Team:ETH Zurich/GFP

From 2013.igem.org

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<h1>Mine Cells </h1>
<h1>Mine Cells </h1>
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Two proteins of interest for our design are produced by mine cells: (i) LuxI and (ii) [https://2013.igem.org/Team:ETH_Zurich/Experiments_4 ''NagZ''].  In both cases, the genes are expressed under constitutive promoters. Aditionally, the synthesis of AHL is also carried on by mine cells. Specifically, the product of ''luxI'' gene is directly involved in the synthesis of the molecule, using as substrates ''S''-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway (Schaefer ''et al.'', 1996).<br><br>
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Two proteins of interest for our design are produced by mine cells: (i) LuxI and (ii) [https://2013.igem.org/Team:ETH_Zurich/Experiments_4 ''NagZ''].  In both cases, the genes are expressed under constitutive promoters. Aditionally, the synthesis of AHL is carried on by mine cells; specifically, the product of ''luxI'' gene is directly involved in the synthesis of the molecule, using as substrates ''S''-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway (Schaefer ''et al.'', 1996). We assume precursor molecules are provided by the cell in non-limiting conditions. <br><br>
The PDEs for the states involved in the sender module are given below:
The PDEs for the states involved in the sender module are given below:
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<h1>Receiver Cells </h1>
<h1>Receiver Cells </h1>
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Receiver Cells are responsible for processing the signal sent by the mine cells. The output is a fluorescence signal whose intensity correlates with the sensed AHL concentration. <br><br>
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Receiver Cells are responsible for processing the signal sent by the mine cells. The output is a fluorescence signal whose intensity correlates with the sensed AHL concentration; for this purpose GFP expression is under control of P<sub>lux</sub> promoter. Once AHL reaches a non-mine colony, it can bind to LuxR protein, and the complex  LuxR/AHL as a transcriptional activator<br><br>
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In both models, at high concentrations of the toxic substance CI is expressed and it inhibits the production of LuxI, which means that AHL is not produced. Once AHL is not there, it can not bind to LuxR, so LuxR can not repress RFP anymore. This results in RFP expression; all the cells turn red. As mentioned, this module of the model acts as a high pass amplitude filter for the toxic substance, or low pass amplitude filter for AHL, since AHL is produced only when the toxic substance levels are low.
GFP is produced when the concentration of AHL is sufficiently high to give rise to the complex formation  
GFP is produced when the concentration of AHL is sufficiently high to give rise to the complex formation  

Revision as of 15:01, 16 September 2013

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Contents

Reaction-Diffusion Model: genetic circuit with GFP as reporter gene

In our first spatio-temporal model, we wanted to find out if (i) a suitable AHL gradient forms at all and (ii) validate the model with experimental data. Essentially we model the receiver cells (E. coli DH5α strain) being transformed with a plasmid containing GFP under the control of pLux promoter. Subsequently, we simulate a 2D spatio-temporal reaction-diffussion system with COMSOL Multiphysics.

AHL: Reaction-Diffusion Equation


The change of AHL concentration over time is influenced by two processes: (i) local chemical reactions and (ii) diffusion; which causes the molecule to spread over the agar plate (Eq. 1).

Equation 1: General partial differential equation for AHL reaction-diffusion. D(AHL(r,t),r) is the diffusive term, R(AHL(r,t)) is the reaction term


For diffusion, we have a partial differential equation (Eq. 2) which describes density fluctuations over time and space. We don't model AHL diffusion into and out cells explicitly; the underlying assumption is that this process is fast. From equation 2, DAHL(AHL(r,t),r) denotes the collective diffusion coefficient for AHL at location r. However, we assume that the diffusion coefficient does not depend on the density, i.e., DAHL is a constant. The value reported in the literature for the diffusion constant corresponds to measurements performed in water at 25oC. Since diffusion in our system happens in agar, we scaled the diffusion constant by a factor Cagar (Fatin-Rouge et al., 2004).

Equation 2: Diffusive term for AHL.


For the reaction component, the change of AHL concentrations over time is given by an ordinary differential equation (ODE), that comprises production and linear degradation. The synthesis of the signalling molecule depends on the product of luxI gene. Now for the degradation, we consider that AHL degrades at different rates depending on the localization, i.e. cytoplasmic or extracellular. Given that the intracellular degradation is driven by enzymatic degradation, whereas the extracellular decay is a non active process. Our model also includes a dilution factor due to the cell growth (Eq. 3).

Equation 3: Reaction term for AHL. DF is the dimensionless dilution factor, where N0 is the initial concentration and Nm is the carrying capacity.


Finally, we need to specify the initial conditions (at time t = 0) and boundary conditions. At the starting point there is no AHL in the agar plate, thus the initial concentration is zero ([AHL(r,t=0)] = 0 M). For the boundary condition, we take into account that there is not flux out of the agar plate.

Equation 4: Neummann Boundary Condition.


ODE's for other species

AHL is the only species in the model that is diffusing in and out cells, hence the change of the other species concentrations over time is given by system of non-linear ordinary differential equations (ODEs), following Michaelis-Menten kinetics. Our system consists of two type of genetically engineered colonies: (i) Mine Cells and (ii) Receiver Cells, that can be treated as modules and communicate only by a single molecule, AHL.

Mine Cells

Two proteins of interest for our design are produced by mine cells: (i) LuxI and (ii) NagZ. In both cases, the genes are expressed under constitutive promoters. Aditionally, the synthesis of AHL is carried on by mine cells; specifically, the product of luxI gene is directly involved in the synthesis of the molecule, using as substrates S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway (Schaefer et al., 1996). We assume precursor molecules are provided by the cell in non-limiting conditions.

The PDEs for the states involved in the sender module are given below:

Equation system 5: System of Differential equations for mine cells.


Receiver Cells

Receiver Cells are responsible for processing the signal sent by the mine cells. The output is a fluorescence signal whose intensity correlates with the sensed AHL concentration; for this purpose GFP expression is under control of Plux promoter. Once AHL reaches a non-mine colony, it can bind to LuxR protein, and the complex LuxR/AHL as a transcriptional activator

In both models, at high concentrations of the toxic substance CI is expressed and it inhibits the production of LuxI, which means that AHL is not produced. Once AHL is not there, it can not bind to LuxR, so LuxR can not repress RFP anymore. This results in RFP expression; all the cells turn red. As mentioned, this module of the model acts as a high pass amplitude filter for the toxic substance, or low pass amplitude filter for AHL, since AHL is produced only when the toxic substance levels are low.

GFP is produced when the concentration of AHL is sufficiently high to give rise to the complex formation

Results: Simulation