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Team:TUDelft/Overview
From 2009.igem.org
Time-delay genetic circuit
In nature, a fundamental “device” in gene regulation circuits is time-delay responses to internal or external stimuli. These devices are involved in essential patterns such as oscillating systems, circadian clocks, cell differentiation and development, for mention some (1). Time-delay circuits have the capability to integrate signals and trigger events after a delay from the initial detection event. There are two approaches to construct a time delay genetic circuit, these are: 1) protein-based transcriptional regulators and 2) RNA-based post-transcriptional regulators (2). In order to gain a better understanding on how certain genetic circuits are build and how they work to derive in complex responses upon stimuli, in the field of synthetic biology, the reconstruction of genetic circuits is one of the more investigate topics. Specially, experiments focused on transcriptional regulation components are relatively well understood and easy to emulate. Although circuits based on transcriptional networks can provide complex behaviors, as the complexity increases it is clear that many natural circuits are not only transcriptional driven but they are controller in other levels such as post-transcriptional regulation (3).
Based on different literature and the requirements of the project (see Why? and How? sections), two genetic configurations, one protein-based and other RNA-based, have been considered which will deliver a time-delay genetic circuit.
Why?
As mentioned in the project description, one important feature performed for our system is the reset of the signal once the message is received. The time between the signal (or self destructive plasmid/conjugation plasmid) has been received and the reset (or destruction of the plasmid) is an important parameter which will allow the signal to be send to the next cell (receiver) through the consecutive conjugation system before “losing” the message. Therefore, it is necessary to construct a device which will give enough time for the two subsequent events happen. This device is termed time-delay genetic circuit in this project. In a final version, the time-delay genetic circuit will have as input the entry conjugation plasmid into one cell and will deliver as output the expression of the endonuclease in charge of the destruction of the mentioned plasmid (figure 1).
Figure 1. Black Box scheme of the Time-delay genetic circuit. The input of the device is the introduction of the “conjugation plasmid” into the cell; the output is the expression of the endonuclease which will destroy the plasmid hence it will reset the signal or “message”. The consecutive arrows after the black box indicates a time-delay in the output signal. In a “message framework” (dashed orange box), two events should happen: reception and delivery of the message, during the input and before the output of the time-delay device.
As a bridge which connect the conjugation system and the self destructive plasmid, the time-delay genetic circuit as described above could not been constructed and tested until the other two sub-projects have been completed. To avoid a shift in the schedule, the device will be tested using IPTG as input and green fluorescent protein (GFP) as output (figure 2). A detailed scheme and description of this device will be showed in the following sections.
Figure 2. Black Box scheme of the Time-delay genetic circuit proof of concept. The input of the device is the addition of IPTG to the medium; the output is the expression of green fluorescent protein (GFP). The consecutive arrows after the black box indicates a time-delay in the output signal.
How?
From the literature review, two different genetic circuit configurations were contemplated: A synthetic transcriptional cascade, which has been showed to perform time-delay behavior in previous studies (4) and an approach based on post-transcriptional regulation which we termed biosynthetic AND gate.
As the conjugation system will have two plasmids (conjugation and helper plasmids), the approach followed in this sub-project was to split the construction of the time-delay genetic circuit in two independent plasmids which in theory should be present in a single cell in order to initialize the time-delay. This can be achieved given two different selection markers and apply the two different selection pressures.
Due to the expected long delay time needed, an optimistic approach will be the combination of both approaches, synthetic transcriptional cascade and biosynthetic AND gate in order to gain a desire phenotype.
Figure 3. Time-delay genetic circuit approach. Two approaches have been considered: 1) (Blue circle) Synthetic transcriptional cascade, the delay is due to a sequential expression of genes, and 2) (Green circle) Biosynthetic AND gate, the delay is due to the need of the presence of two different events and post-transcriptional control. In order to achieve a long delay both approaches could be ultimately combined.
