Project Description
This problem lies at the interface of genomics and design, and is being considered by many leading researchers and institutions. We are attempting to learn how biological systems function by putting together simple ones and seeing if we can manipulate them in meaningful and predictable ways. The overall goal is to develop the tools and theory to be able to construct a freestanding biological entity - a synthetic organism. However, as a step toward this goal, we are producing a semi-synthetic system: a metabolically modified organism.
Our interim target it a modified bacterium containing biological oscillator comprised of a linked set of three promoter/repressor pairs. Each promoter controls the transcription of the repressor protein for the next promoter in sequence. Thus, when a given promoter is active, it produces the repressor for the next, i+1, and shuts off that module. But the i+1 promoter controls the i+2 protein, and now that is it shut down the next, i+2, promoter is tuned on. When this module is linked back to the initial promoter, this will turn off this initial transcript, allowing the i+1 module to turn on, and turning off the i+2 module, which then allows i back on . . . we get a rolling game of Rock-Paper-Scissors, with a period and phase dependent on the various intrinsic biological rates of the process. A figure illustrates this process.
This description may alternatively seem unavoidable or improbable, depending on your particular perspective. A rigorous analysis starts from an understanding of the basic parameters that describe each genetic element and its interactions with the system. The particular topology in which the three modules are linked results in a set of six differential equations that describe the time dependent evolution of the. To better understand this system, and assist us in the design of our system, we developed a computational environment that accepts these parameters and simulated the behavior of the system produces. This shows that only shows that particular values of decay and transcription rates allow the system to oscillate, with other values locking the system in some stable state -much as a transistor radio circuit will only work with particular values of resistors and capacitors. This information was used, for instance, to modify the decay rates of certain proteins in our system.
This project is not simply a modeling exercise: we are currently building this system and produce the actual bacterium that will flash different colors in response to this oscillation. This involves the synthesis of three plasmids containing 21 individual genetic elements - some of which had to be modified to alter their default (i.e. native) behaviors to produce parameter values compatible with oscillation, as describes by the model simulation described above. The assembly of this synthetic genetic system is a moderately complex task, and a large part of the effort this semester has been devoted to developing a plan to and implementing a plan to do this. Our efforts in this regard are about 90% complete at this date.
This target was previously referred to as an "interim target": our final target involves elaboration to incorporate a communication system between individual cells, so that the population of bacteria will oscillate in unison. Currently the individual oscillators within each bacterium eventually get out of phase, resulting in an averaged, non-oscillatory macroscopic signal. Theoretical work suggests that it is possible to link these circuits in individual cells by use of extracellular signaling pathways that bacteria use to sense their own density. This requires additional design work to incorporate those pathways into our circuit, analysis using the modeling software to optimize the linkage and ensure the modified oscillation is robust and concerted, and to design the genetic details of the circuits as they occur on the project plasmids.
