Team:KULeuven/Model/Cell Death

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Pictogram celldeath.png

Contents

Cell Death

Position in the system

The Cell Death subsystem receives input from two other subsystems, namely:

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.

Describing the system

Cell Death.jpg

ODE's

Parameters

Remark: update parameters to repressive promotor

Parameter values (Cell Death)
Name Value Comments Reference
Degradation Rates
dLuxR 0.0010 s-1
dLuxR_HSL 0.0010 s-1 complex of HSL and LuxR degrades, giving back HSL
dRNA_LuxR 0.00227 s-1 link
dCcdB 7.7E-5 s-1 link
dRNA_CcdB 0.00231 s-1 link
dHSL 1.02E-6 s-1 link
Association/Dissociation/Reaction Rates
kass (HSL+LuxR) 1E6 s-1 association rate of HSL with LuxR. Chosen to be relatively (to the other rate constants) high and such that Kdiss (HSL + LuxR) equals 10-6
kdiss (HSL+LuxR) 1 s-1 dissociation rate of the HSL-LuxR complex
kass (HSL+lactonase) 1E6 s-1 association rate of HSL with lactonase
kdiss (HSL+lactonase) 446.5 s-1 dissociation rate of the HSL-lactonase complex
kcat (HSL:hydroxy-acid) 29 s-1 catalytic transformation of HSL to an hydroxy-acid, lactonase is the enzyme link
Dissociation Constants
KHSL_LuxR 1E-6 [M]/L kdiss / kass (HSL+LuxR) link
KHSL_LuxR 4.05E-6 [M]/L binding HSL_LuxR on LuxPromotor link
Hill Cooperativity
nHSL_LuxR 2.08 link
Transcription Rates
kmRNA_LuxR (constitutive promotor) 0.025 s-1 see Constitutive Promotors & E. coli transcription Rates
kmRNA_CcdB 0.025 s-1 maximal transcription rate for CcdB RNA (no LuxR repressor present)

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

Models

CellDesigner (SBML file)

Cell Death

Matlab (SBML file)

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

Cell Death

New Cell Death

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.
Hybrid promotor.PNG
  • 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

Cell Death new.jpg

ODE's

todo!!!

Parameters

Parameter values (Cell Death)
Name Value Comments Reference
Degradation Rates
dLuxR 9.627044174E-5 s-1 no LVA tag, so longer lifetime
dcomplex 9.627044174E-5 s-1 complex of HSL and LuxR degrades, giving back HSL
dRNA_LuxR 0.00227 s-1 link
dccdB 7.7E-5 s-1 stable in the absence of ccdA link
dRNA_ccdB 0.00231 s-1 link
dHSL 1.02E-6 s-1 very stable in the medium, average lifetime of 185h link
Association/Dissociation/Reaction Rates
kass (HSL+LuxR) 0.002372 s-1 association rate of HSL with LuxR (estimate from KM but recalculated to remove molar dimension)
kdiss (HSL-LuxR) 1.0 s-1 dissociation rate of the HSL-LuxR complex (estimate from KM but recalculated to remove molar dimension)
kass (HSL+lactonase) 0.002372 s-1 association rate of HSL with lactonase (estimate from KM but recalculated to remove molar dimension)
kdiss (HSL-lactonase) 4470.0 s-1 dissociation rate of the HSL-lactonase complex (estimate from Kd but recalculated to remove molar dimension)
kcat (HSL>>hydroxy-acid) 29 s-1 lactonase catalyzed transformation of HSL to a hydroxy-acid link
Dissociation Constants
KHSL_LuxR 1E-6 [M] HSL binding to LuxR link
KHSL_LuxR-promoter 4.05E-6 [M] binding of HSL_LuxR complex to the Lux Promotor link
KHSL_lactonase 4.47E-3 [M] KM of lactonase with 3OC6HSL link
Hybrid Promoter
KC2 P22 2.6E-10 [M] Dissociation constant for P22 c2 promoter binding link
KHSL_LuxR 4.05E-6 [M] Dissociation constant for HSL-LuxR complex promoter binding link
Hill 2 Hill coefficient for P22 c2 binding link
kleaky 5E-4 s-1 transcription rate from the hybrid promoter in the unactivated ON state
kmax 0.0030 s-1 maximal transcription rate for the fully activated ON hybrid promoter (might be higher)
Terminator eff 60.8% percentage of transcription termination at the B0014 double terminator in front of ccdB link
Translation Rates
kLuxR translation 0.556 s-1 Translation rate for LuxR, B0034 RBS (relative efficiency 1.0) link
kccdB translation 0.00556 s-1 Translation rate for ccdB, B0033 RBS (relative efficiency 0.01) link

Models

CellDesigner (SBML file)

Cell Death

Matlab (SBML file)

Cell Death

Simulations

To simulate the cell death, the input signal TetR is all the time low (5E-5) except from 50000 till 80000 seconds,from 180000 till 190000 seconds and from 250000 till 270000 seconds (0.0125). 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.