, and prototypes of artificial cells have also been engineered with his system.
“The construction of biological systems in test tubes using DNA programs provides a means to study biochemical processes in isolation, with a greater level of control and a greater freedom of design compared to in vivo,” Noireaux explains.
In vivo assays often take days to weeks to run due to cell transformation and cultures. With TXTL, gene circuits can be prototyped in a matter of hours. However, the throughput capacity and cost per reaction remains a bottleneck. In addition, TXTL reactions are complex, being comprised of a dissimilar collection of reaction components, including salts, energy buffers, DNA, and cofactors. These numerous component variations make it impractical to screen TXTL reactions manually.
“The sheer number of potential reactions makes effective implementation and efficient use of reagents critical, while maintaining reproducibility and achieving high-throughput capability,” Noireaux says.
Introduced to the Echo® Liquid Handler during a summer course at Cold Spring Harbor Laboratory, Noireaux was immediately smitten.
“It was exactly the machine I needed. It allows my lab to change the biochemical solutions in a very fine manner, and to miniaturize the volume of TXTL reactions, significantly reducing the use of precious reagents, and thus the cost per reaction,” Noireaux says. “More reaction conditions can be assembled at lower volumes than possible manually, with equivalent or superior results. It improves TXTL reaction assembly considerably.”
The Echo® 550 Liquid Handler has changed the way Noireaux’s small lab operates, he says. Output has increased 5–10 fold, while manual work has decreased dramatically.
“Without exaggerating, for such small labs, the Echo system is a lab saver,” Noireaux says.
It has also made it much easier to host undergraduates in the lab.
“Education is a very important aspect for me,” Noireaux says. “Students learn how to use the Echo system — it’s so easy, with no pipetting required — and for example I’ve seen an undergraduate do a few 1,000 reactions per week. By hand, this would have taken months.”
“If you want to accelerate your work, if you want to stay competitive, if you want to do highly reproducible, high-throughput science, you need an Echo liquid handling machine, especially for labs working with TXTL systems,” Noireaux adds. “It’s really by far the machine that people need when they want to do cell-free work.”
Noireaux is exploring additional applications of his system, including CRISPR technologies. In a recent paper in Cell Press, in collaboration with the Chase Beisel lab at North Carolina State University, he describes how his E. coli TXTL system can be used to vastly improve the speed and scalability of CRISPR characterization and validation.
“We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases,” he writes.