Team:Imperial College London/M1

From 2009.igem.org

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  After the transformed cells are cultured to the desired optical density (OD=0.7), module 1 is triggered by the introduction in the media of IPTG. A walkthrough of the genetic circuit is available [[Team:Imperial_College_London/M1/Genetic|here]]
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Protein production should only be initiated once the growing cell culture has passed a certain threshold of development. As protein production can be detrimental to the cell through a drain of resources, the cell density must be sufficient so that inducing protein production will not halt proliferation. <br>
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In <i>the E.ncapsulator</i> the protein production has been put under the control of an external inducer, IPTG. The cell density will be measured, and upon passing the required threshold, IPTG can be added to kickstart the production of our protein of interest.
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A walkthrough of the genetic circuit is available [[Team:Imperial_College_London/M1/Genetic|here]]
==How:==  
==How:==  

Revision as of 14:26, 2 September 2009

Contents

Overview

What:

  Module 1 is the protein synthesis step aimed at the delivery of the protein of interest to the gut. It is essential to note that the E.ncapsulator is versatile with regard to the protein being produced and thus to diseases it targets or the functions it adds.
To demonstrate a proof of principle for the iGEM competition, we have decided to produce two different enzymes: cellulase and PhenylAlanine Hydroxylase (PAH).   This module is the key customisable component of our system, allowing the scientist to develop E.ncapsulator delivery systems bearing the enzyme of choice. This customisation is achievable by replacing only one gene in the genetic circuit.

Why:

Cellulase

Ruminants survive on a diet high plant matter through the breakdown of cellulose and xylan by their gut microflora. Non-ruminants, such as humans, are unable to release much of the energy found in these compounds, and so must rely on an omnivorous diet. Of course, the transfer of energy between trophic levels is notoriously poor. If non-ruminants were able to obtain more energy from plant matter, the net effeciency of energy transfer would be raised reducing the volume of food required in a diet. In addition, polysaccharides such as cellulose form viscous gel-like structures that trap starch, proteins and fats which would otherwise be accessible to the animal's digestive enzymes and transport systems.


PAH

Phenylketonuria (PKU) is an autosomal recessive genetic disorder where the body is unable to break down the amino acid phenylalanine. This is because of a deficiency of an enzyme called phenylalanine hydroxylase (PAH), which converts phenylalanine to tyrosine. Absence of PAH causes phenylalanine to break down into phenylketone. A chronically high level of phenylalanine, called hyperphenylalaninemia will result. Phenylalanine then accumulates in blood and tissues and results in characteristic symptoms of PKU. Left untreated, PKU can lead to problems with brain development resulting in progressive mental retardation and brain damage.

Current treatments for PKU are centred around following a very strict low-protein diet. By engineering the E.ncapsulator to manufacture PAH, we hope that our cells can be taken as a dietary supplement to replace the missing PAH. This will break down phenylalanine from the diet, before it is absorbed into the bloodstream.

When:

Protein production should only be initiated once the growing cell culture has passed a certain threshold of development. As protein production can be detrimental to the cell through a drain of resources, the cell density must be sufficient so that inducing protein production will not halt proliferation.
In the E.ncapsulator the protein production has been put under the control of an external inducer, IPTG. The cell density will be measured, and upon passing the required threshold, IPTG can be added to kickstart the production of our protein of interest. A walkthrough of the genetic circuit is available here

How:

II09 CellulaseBacterium.png

 Module 1 in our design defines the application of our E.ncpsulator vector. In fact, module 1 determines the protein that is produced by the bacterium and that is aimed for delivery to the gut. It is essential to note that the E.ncapsulator project is versatile with regard to the protein being produced and thus to diseases it targets or the functions it adds.
To make proof of principle for the iGEM competition, we have decided to produce two different enzymes: cellulase and PhenylAlanine Hydroxylase (PAH).

Cellulase is an enzyme able to catalyse (degrade) cellulose, a material widely available in our diet. However, the human body is unable to degrade cellulose into glucose naturally. By producing a special cellulase (able to degrade cellulose to glucose on its own) in our E.ncapsulator system, we would perform the delivery of cellulase enzyme to the digestive region of the gut, thus enabling the degradation of cellulose into glucose. In other words, people ingesting E.ncapsulated pills of cellulase would be able to derive energy from eating grass, wood or other cellulose-containing materials.


PenylAlanine Hydroxylase (PAH) is a liver enzyme that degrades essential amino acid Phenylalanine into Tyrosine. PhenylKetonUria (PKU) is a condition associated to the creation by the body of a defective version of the PAH enzyme. As a consequence, individuals with this disease are unable to breakdown phenylalanine which causes its accumulation in the blood and brain ultimately causing severe damages to the brain resulting in mental retardation. Current treatment of this disease is limited severe diet restriction and one drug ([http://en.wikipedia.org/wiki/Kuvan Kuvan]). The problem is that Kuvan is in fact a drug that delivers BH4 (tetrahydropbiopterin), a cofactor to PAH, and therefore only treats roughly half of Phenylketonuriacs ([http://www.ncbi.nlm.nih.gov/pubmed/14726806 Matalon et al., 2004]). By using the E.ncapsulator system to deliver PAH to the gut, we are enabling a better control of the disease and providing a complementary solution to it.

 This module is the key customisable component of our system, allowing the scientist to develop E.ncapsulator delivery systems bearing the enzyme of choice. This customisation is achievable by replacing only one gene in the genetic circuit.


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Module 1: Genetic circuit

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