Bacteria learn how to take a pulse: programming microbes to convert digital light signals to analog gene expression.

What do telecommunications, power delivery, and your audio system* all have in common? For starters, their underlying electrical systems use digital pulses, alternating ON and OFF states over time. These pulse patterns and the way they change, known as pulse width modulation or PWM, can encode and transmit information. Now, research from a team of British and American scientists have made a surprising addition to the list of systems that can decode information in pulse widths: Escherichica coli (E. coli), a bacteria normally found in our gut. 

In an article recently published in the Journal of Molecular Biology, the research team describes genetic modifications to E. coli that enable it to read the pulse width modulation of alternating green and red lights. The gene expression of a reporter protein represented an analog output in response to this digital pattern of light color. In essence, scientists have been able to replicate in bacteria a process important in electrical engineering.

The creation of a system in E. coli capable of decoding PWM information is a significant step forward in the field of synthetic biology. This field, which sits at the intersection between biology and engineering, attempts to design artificial sensing and gene regulatory networks in bacteria. Perhaps most exciting, however, is the potential to use microbes like this one described in this study as an interface between digital signals from machines and the biological activity of cells.

For more detail and commentary about this study, please select 'Read More'. Which ways do you think PWM sensing in E. coli should be used? How would you continue this study? Comments are welcome below!

*not all audio systems utilize PWM, if I am not mistaken

Bringing Biological Pulse Response into the Light

Pulse width modification may sound like an unapproachable and esoteric topic from an electrical engineering textbook, but in reality it is deceptively simple. Alternating between an ON and OFF state in rapid succession, like repeatedly flipping a light switch very quickly, will produce a result that is neither the full ON or full OFF result. In the light switch analogy, the room would be neither bright or dark, but rather half lit. This averaged signal is simply proportional to the fraction of the time spent in the ON state. Note here that it is possible to change both the frequency (how rapid the switch is flipped) and the pulse width (the relative fraction of time spent in ON versus OFF; these don't need to be equal).

Dr. Travis Bayer, a Professor at the University of Oxford, led the study that sought to introduce pulse width modification in E. coli. To accomplish this, the researchers imported into E. coli a light sensing protein duo, the two-component ccaS and ccaR system, which is naturally found in another bacteria. These proteins were programmed to control the level of gene expression for a reporter protein (GFP) in response to light. The levels of the GFP reporter can easily be measured, allowing researchers to track the output of their system in response to the input light.

The two-component ccaS and ccaR system is naturally used to regulate gene expression in photosynthetic organisms, helping them alter their light absorbing pigments to match the levels of green and red light. Red light switches ccaS into an inactive state, and green light switches this protein back into an active form, when it can 'instruct' ccaR to bind DNA and promote expression of the target gene. The ccaS protein can only adopt an ON or OFF state, but ccaR and other molecular processes must convert this state into the output of the target or reporter gene.

To turn on GFP gene expression, researchers needed only to expose the microbe to green light; red light was used to turn expression off. Thus, a simple colored LED light, coupled with the ccaS/R proteins, acted as a flippable switch for the GFP gene. To achieve intermediate levels of GFP expression, the bacteria were subjected to alternating pusles of green and red light. Like the pulse-width modification effect observed in electronics, the resulting expression reflected the average amount of time spent in the ON state. Thus, the flippable switch for expression was converted into a dimmer switch, simply by choosing the right frequency and the desired time for each pulse (i.e. pulse width). To create the averaging effect, the frequency of the pulses must be faster than the dynamics of the entire system (not just the ccaS protein, but also the downstream molecular events to produce GFP). 

The ability to achieve an intermediate level of gene expression may not seem like an important innovation. Indeed, many molecular biologists use control systems that increase gene expression in response to increasing concentration of a small chemical (i.e. a molecule like Lactose, that induces Lac gene expression). However, chemical induction of gene expression usually does not allow for fine-tuning with the precision observed in this study. Furthermore, intermediate levels are often achieved with chemical induction only at the population level; individual cells are still either fully ON or fully OFF, the ratio of each cell in the population is what changes.

The pulse-width modification system developed by the Bayer laboratory group and colleagues can produce intermediate levels of gene expression both at the population and individual cell level. An important experiment, found in the supplementary materials to the article, demonstrates that each cell adopts the intermediate expression that reflects the time of the green light pulses. Finally, the researchers show that this same approach can be used to control the expression of genes other than GFP, suggesting a wide utility of pulse width modification. Indeed, several natural genetic networks have been described that appear to use pulse width modification in controlling cell behavior.

Teaching an old microbe newer and newer tricks

Introducing pulse width modification into the molecular biology of gene expression is a significant advance. More than just a biologist's parlor trick, this is an important addition to the synthetic biology toolkit. It is a way that researchers can dial in to a specific level of gene expression, which can be important for programming ever more complex cellular behaviors. In addition, the creation of this artificial genetic network will inform the study of natural examples of pulse-width modulation used by bacteria to sense and respond to their environment.

This work also represents another example of synthetic biologists attempting to generate analog signals and responses in cells, instead of imposing digital logic on genetic networks. Another recent study by the Sarpeshkar group at MIT constructed a genetic circuit that operated in an analog fashion, but in a way different from that featured in this study. Together, these studies demonstrate how exploring different paradigms for gene expression control can lead to technical innovations in the field of synthetic biology.

In future studies, it would be great to see a more rigorous and systematic characterization of the pulse-width modulation system. How does the context of the cellular environment, and the efficiency of transcription and translation effect the pulse-width modulation system? In other words, how robust is gene expression under PWM control? Does green fluorescence from the GFP produced impact the ccaS/ccaR system in individual and nearby cells? Are their limits or constraints to what output may be connected to a PWM control system? How would exposing green light and red light to the cells simultaneously impact this system? Which iGEM teams next year will utilize PWM in their design?

Please share your thoughts as a comment below. Answer to the above questions, and other questions, comments and criticisms about the research are all welcome!

Selected References and Links

Davidson, E., Basu, A., Bayer, T. (2013) Programming Microbes Using Pulse Width Modulation of Optical Signals. Journal of Molecular Biology Vol. 425 (22) pp. 4161-4166.

[PMID: 23928560]

The article which is the focus of this summary / press release

Daniel R., Rubens, JR., Sarpeshkar, R., Lu TK (2013) Synthetic analog computation in living cells. Nature Vol 497 (7451) pp. 619-23

[PMID: 23676681]

A study that achieves analog computation / gene expression in bacteria, but done using chemical induction and a clever 'shunt' plasmid system.

Hirose, Y., Rockwell, N., Nishiyama, K., Narikawa, R., Ukaji, Y., Inomata, K., Lagarias, J., Ijeuchi, M, (2013) Green/red cyanobacteriochromes regulate complementary chromatic acclimation via a protochromic photocycle. PNAS Vol 110 (13) pp. 4974-79
[PMID: 23479641]
[PMC: 3612671] (Free Article)
A study which examines how the ccaS system (as well as other similar cyanobacteriochromes) behaves in nature, including how it responds to a mixture of green and red light

Tabor, J., Levskaya, A., Voigt, C. (2011) Multichromatic control of gene expression in Escherichia coli. Journal of Molecular Biology Vol. 405 (2) pp. 315-24
[PMID:  21035461]
[PMC: 3053042] (Free article)
An earlier study which utilized the ccaS - ccaR system in E. coli. In this study, the ccaS/R system is used in an orthogonal system alongside a red light activated system (with light regulated by time or space).