The Name is Coli. Escherichia coli.

Fabian Ortega | fabian.ortega@yale.edu March 14, 2012

Colored E. Coli grows on plates before being transferred to a membrane. Photo courtesy of Manuel Palacios.

We have witnessed the use of invisible ink in our favorite Mission: Impossible and James Bond films – now, scientists have discovered a cutting-edge method to encode secret messages with a simple yet brilliant molecular biology technique. Recently, scientists have been able encode messages in molecules, such as DNA and protein, and now, they have found a way to use bacteria. In June 2011, Manuel Palacios, a chemist working at Tufts University, published a study that explained Steganography by Printed Arrays of Microbes (SPAM), the novel technique that allows one to encode messages in the bacterium Escherichia coli.

Escherichia coli (E. coli) is a Gram-negative bacterium particularly useful in molecular biology. In a technique known as transformation, a scientist can introduce foreign DNA into E. coli cells. This DNA molecule allows the bacterium to synthesize specific proteins. E. coli’s potent genetic machinery has been exploited by industrial microbiology to mass-produce useful peptides such as insulin, human growth hormone, and blood clotting factors, among many others.

With this ideal model organism, Palacios began his experiment by transforming E. coli with DNA that codes for fluorescent proteins. This means that after the E. coli take up the foreign DNA, they begin synthesizing the appropriate proteins. Now to visualize these proteins, one only needs to shine the E. coli cells with a light-emitting diode, which would cause these bacteria to glow. Such diodes are actually readily accessible and available in everyday items, such as in a simple iPhone app. In his experiment, Palacios used many colors, amongst them cyan, green, yellow, orange, tomato, and cherry. After transforming the bacteria, he transferred them to a membrane, which can then be mailed.

However, how does one read the message? The array of colorful dots is based on a dot-only binary Morse code, in which two dots represent an alphanumeric symbol: A-Z and 0-9. For example, an orange dot followed by a cyan dot represent the letter a, and so on, creating a system that resembles a decoder ring. Even though the process of creating and reading the message may seem straightforward, Palacios designed a system with several layers of defense: the message needs to be “developed” under specific conditions. To this end, E. coli cells are especially useful communication tools because they create messages that are both time-sensitive and environment-sensitive.

When the E. coli are mailed, it takes about 24 hours for them to be able to glow, which is the time it takes for the bacteria to synthesize the fluorescent proteins. Thus, the real message may only appear after a period of 24 hours. After this time period, the message will be incomprehensible because the colonies have started to die from the lack of nutrients. Like any living organism, E. coli requires a food source to produce energy for survival. In other words, this is a self-destructing message, very à la 007.

When an E. coli message is illuminated by a diode, the various strains of bacteria glow in a pattern that can encode information. Photo courtesy of Manuel Palacios.

Finally, a person may only be able to read the message if the bacteria are exposed to a specific antibiotic medium, such as ampicillin. Similar to the time-sensitive limitations, if the message were read in the absence of antibiotics, then it would be undecipherable. However, in the presence of a certain antibiotic, one specific colored-colony (or multiple ones) would die, thus clearing up the hidden message.

Even though this newly developed system seems practical, it is still in its infancy. For now, any cryptographer would be able to crack these E. coli-encoded messages. For example, the specialist could grow several batches of the message and apply different antibiotics at different time points. This becomes a manageable task because there are a limited number of antibiotics and all E. coli are likely to die within 48 hours. Yet, Palacios remains unsatisfied. He wants to expand his idea of watermarking E. coli to more complex organisms, such as yeast. And with more complex model organisms, more sophisticated messages can be encoded and more useful applications can be derived from this method. Ultimately, the ways that this communication system can be improved are as limitless as molecular biology itself.