Cells live, grow, reproduce and respond to their environment using complex cellular networks including components like positive and negative feedback loops, feed-forward regulation and cell-cell communications. Using the basic regulatory motifs we currently understand, researchers have started to build artificial systems that mimic these cellular networks. In the beginning these were made to further our understanding of natural cellular networks but since the emergence of synthetic biology, researchers have begun developing networks with novel functions. These artificial networks are referred to as synthetic gene circuits or engineered gene circuits.
When it comes to thinking of a cell as a part or a gene as a circuit, we have to get back to the fact that synthetic biology is the application of engineering principles to biological systems. Gene circuits are often compared to electrical circuits where you have an on or off state corresponding to whether a gene is being expressed or repressed respectively. Electrical circuits are made of a collection of parts such as resistors and capacitors which are analogous to the growing database of biobricks. By putting the right parts together, you’re able to make useful systems like biological switches, oscillators, logic gates, metabolic controls, new translational machinery, population controls and pattern formations.
Simple Synthetic Gene Circuits
A well-known synthetic gene circuit is the genetic toggle switch which is most simply put, an on-off switch for a specified reporter protein. It’s made of two repressors, TetR and LacI which will repress each other depending on certain chemicals in the environment. To express the reporter (the on state), the chemical IPTG is added to inhibit lacI and allow expression of tetR and the reporter protein. To repress the reporter (the off state), aTc is added to inhibit tetR and allow expression of lacI and repression of the reporter.
(Figure 1. Adapted from Tu et al figure 20.1)
Theoretically, circuits like this could be used to stabilize or destabilize a cell or a system of circuits where the reporter may act in either fashion. This property is known as bistability.
Another simple synthetic gene circuit is known as the repressilator which is made of three sequential repressors which repress each other in turn. Personally I think the name sounds like something Dr. Doofensmirch would think of from Phineas and Ferb.
The first repressor is TetR which inhibits the second repressor lamba-cI which inhibits the third repressor lacI which loops around like a negative feedback loop to inhibit TetR. This may seems like some sort of circuit of futility but mathematically, researchers predicted it would allow sustained oscillating states in the system. Using repressors with strongly binding promoters and repressors (low ‘leaky’ transcription), cooperative repression characteristics (not interfering with one another) and similar protein and mRNA half-lives, researchers were able to realize their calculations and produce oscillatory behavior in individual cells. This might seem like no big deal but it’s a step towards more complex oscillators such as those dependant on circadian rhythms.
Aspects of Cellular Noise
The main hurdle to overcome for the creation of reliable gene circuits is the cellular ‘noise’ from other cellular processes. Cellular noise includes things like compounds taken up from the environment, mRNA and mRNA fragments and proteins regularly found in the cell. The noise is specifically in reference to how the compounds interact with the gene circuit and effect it’s functioning. Think of it like the static noise you hear on the radio when you’re trying to find a certain radio station, it get really hard to hear the station you want until you can tune out the other junk.
Noise is especially a problem with the repressilator where only 40% of cells are able to perform the desired oscillations and even sibling cells have oscillations that out of synch. Natural oscillations have been shown to be much more reliable and robust but researchers are unsure how they function so well despite the cellular noise.
One motif shown to resist noise is the negative feedback loop where the output of the system dictates the future output, in this case reducing it. The system essentially has a limit to the amount of gene product it will accumulate by either reducing the expression directly or by increasing consumption of the product by other systems.
An example of this motif is bacterial chemotaxis where bacteria sense a factor in the environment that they perceive as positive and swim towards it. The chemotaxis depends on the receptors for said factor where they will sense any changes in factor concentration in the environment. When the receptors sense a steady concentration of the factor they essentially clear themselves in order to sense smaller variations in the environment.
A simple analog to this would be if I asked you to name the colours in figure 3a. You would reasonably say they are purple, red and green. If I asked you to name the colours in figure 3b however you may classify them further as pastel green, olive green and forest green. Before you would have just called them green but where the differences between the colours become smaller, you have to make more specific distinctions between them.
(Figure 3. An analog to how receptors ‘clear’ themselves to sense smaller variations in concentration)
An interesting aside to the problem of cellular noise is that although it’s mainly disruptive, it can also be helpful. In 2008, researchers were able to make an oscillator from an activator-repressor pair that showed more synchronicity and more powerful oscillations than they’d mathematically predicted. When they looked into it they found that both the activator and the repressor contained destabilization sequences that made them susceptible to proteolysis. High levels of either product initiated the proteolysis, effectively clearing the cell of the products and creating more precise oscillations than researchers expected. Looking into how we can exploit these interactions further will surely lead to synthetic gene circuits functioning at levels we never expected to develop.
Originally researchers wanted to create gene circuits from scratch that would function as independently as possible from the natural networks present in the cell or even replace natural networks but from examples like the activator-repressor oscillator, researchers have found that working with natural networks and integrating the two provided a lot more progress in the understanding and manipulation of gene circuits. The two synthetic gene circuits I’ve discussed in detail so far are from the early 2000’s and since then there has been a completely new generation of synthetic gene circuits that explore the integration of natural and synthetic much more.
Applications
There’s a lot more on this topic I could discuss but I’d like to mention a few applications of synthetic gene circuits before this post runs too long. The most common application is the production of specified proteins for drug development where bacteria are engineered to produce a protein they might not usually make or they might be engineered to produce more of a protein than usual. A lot of proteins are found in plants and we originally had to grow those plants and collect the proteins from them to process them further into medications but bacteria grow much faster than plants and are much easier to maintain. The main thing to consider is that even if you’re able to engineer the proper circuits, you also have to provide enough substrate for the protein to be made and to balance the production of the protein with the bacteria’s natural metabolism so as not to harm it. It’s similar to how the ideal parasite doesn’t harm its host, only taking what it needs and benefiting from the host’s protection.
Other applications include use in gene therapy for diseases like cancer and for the reduction of pollutants in the environment. If you’re interested in reading further about synthetic gene circuits, check out the sources below.
Synthetic Biology: Integrated Gene Circuits
Engineering Gene Circuits: Foundations and Applications
Introduction of Customized Inserts for Streamlined Assembly and Optimization of BioBrick Synthetic Gene Circuits
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