By seeding sheets of what look like paper with encrypted patterns of bacteria engineered to glow in certain conditions, researchers have developed an invisible ink for the biotech age.

Among the potential uses are secret, forgery-resistant bacterial barcodes and watermarks, though imagination soon arrives at more entertaining possibilities.

“Obviously, the secret agent kind of application jumps out,” said chemist David Walt of Tufts University, who developed the system with fellow Tufts chemist Manuel Palacios. “Somebody embedded in an environment where they need to get a message out but don’t want to be caught.”

The system, which Walt and Palacios named InfoBiology — individual messages are called SPAM, short for “Steganography by Printed Arrays of Microbes” — is described Sept. 26 in Proceedings of the National Academy of Sciences. It builds on principles displayed in Walt’s earlier work on fuses that convey information as they burn, producing a simple form of chemistry-based communication.

The code used by Walt

“We were sitting around the lab, thinking how we could do the same thing with biology,” said Walt. “We were familiar with work done earlier, where people put codes in DNA. You can synthesize DNA in codes where letters correspond to different combinations of bases, then sequence it out and read the code. But that requires some pretty sophisticated instrumentation. We thought about doing this with a real simple readout: color. That’s when the idea of using fluorescent proteins came up.”

Fluorescent proteins, which glow under ultraviolet light and are produced when a selected gene becomes active, are ubiquitous in genetic research, where they’re used to monitor gene activity.

Walt’s team designed seven strains of E. coli, each a different color. For that bacterial library of seven characters, a simple cipher was generated: One green unit and one orange, for example, would equal an “I,” while a red and green together meant “S.”

The researchers then seeded their bacteria in coded dot patterns on a plate of agar. Once the bacteria grew, the researchers pressed a nutrient-rich nitrocellulose sheet onto the plate, thus seeding it with the same pattern of bacteria.

“Once you put bacteria on these nitrocellulose membranes, which really look like paper, you end up with a very stable message,” Walt said.

When the sheet is pressed against a fresh plate of agar, the bacteria transfer again. Viewed under a fluorescent light, the plate can be decoded by anyone with the cipher. Additional levels of security can be added by targeting fluorescence promoters to genes of particular purpose.

Walt’s group added fluorescence to antibiotic resistance genes, so the message only became apparent when the agar plate was dosed with ampicillin. Most any gene involved in responding to a stimuli — extreme cold or heat, for example, or other nutrients and compounds — could be used the same way, said Walt. It would also be possible to use E. coli engineered to lose their fluorescence properties over time.

“These mutants would add an inherent security measure by self-deleting the message as it develops,” wrote Walt’s team, “similar to the way the Mission Impossible recording self-destructed.

A step-by-step schematic of the InfoBiology process. Image: Walt et al./PNAS

Image: A fluorescing SPAM array. (Manuel Palacios)

Citation: “InfoBiology by printed arrays of microorganism colonies for timed and on-demand release of messages.” By Manuel A. Palacios, Elena Benito-Peña, Mael Manesse, Aaron D. Mazzeo, Christopher N. LaFratta, George M. Whitesides, and David R. Walt. Proceedings of the National Academy of Sciences, Vol. 108 No. 39, Sept. 27, 2011.

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