In the field of synthetic biology, researchers aspire to program cells to behave like little robots for sensing environmental toxins, delivering drugs, and more. For such computation, a system needs to perform logic and store data in memory, but most existing cellular genetic circuits can only do one or the other efficiently.
Now, Lu and colleagues have built the first circuits in bacterial cells that combine both basic aspects of computing: logic functions and memory, encoding data permanently in DNA that is passed on through 90 generation of bacteria. The research was published this week in Nature Biotechnology (1).
In 2009, Lu, Boston University synthetic biologist James Collins, and Harvard geneticist George Church designed a synthetic gene circuit in E. coli that could count up to three. Their system utilized a chain of enzymes called recombinases that activated three genes in a row, like a line of dominoes, to count (2).
Using that work as inspiration, Lu designed new circuits that use the recombinases to cut out stretches of DNA, flip them, and insert them back into the genome. In this context, flipping a piece of DNA up or down is like turning it on or off, or a ‘1’ or ‘0’ in binary code. If two bits of DNA bookend a piece of regulatory DNA, such as a promoter that activates a gene, the bits act as a two-input logic gate, detecting multiple inputs and then outputting an answer. In this case, the output would be whether the gene is activated or not. Because the actual DNA of the bacterial genome is stably altered, the input is stored as permanent memory. The team demonstrated that the cells retained the memory for at least 90 generations.
Applying their technique, the team built a system in which recombinases targeted DNA sequences around promoters for a gene controlling green fluorescent protein (GFP) production. They created a total of 16 two-input Boolean logic functions in individual cells. In one instance, for example, the recombinases flip both target DNA sequences “on,” the promoter activates the GFP gene, and the cell expresses GFP and begins to glow. Once the sequences are flipped, they can’t be returned to their original state. So if Lu wanted to read the cell’s history of inputs, he could either measure the cell’s GFP output and deduce the inputs, or sequence the cell’s DNA where the data is stored.
A genetic circuit with logic and memory could be used to track whether disease-related genes are turned on or off in an organism as a way to study disease progression, said Lu. Or in biotechnology, the system could be used to program a cell once to produce a drug at a certain level, eliminating the need for constantly stimulating the cell, and future offspring of the cell would also produce the drug.
Lu is currently working to extend the system to eukaryotes, where he imagines a genetic circuit able to track the complex process of information in stem cells that leads to cell differentiation, then recapitulate that process to produce desired types of cells.
“We want to make differentiation more effective for therapeutic purposes or just to provide this as a tool for biologists to study those pathways,” said Lu.
1. Siuti, P., J. Yazbek, and T.K. Lu. 2013. Synthetic circuits integrating logic and memory in living cells. Nature Biotech, doi:10.1038/nbt.2510.
2. Friedland. A.E, T.K. Lu, X. Wang, D. Shi, G. Church, and J.J. Collins. 2009. Synthetic gene networks that count. Science, 324:1199-1202.