Why?

As mentioned in the project description, one important feature of our system is the reset of the signal once the message is received. The time between when 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 provide enough time for the two subsequent events happen. This device has been termed the time-delay genetic circuit in this project. In the final version, the entry of the conjugation plasmid into one cell will serve as the input to the time-delay genetic circuit while the output will be the expression of the endonuclease in charge of the destruction of the signal 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 and hence 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 connects the conjugation system and the self destructive plasmid, the time-delay genetic circuit described above could not been constructed and tested until the other two sub-projects had 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.

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