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 [http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=124983&blobtype=pdf link]
dCcdB 7.7E-5 s-1 [http://www.ncbi.nlm.nih.gov/pubmed/8022284?dopt=abstract link]
dRNA_CcdB 0.00231 s-1 [http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=124983&blobtype=pdf link]
dHSL 1.02E-6 s-1 [http://aem.asm.org/cgi/content/abstract/71/3/1291 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 [http://www.jbc.org/cgi/reprint/M311194200v1.pdf link]
Dissociation Constants
KHSL_LuxR 1E-6 [M]/L kdiss / kass (HSL+LuxR) [http://jb.asm.org/cgi/content/full/189/11/4127?view=long&pmid=17400743 link]
KHSL_LuxR 4.05E-6 [M]/L binding HSL_LuxR on LuxPromotor [http://parts.mit.edu/igem07/index.php/Tokyo/AHL_assay link]
Hill Cooperativity
nHSL_LuxR 2.08 [http://parts.mit.edu/igem07/index.php/Tokyo/AHL_assay 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)

[http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T39-3Y6HKD6-BK-1&_cdi=4941&_user=877992&_orig=search&_coverDate=08%2F30%2F1995&_sk=998379998&view=c&wchp=dGLbVtz-zSkzS&md5=49efd14150c71e668eabdef225220ce3&ie=/sdarticle.pdf 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


Simulations

todo!!!

Sensitivity Analysis

todo!!!

New Cell Death

Extensions to previous system

Todo!!!

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 [http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=124983&blobtype=pdf link]
dccdB 7.7E-5 s-1 stable in the absence of ccdA [http://www.ncbi.nlm.nih.gov/pubmed/8022284?dopt=abstract link]
dRNA_ccdB 0.00231 s-1 [http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=124983&blobtype=pdf link]
dHSL 1.02E-6 s-1 very stable in the medium, average lifetime of 185h [http://aem.asm.org/cgi/content/abstract/71/3/1291 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 [http://www.jbc.org/cgi/reprint/M311194200v1.pdf link]
Dissociation Constants
KHSL_LuxR 1E-6 [M] HSL binding to LuxR [http://jb.asm.org/cgi/content/full/189/11/4127?view=long&pmid=17400743 link]
KHSL_LuxR-promoter 4.05E-6 [M] binding of HSL_LuxR complex to the Lux Promotor [http://parts.mit.edu/igem07/index.php/Tokyo/AHL_assay link]
KHSL_lactonase 4.47E-3 [M] KM of lactonase with 3OC6HSL [http://www.jbc.org/cgi/content/full/279/14/13645#TBL2 link]
Hybrid Promoter
KC2 P22 2.6E-10 [M] Dissociation constant for P22 c2 promoter binding [http://www.jbc.org/cgi/content/abstract/258/17/10536?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&andorexactfulltext=and&searchid=1&FIRSTINDEX=0&sortspec=relevance&firstpage=10536&resourcetype=HWCIT link]
KHSL_LuxR 4.05E-6 [M] Dissociation constant for HSL-LuxR complex promoter binding [http://jb.asm.org/cgi/content/abstract/JB.01443-07v1 link]
Hill 2 Hill coefficient for P22 c2 binding [http://www.jbc.org/cgi/content/abstract/258/17/10536?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&andorexactfulltext=and&searchid=1&FIRSTINDEX=0&sortspec=relevance&firstpage=10536&resourcetype=HWCIT 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 [http://partsregistry.org/Part:BBa_B0014 link]
Translation Rates
kLuxR translation 0.556 s-1 Translation rate for LuxR, B0034 RBS (relative efficiency 1.0) [http://partsregistry.org/Part:BBa_B0034 link]
kccdB translation 0.00556 s-1 Translation rate for ccdB, B0033 RBS (relative efficiency 0.01) [http://partsregistry.org/Part:BBa_B0033 link]

Models

CellDesigner (SBML file)

Cell Death

Matlab (SBML file)

Cell Death

Simulations

todo!!!

Sensitivity Analysis

todo!!!