Team:KULeuven/Model/Cell Death

Position in the system
The Cell Death subsystem receives input from two other subsystems, namely:


 * Inverter
 * Pulse Generator

LuxR is the component repressing the regulation of CcdB, the toxic product causing cell death. There the LuxR production is constitutive, no protein controls the gene regulation of LuxR, but the amount of LuxR available to repress the transcripion of the CcdB gene is controlled by HSL (Homoserine lactone).

If the inverter subsystem produces HSL (occurs when no light is detectable), this will forms a complex with LuxR. This will diminish the amount of LuxR available to repress the CcdB transcription and initiate cell death. When waiting long enough the amount of HSL becomes critical.

If however the pulse generator becomes active (by the filter), it will produce a pulse of lactonase, which will then bind to the HSL, reacting to an hydroxy-acid. As opposed to HSL, this hydroxy-acid will no longer form a complex with LuxR. This increase in LuxR lowers the CcdB production. The challenge is to generate a pulse of lactonase high enough to neutralise all HSL present in the cell.

ODE's
  

Parameters
Attention: old system, old parameters!

This paper says that the synthesis of only a few molecules of CcdB protein is probably lethal.

All references for parameters can be found below, in the new section.

Matlab (SBML file)
Remark: not yet up to date to latest (final) version

Extensions to previous system
During the summer we switched from the above system to a new one which you can see just below, take a look at this figure as it will help you understand the regulation that is present. This new system has a few novelties.


 * First of all, transcription begins at a new hybrid promoter we made: BBa_K145150. This promoter is repressed by c2 P22, which is produced by the memory in the OFF state, making premature activation and cell death impossible. Besides this repression, the promoter is activated by the HSL-LuxR complex originating from a previously activated timer. The promoter behaves as shown schematically below.




 * Second, LuxR is now no longer constitutively produced but is placed behind the beforementioned hybrid promoter. This construction mimicks more closely the natural system where LuxR is upregulated when a threshold amount of HSL is present. Plus it also increases the time it takes to activate ccdB, lengthening the timer. The system will now auto-activate if enough HSL is present and the memory is in the ON state.


 * Third, the ccdB coding region is also downstream of the hybrid promoter and is thus also subject to the regulation explained above; c2 P22 repression and HSL-LuxR auto-activation. One difference is that the polymerase must first read through a bad terminator with about 60% efficiency before reaching this coding region. Another difference is in the ribosome binding sites preceding both coding regions. Where LuxR can be translated from a RBS with a relative efficiency of 1.00, the ccdB frame can only be read from a 0.01 efficiency RBS.

Describing the system
see also: Project:Cell Death



ODE's
  

Simulations
To simulate the cell death, the input signal TetR is all the time low (5E-5 s-1) except from 50000 till 80000 seconds,from 180000 till 190000 seconds and from 250000 till 270000 seconds (0.0125 s-1). The left figure shows us that a high input signal will lower the amount of HSL (the HSL will be converted into hydroxy acid by lactonase). This will increase the amount of free LuxR because there is not enough HSL to form the HSL-LuxR complex. During the pulse, the right figure shows us an increase of the amount of CcdB. Whenthe memory switches from state zero to state one, the amount of CIIP22 decreases which makes it possible to produce a small amount of CcdB (background signal). When the input signal is low again, the amount of HSL increases. This extra HSL will form with LuxR the complex which lowers the amount of free LuxR. The higher amount of the complex promotes the production of CcdB which increases to a (hopefully) deathly level. A new pulse lowers the amount of HSL and the complex and increases the amount of LuxR for the same reasons as before. The second pulse is smal in length and decreases the amount of CcdB only partially. The third pulse is long enough for a total reset of the entire system.

All graphs have amounts (number of molecules in the cell) plotted vs time, measured in seconds.

 