Team:NTU-Singapore/Project/Prototype/Degrade

The Gunner

The Degradation will focus exclusively on breakdown of cholesteryl esters found in the lipid core.

As we have elaborated earlier in the Prototype Design section, this device consists of mainly a pTet-regulated sequence, with cholesteryl esterase (CHE) and LuxI as the major products.

Let us now see how exactly the device works.

Background

The thickening of an artery wall in atherosclerosis is mainly due to the build-up of fatty materials. Accumulation of cholesteryl esters (CEs) in macrophage foam cells is critical to atherosclerotic plaque formation. CEs are dehydrolyzed between the carboxylate group of a fatty acid and the hydroxyl group of cholesterol. They are less soluble in water than cholesterols, and form insoluble aggregates.

In order to alleviate the symptoms of shrinking blood vessel, and eventually cure atherosclerosis, CEs must be degraded and removed from the site of the plaque.

Previous works show that various cholesteryl ester hydrolases (CEHs) have been successfully cloned and expressed in both murine and human.

Further studies demonstrate that CEHs not only reduce high-fat, high-cholesterol diet-induced atherosclerotic lesion in mice significantly, but also eliminate CEs from macrophage foam cells in human.

So a prominent enzyme that can help degrade CEs should be incorporated in our system. Cholesteryl esterase (CHE), systematically known as steryl-ester acylhydrolase, is chosen to perform the crucial task because it is the prokaryotic homolog of CEH.

Mechanism

CHE catalyzes the hydration reactions of CEs into free cholesterol and fatty acids with high specificity.

Cholesteryl Ester + H2O → cholesterol + RCOOH

A device that could produce CHE is introduced to achieve the function of degrading and eliminating cholesteryl ester.

The gene is designed to work under the control of promoter pTet. pTet is a well-known promoter that is constitutively ON and repressed by TetR. TetR repression is inhibited by the addition of tetracycline or its analog, aTc.

Since TetR production is controlled by pNO, and it is suppressed at low NO concentration (indicating inflammatory condition at the atherosclerosis site), therefore the degrading device can be activated when NO concentration is low. With the proper working of pNO and pTet inverter, CHE synthesis can accomplish designed objectives with both temporal and spatial specificity.

More about Cholesteryl Esterase

The 936 base pairs CHE gene is obtained from Pseudomonas aeruginosa, who produces the extracellular enzyme naturally. CHE suits more with our E. coli model system for similar expression mechanisms employed between the two organisms.

Construct Design

CHE gene was synthesized by GeneArt in accordance with the standard of Registry of Standard Biological Parts. Ligations were carried out to get the testing and final construct in our system.

Parts that are related to the degrading device, and have been successfully constructed are :

• CHE
• RBS-CHE
• CHE-T
• RBS-CHE-T
• RBS-CHE-RBS-GFP-T
• pLux-RBS-CHE-T
• pLac-RBS-CHE-T

All the parts have been verified by running agarose gel electrophoresis of double digestion products as well as PCR products. Bands with correct sizes are observed.

Characteristic Equations

Transcription of CHE

We first assume that rate of mRNA production for both CHE gene sequence and LuxI gene sequence are equal.

Repressive transcription of CHE and LuxI are represented by the following two equations:

Translation of TetR

Deterministic first order ODE is used again to represent the translation of TetR protein.

Where Ktl is the translation rate of mRNA & Dp is the degradation rate of TetR protein.

Modelling & Simulation

We make the following assumptions:

1. Biological systems of transcription and translation are assumed to be linear and time-invariant
2. Concentration of the transcription factor, NorR in E.coli is assumed to be in excess and constant. Hence, [NorR] is not taken into account in our model
3. There is no time lag for NO diffusion from ext. environment into the cell
4. Constant Degradation rates for mRNA as well as protein

Instead of modelling with TetR, we are using GFP as our output. This is part of our effort in trying to predict the wetlab characterization of pNO.

The following is our Sense device represented as a Simulink system. Please click on it for a larger view.

Device Construction

Device Characterization

Literature / References

Please proceed here to view our full list of references.