Circadian rhythm is the invisible manager of living organisms, regulating the processes in our bodies according to day and night. Scientists have been busy developing similar treatments that can work naturally in a human body. And the communication protocol has a lot of similarity with a popular spectator practice in sports games.
Our bodies, as well as those other organisms, regulate based in a timed-cyclic manner. Michael Elowitz and Stanislas Leibler of the Princeton University created a biological clock within Escherichia coli. The findings were reported in their papers in the Nature journal. The circuit was built by attaching three pieces of DNA, those that prevent the production of proteins by other genes. The novelty of this circuit is that it switches genes ON and OFF in an oscillating wave-like manner. One gene (which is ON), switches the next one in the chain OFF, which in turn switches the adjacent gene to ON, and so on. The working of the circuit could be verified visually, as the protein which produces a green fluorescence was the one being managed. The end result was cells glowing in waves of green.
Timothy Gardner of the Boston University led a team in exploring further opportunities in this field. They constructed a component called the bistable or the flip-flop. They work such that when it is switched ON, it remains in that state, even with less or no stimulus. This works similar to a music player. The ‘Play/Pause’ button works in the same way. Once you push the button, it starts playing music. The same button when pressed again, pauses the song but doesn’t switch off the player itself. Upon pushing the button, the song resumes. This is a bistable state (ON and ON-STANDBY) achieved by this team who experimented on the E.coli bacteria, similar to the project above.
These projects demonstrated the ability to govern biological processes by building components from DNA but working on an electrical logic. Over the next few years, switches, pulse generators, timers, oscillators, counters and logic calculators have been built. These have been used to control genes, protein functioning, cell-cycles, metabolism and inter-cell communication.
Insulin and Mexican waves
Electrical circuits are designed, like most physical engineered products, using forward-engineering. They follow physical laws, are built by connecting components in a controlled environment. Genetic circuits are different. The components are evolved and work within highly complex cellular environments. This leads us to explore other developmental methods than previously discussed. One such common interest in engineering and genes is time-keeping.
Clocks are an integral part of anything science. Oscillatory clocks are of special interest to engineering. Biological clocks are central to getting the right things done on time, in living organisms. It is said that these internal clocks have evolved separately, with every organism having its own set of timed activities that govern its internal activities. The way they work is through autoregulatory gene network that drives the rest of the architecture. The feedback (positive and negative) loops are coupled with segmented clock networks and both work in unison.
Such complex nature of biological clocks is what makes them an ambitious target for the application of synthetic biology and computational analysis. However, it is so intricate that such networks cannot be engineered on the go. Circadian rhythms are helpful in keeping up with the day-night cycle as well as to meet the necessary changes the 24-hour cycle demands. Drosophila was used to test synthetic networks by a team, led by Jeff Hasty at the University of California. The genetic makeup mimicked circadian rhythms of much higher organisms, making this a viable testing ground to test an oscillation generator.
To demonstrate a negative feedback loop, consider three friends standing in a triangle. The rule is to do the opposite of what the person to your left is doing. If the person to your left is sitting, you stand and vice-versa. When the first friend stands, the second one sits, while the third friend stands. This is followed by the first friend sitting, which makes the second one to stand and the cycle goes on. When the distance is added between the friends and constraints are introduced (friend 2 sings while standing), you got yourself a biological oscillator (a model of the clock). This model helps illustrate how genes interact with each other across a system.
Moving forward, let’s translate the above example to the realm of genes. When gene A is active, gene B is shut. Gene C is now activated which shuts down gene A. Carrying this design, if one of the genes when active, is made to output fluorescence, one notices a flash every second round. Note that the duration and nature of these added features can be varied. Complexity can be heightened by introducing other loops and mechanisms. Now, this might seem like a naïve method of mimicking actual biological oscillators. But, this project has an ace up its sleeve.
The gene set-up could also communicate with its neighbors. This meant all similar gene groups are in sync. The behavior is analogous to a Mexican wave. If you’re in one, you stand when the person beside you sits. But it just isn’t you alone. An entire row of people joins you. Similarly, the gene groups would behave, except, the numbers in a stadium dwarf in front of the gene groups used. The complexity of operating such a set-up is equivalent to turning all the traffic lights in the world to green, at the same time!
One of the first things that come to mind, when we think of its benefits, is diabetes. It occurs when the body is unable to regulate the glucose level in the blood while the kidneys produce an excess of urine. It is either due to low insulin production or when insulin isn’t used the way it has to be. Glucose is the fuel for all organisms since they are the source of energy in cells. Maintaining the right level of glucose is paramount since anything too high or low might prove to be detrimental. Thanks to evolution, we have developed methods to ensure a constant availability of glucose to cells. Insulin is central to this process, monitoring the glucose level and guiding tissues to store excess glucose as fat. The liver plays a role here as well. It must be noted that when glucose levels are low, then the opposite occurs. As such, insulin ensures a steady amount of glucose in the bloodstream and all of this is possible due to a feedback loop.
If you are thinking that insulin steps in only when you are having that sweet dish, you are wrong. Insulin works in oscillations with a time frame of three to six minutes, constantly monitoring the situation. When this mechanism breaks down, diabetes sets in. If a lab-grown substitute is to work, the oscillatory pattern needs to be maintained. Hopefully, the synthetic circuit that was discussed earlier looks promising. It needs to be noted here that the testing normally involves the lower level of organisms. A feature of a human body is its ability to fend off any foreign organism that it deems to be a threat. So the primary challenge will be to ensure that it is compatible with the body and that it integrates well with the cellular neighborhood, where the gene circuit carrier is parked.
This series is inspired by Chapter 9 – “Logic in Life” of the book “Creation: How Science is Reinventing Life Itself” by Adam Rutherford. [Rutherford, A. (2013).Creation. New York: Current.]
Cookson, N., Tsimring, L. and Hasty, J. (2009). The pedestrian watchmaker: Genetic clocks from engineered oscillators.FEBS Letters, 583(24), pp.3931-3937.
Forward engineering is the process of building a system, based on a set of specifications and observing the ability of the system to meet the expectations- Bcp.psych.ualberta.ca. (2018).University of Alberta Dictionary of Cognitive Science: Forward Engineering. [online] Available at: http://www.bcp.psych.ualberta.ca/~mike/Pearl_Street/Dictionary/contents/F/foreng.html
Diabetes is a disorder of metabolism – the way the body uses digested food for energy. With the help of the hormone insulin, cells throughout the body absorb glucose and use it for energy. – PubMed Health. (2018).Diabetes Mellitus – National Library of Medicine – PubMed Health. [online] Available at: https://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0024704/