Team:KULeuven/Model/Cell Death

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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

ODE's

Parameters

Remark: update parameters to repressive promotor

*Parameter values (Cell Death)*
Name	Value	Comments	Reference
Degradation Rates
d_LuxR	0.0010 s^-1
d_{LuxR_HSL}	0.0010 s^-1	complex of HSL and LuxR degrades, giving back HSL
d_{RNA_LuxR}	0.00227 s^-1		[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=124983&blobtype=pdf link]
d_CcdB	7.7E-5 s^-1		[http://www.ncbi.nlm.nih.gov/pubmed/8022284?dopt=abstract link]
d_{RNA_CcdB}	0.00231 s^-1		[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=124983&blobtype=pdf link]
d_HSL	1.02E-6 s^-1		[http://aem.asm.org/cgi/content/abstract/71/3/1291 link]
Association/Dissociation/Reaction Rates
k_ass (HSL+LuxR)	1E6 s^-1	association rate of HSL with LuxR. Chosen to be relatively (to the other rate constants) high and such that K_diss (HSL + LuxR) equals 10^-6
k_diss (HSL+LuxR)	1 s^-1	dissociation rate of the HSL-LuxR complex
k_ass (HSL+lactonase)	1E6 s^-1	association rate of HSL with lactonase
k_diss (HSL+lactonase)	446.5 s^-1	dissociation rate of the HSL-lactonase complex
k_cat (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
K_{HSL_LuxR}	1E-6 [M]/L	k_diss / k_ass (HSL+LuxR)	[http://jb.asm.org/cgi/content/full/189/11/4127?view=long&pmid=17400743 link]
K_{HSL_LuxR}	4.05E-6 [M]/L	binding HSL_LuxR on LuxPromotor	[http://parts.mit.edu/igem07/index.php/Tokyo/AHL_assay link]
Hill Cooperativity
n_{HSL_LuxR}	2.08		[http://parts.mit.edu/igem07/index.php/Tokyo/AHL_assay link]
Transcription Rates
k_{mRNA_LuxR} (constitutive promotor)	0.025 s^-1	see Constitutive Promotors & E. coli transcription Rates
k_{mRNA_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)

Matlab (SBML file)

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

Simulations

todo!!!

Sensitivity Analysis

todo!!!

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. This new system has a few novelties. First of all, transcription begins at a new hybrid promoter we made: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K145150 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 enoguh 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 coing 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.