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Bacterial Op Amps
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A novel genetic circuit design was modelled to emulate resistor units to be used with biological op-amps. The resistor units were set up inside synells, which exploits quorum sensing to interact with the existing biological op-amp architecture via the positive feedback loop.
The purpose of this innovation is to investigate the efficacy of a novel resistor unit in a biological op-amp through simulation.
If the initial values of the bacterial system are modified, then the positive feedback loop of substrate will be modified so that substrate levels decrease sharply because of an increase in degrading enzymes.
Bacteria are prokaryotic organisms measuring a few micrometers in length and exist together in the millions. Bacteria perform a variety of different functions such as growth and division,signalling, and differentiation. One of the ways bacteria divide is binary fission, in which one bacterium splits in two identical bacteria. Binary fission creates a culture of identical bacteria. The bacteria species of interest in this project is Pseudomonas Putida, a root colonizing, plant growth-promoting organism, because it is gram-negative, which means that it uses N-acyl homoserine lactones as a signalling molecule for quorum sensing.
Transcription is the process in which DNA is transcribed to RNA, which occurs in several steps. First, an enzyme called RNA Polymerase(RNAP) binds to primers on a strand of DNA. The RNAP then moves across the strand of DNA, separating the two strands. The RNAP codes for messenger RNA(mRNA) by adding complementary nucleotide bases to the strand. The mRNA, which is now almost identical to the coding strand; uracil replaces thymine, and the mRNA then gives directions to ribosomes. Transcription can also be encouraged through the use of transcriptional activators, which bind to the RNAP and encourage transcription. Moreover, small molecules called coactivators will amplify the effects of the transcriptional activator. Inducers also can control whether the transcriptional activator performs at all; when an inducer binds to a transcriptional activator, it can turn the activator “on”. The performance of the transcriptional regulator is proportional to the amount of coactivators or inducers that bind to it; an increase in inducers will equate to an increase in transcription.
Quorum sensing is the cell-cell communication system in which bacterial cultures respond to fluctuations in population density. This process in Pseudomonas Putida is facilitated through the use of AHL, an autoinducer and coactivator. First, bacteria produce AHL and push it into extracellular space. Once there, bind to receptors and transcriptional activators(PpuR) of other bacteria which control transcription of target genes. Once the AHL binds to the PpuR, it forms a PpuR-AHL complex, and these complexes encourage transcription. By doing so, it encourages the production of a synthase(PpuI), which is the final protein responsible for intracellular AHL synthesis. Meanwhile, the PpuR-AHL complex dissociates into PpuR and AHL, and both PpuR and AHL are fed back into the system. The AHL that goes back into the system creates a positive feedback loop(AHL-positive feedback loop), and AHL produced by PpuI enters into the systems of other bacteria to produce more AHL. Without a regulating factor, there would be too much AHL and there would be no controlled gene expression. That’s why AHL is regulated by a lactonase, whose production is induced by the PpuR-AHL complex. Normally, lactonase is activated after a delay; the AHL must start completing the AHL-positive feedback loop before the lactonase can start being produced and start degrading AHL. Based on the amount of AHL that binds to PpuR, the bacterial culture can measure the entire cell density and can then coordinate gene expression. The main processes of quorum sensing can be summarized to synthesis of AHL, recognition of extracellular AHL, and response in the form of virulence in bioluminescence, and other group behaviours.
Due to the modelling nature of this project, the procedures are primarily the modelling work done on a computer. In this project, a system of delay differential equations that model the dynamics of major factors(AHL, PpuR, PpuR-AHL, lactonase, substrate, population size) of quorum sensing is modelled and manipulated. First, a full model of the dynamics is constructed to get an intuition for the system as well as understand the relationship between certain factors such as AHL, PpuR, Lactonase, and the PpuR-AHL complex. The equations are given by many, many terms and variables that each represent a factor of the quorum sensing system. The full model gives a lot of information about the dynamics of each quorum sensing factor, and provides a graph of each factor. However, this project is primarily interested in what the dynamics of AHL for a single cell and the effects of modifying them. Consequently, the equations can be modified.
In terms of the biology, the AHL-positive feedback loop is hijacked by introducing synthetic minimal cells (Often shortened to synells, are small structures able to perform a function determined by the DNA implanted in the cell). This synell would contain the gene aiiA, which encodes lactonase producing proteins. The synell then seeks out the AHL-positive feedback loop and begins degrading the produced AHL. This is modelled on the computer by subtracting the AHL concentration differential equation by a rate, q, which is multiplied by the concentration of AHL. Since the lactonase is being introduced into the system through a synell, it eliminates the need for delay differential equations, simplifying the model to a system of ordinary differential equations.
In this project, the AHL-positive feedback loop was interrupted with the use of synells, which allow for compartmentalization of genetic cascades(chain reactions of gene expression - a predetermined output for given input/stimulus) as well as internal communication without crosstalk. The synell is predetermined to act as a middleman, in which the controlled amount of AHL that passes through the feedback loop is degraded by a lactonase, produced by aiiA, a gene that leads to the production of that lactonase, before the remaining AHL passes on. This leads to AHL production being significantly reduced due to the synell. This synell behaviour is very similar to the behaviour a resistor exhibits on a voltage. The synell behaviour of degrading some AHL and letting the rest participate in the AHL-positive feedback loop was modelled and a simulation produced graphs that reflect this behaviour very accurately. Although this method would work well, care must be taken so that the rate of AHL degradation doesn’t surpass the rate of AHL production. For that reason, a variety of rates were tested, and their graphs analyzed to see which rate was viable. The amount of AHL drops suddenly, this is clearly seen in the graph. All AHL concentrations, regardless of degradation rate, see a steep drop as soon as the AHL-positive feedback loop is initiated. In addition, degradation rates at values of 1.5 to 0 are viable values for simulation, but anything beyond that is extraneous. In summation, the behaviour of the synell reflects the behaviour of a resistor, in how it significantly decreases AHL concentration, much like how a traditional resistor does with voltage.
In this project, a synell interrupted the AHL-positive feedback loop and degraded a certain amount of AHL through the use of an AHL degrading enzyme lactonase, effectively creating an AHL resistor. The result of this project supports the hypothesis, if initial values of a bacterial system is altered, can the positive feedback loop of substrate be modified, for a couple of reasons. First, the initial values of the bacterial system were modified, as lactonase is normally activated with a delay; in this project, lactonase was introduced through the synell with almost no delay. Second, modifications were clearly made to the AHL-positive feedback loop; a significantly reduced constant amount of AHL was given back into the system from the AHL resistor. In summation, the AHL resistor modified the initial values of the bacterial system by introducing lactonase into the AHL-positive feedback loop, which allows the feedback loop and gene expression to be controlled.
Applications of simulating analog computer components with quorum sensing are far-reaching in the field of analog computing and more. In this project, the effects of an AHL resistor on the existing op-amp architecture has been modelled. Analog computing in biological applications will be revolutionized from the possible use of quorum sensing; controlling certain chemicals and hormones in the human body can be made possible with this technology.
Future research can be done for AHL capacitors, which spread out AHL degradation over a longer period of time than a resistor. AHL resistors and AHL capacitors combined could create op-amps similar to the ones currently in use. Short-term future research would be getting values for variables, creating a visual representation of the bacterial culture, and extending modelling to model the effects of a variable resistor.