In the first article (here), we have explored what Turing patterns means in a biological sense by given examples of how it occurs in nature. The series of the articles is building up to genetically modifying organisms to forward engineer Turing patterns. But first, a clear understanding of genes and gene regulatory networks must be given.
Gene Anatomy
At a crude level, in any living organisms –with some rare few exceptions–, The DNA acts as the blueprint for computations performed in the cell. It consists of nucleic acid monomers linked together to form polymer chains. There are four main types of nucleic acids, adenine (A), guanine (G) cytosine (C), and thymine (T) [3]. DNA molecules usually come in double-helix strands of polymer chains, as shown in figure 1. The two polymer chains are linked together via hydrogen bonds in the given structure: an adenine (A) base is always linked to a thymine (T) base, and cytosine base (C) is always linked to a guanine (G) base.
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The whole genome –complete set of DNA– consist of smaller regions called genes. Genes are sequences of DNA that code for a specific protein, or part of a specific protein (usually). The first process of making a protein is called transcription, where a single-stranded copy of a gene is synthesised through RNA polymerase to form mRNA (messenger RNA). RNA polymerases are enzymes (large proteins) that bind to a specific region upstream of the targeted gene, that region is the promoter region (Figure 2). Promoters contain RNA polymerase binding sites to initialise transcription, as well as binding sites of different transcription factors, they are crucial for controlling the rate of protein synthesis. mRNA travels outside of the nucleus (in eukaryotic cells) to be made into a chain of amino acids, called proteins, through the ribosome [4]. This whole process is known as the central dogma of biology, it is summarised graphically in figure 3.
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| Figure 2: A simplified view of the structure of the gene. The regulatory region -promoter- comes always before the actual gene. Extrons are coding part of the gene while introns are the noncoding part. [2] |
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| Figure 3: A schematic showing how information is transcribed from DNA to form mRNA (messenger RNA), which is translated to proteins via the ribosome [5]. |
Circuits, From Electronics to Biology
Now that we have briefly understood the central dogma of biology, it is worth dipping into electronics and electric circuits briefly before diving into genetic circuits. Formally, a circuit is a self-sufficient loop that can perform a specific function, which can take inputs and produces outputs. The concept is well established in electronics; where say, one is interested in creating a simple blinking LED circuit, the components required for building such a circuit are: A power source, an LED light, resistors, capacitors and some NPN transistors [3] (figure 4). In biology, one can treat the different parts of the genes as individual components with characterised functions similarily on how it is characterised in electronics.
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| Figure 4: An example of a schematic for creating a blinking LED light circuit [3] |
One of the most obvious examples of a genetic circuit with a feedback loop to observe on our bodies is the mechanism of controlling body sweat. There must be biological sensors measuring the temperature of the body to decide when to secrete sweat molecules in order to bring the body temperature down; creating a feedback loop mechanism. The mechanism of sweat secreting, along with many other regulation mechanisms within the body, are governed by different gene regulatory networks. The purpose of gene regulatory networks, as the name suggests, is to regulate gene expression (e.g. control the process of gene synthesis, and thus limiting the concentration of proteins). In this article, I will be using gene regulatory networks and genetic circuits interchangeably, although the former is technically more accurate.
There are different formulas for regulating different proteins, but the key parameters shared between them are promoters, transcription factors and a sequence of nucleic acids forming a gene. One of the ways of controlling the concentration of a targeted gene is through the use of transcription factors, which themselves can be synthesised by the cell and regulated by other transcription factors. One type of transcription factors is called an activator, which increases the synthesis of a targeted gene. On the other hand, inhibitors are transcription factors that limit the transcription process of a targeted gene, thus decreasing the concentration of the protein of interest. Controlling when and how transcription factors are synthesised means controlling the synthesis of any gene of interest, which is best illustrated by the following example of the toggle switch genetic circuit.
Genetic Toggle Switch
One of the simplest and earliest examples of synthetic circuits was done by Gardner, Cantor and Collins [4]. A toggle switch is well known device to control the flow of electricity in electronic or electrical circuits, to switch a light bulb or an LED on/off, for example. In the context of genetics, the desired function of a genetic toggle switch is to turn any gene on or off on our desire. As for electrical toggle switches, many use mechanical devices to switch the circuit on/off. In genetics, however, we make use of inducers block or unblock the synthesis of a gene by blocking the controlling promoter of that gene. To be more specific, the promoter region is blocked so the RNA polymerase cannot bind to initiate transcription. These inducers come in many different forms and I won't get into the technical details of it yet. This toggle switch was constructed using two promoters and their corresponding repressor genes, along with external inputs for control (Figure 5).
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Figure 5: The toggle switch is constructed of two promoters with their corresponding repressors genes [4].
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The two promoters used were both constitutive (allows continuous transcription of the downstream's gene). The downstream repressor gene of a promoter inhibits the other promoter. More concretely, Repressor 2 inhibits Promoter 2 and Repressor 1 inhibits Promoter 1. Inducers are used to inhibit the repression of a certain promoter. A reporter gene is attached downstream of Promoter 2, reporters are used to verifying whether a switch is working as expected, a typical reporter gene is a green fluorescent protein (GFP). An on state (repressed Promoter 2) should show no green fluorescent protein, while an off state (Inducer 2 activated) should express GFP. The mathematical model describing the dynamics of this circuit is:
U and V are the concentration of repressor 1 and 2, respectively. α1 and α2 are the synthesis rate of U and V, respectively. β and γ are the cooperativities of repression of promoters. If you are interested in how the model was derived you can check out the paper itself [4]. I have simulated the model above while controlling the ratio of synthesis of α1 and α2, which will correspond to different concentration of repressor genes, leading to toggling of GFP protein on when repressor 1 (U) is high and off when repressor 2 (V) is high.
References
[1] L. Pray, Discovery of DNA structure and function: Watson and crick," Nature Education, vol. 1, no. 1, p. 100, 2008.
[2] T. E. of Encyclopaedia Britannica, \Gene," 2019.
[3] Dahl, Ø., 2017. Blinking LED Circuit With Schematics And Explanation. [online] Build Electronic Circuits. Available at: <https://www.build-electronic-circuits.com/blinking-led-circuit/> [Accessed 7 April 2020].
[4] Gardner, T., Cantor, C. and Collins, J., 2000. Construction of a genetic toggle switch in Escherichia coli. Nature, 403(6767), pp.339-342.
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