Team:Imperial College London/M1

   Module 2: Encapsulation =Module 1: Protein Production =    Module 2 Timeline

Overview
The E.ncapsulator has been designed to produce and deliver protein biopharmaceuticals to the intestine. Module 1 encompasses the protein production phase of the system. The E.ncapsulator is engineered to allow the production of any protein or polypeptide.

Rationale
In order to perform this function successfuly, the polypeptide (amino acid polymer) must be synthesised at a rate that will be sufficient to facilitate its accumulation inside the cytoplasm of the cell. With our generic design, it is possible to synthesise any polypeptide, so we have a thoroughly reusable system.  About the difference between enzymes and peptides.

Engineering a cell to produce a protein
DNA is transcribed into mRNA which is in turn translated into protein. By knowing the amino acid sequence of the polypeptide of interest, we work backwards and convert this into a DNA sequence coding for production of the protein.

In our case this has been developed further by optimising the DNA sequence for E.coli. By combining this coding sequence the other necessary genetic components, we can engineer the chassis to manufacture the protein.

Genetic circuit
This is the remaining part of the Module 1 genetic circuit

 About the genetic circuit

Polypeptide Showcase
To demonstrate The E.ncapsulator's versatility, we have chosen to showcase it with both enzymes and peptides. These two classes of polypeptide have very different properties that we have considered and catered for in The E.ncapsulator's design.

Enzyme Production
One of the challenges involved in enyzme production is the need for resistance to the proteases found within the small intestine. Although The E.ncapsulator is capable of delivering the enyzmes safely through the harsh environment in the stomach, upon release into the small intestine, the cells would be susceptible to breakdown by the proteases naturally found in the gut enviroment. For this reason, we have used protease resistant forms of the enyzmes to be produced. We have chosen two enzymes to showcase The E.ncapsulator's protein production module. These are:

 About cellulase and our proposed solution 
 * Cellulase - an enzyme that breaks down the tough fibrous molecule cellulose into cellobiose. In delivering cellulase to the gut we aim to increase the nutritional value of food consumed.

<img style="vertical-align:bottom;" width=50px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09Learnmore.png"></a> About the genetic condition phenylketonuria, PAH and how we modified it
 * Phenylalanine Hydroxylase - an enzyme that breaks down the amino acid phenylalanine into tyrosine. In delivering this enzyme to the gut we aim to provide a treatment to those suffering from the metabolic condition, phenylketonuria.

Peptide Production
The delivery and production of short chain peptides is a different challenge altogether. All peptides when synthesised always start with the amino acid methionine. If synthesised directly, this can mean that the peptide no longer has the same bioactivity. The body naturally has a mechanism by which larger polypeptides are degraded into smaller functional peptides.

Using this mechanism, we have designed a universal adapter for short chain peptide production and delivery, by which any peptide can be produced and delivered to the gut.

<img style="vertical-align:bottom;" width=50px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09Learnmore.png"></a> About our unique strategy for universal manufacture of peptides 

To demonstrate this, we have chosen to showcase a short chain peptide, opiorphin:
 * Opiorphin - is a small pentapeptide (5 amino acids) that is naturally produced by the body, and plays a role in pain relief and as an anti-depressant. By delivering this small peptide into the gut, we hope to offer a natural alternative to addictive drugs.

<img style="vertical-align:bottom;" width=50px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09Learnmore.png"></a> About opiorphin, and the remarkable effects of this small pentapeptide 

Wet Lab
We have planned protocols for the testing of each of our enzymes of interest, BBa_K200028 and BBa_K200007 (see Wet Lab Protocols). However, up till now, we have been unable to ligate them into a testable construct.

Dry Lab
Our Dry Lab team focused on analysing enzyme kinetics theories, such as the well-know Michaelis Menten kinetics. One of the main aims of the dry lab was to characterise the dynamics of the system, making sure enzyme delivery was suitable.

Enzyme kinetic simulations were run to model our system. These are some of the general findings: And finally:
 * For lower values of [S0], the rate of formation of the product is directly proportional to the amount of [S0]. However, for higher values of [S0], rate of formation of product saturates at a maximum rate.
 * Michaelis-Menten approximation only holds for very small values of [E0].
 * Increasing K3 values will increase rate of formation of product.
 * Michaelis-Menten assumption can be applied to our system because for us, KM >> [E0]

Conclusion
Engineering of bacteria to produce proteins is by no means a new development. However, due to the way in which we have designed our chassis to manufacture a protease resitant strains of the enzymes, as well as the unique method for the production and delivery of small peptides, we believe that The E.ncapsulator offers a novel approach to polypeptide production.

Project Tour
<img width=150px src="http://i691.photobucket.com/albums/vv271/dk806/CIL.jpg"></a><img width=150px src="http://i691.photobucket.com/albums/vv271/dk806/AIR.jpg"></a>

Module 1 Contents
<img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Homepageimage3.png"></a><img style="vertical-align:bottom;" width="18%" src="http://2009.igem.org/wiki/images/e/ea/II09_Homepageimage3.png"></a><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_geneticcircuit1.png"></a><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Wetlabmainimage9.png"></a> <img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Drylabmainimage6.png"></a> <!--

Peptide Delivery
Many peptide drugs are suseptible to breakdown in the stomach making them suitable candidates for encapsulation.

When synthesised, all polypeptides begin with the same amino acid: methionine. However, many polypeptides are subsequently chopped into smaller functional peptides that do not begin with methionine.



If we were to use natural polypeptide processing pathways, we would be forced to equip The E.ncapsulator with different enzymes for different polypeptides. We have avoided this inelegant solution and instead created a universal processing pathway that is compatible with all peptides. What is more, our system does not require the expression any additional genes!

<img style="vertical-align:bottom;" width=90px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png"></a>  About how our universal peptide processing system works.

Opiorphin
To showcase peptide delivery, we have selected the pentapeptide (five amino acids) opiorphin. This peptide is naturally found in human saliva and plays a role in pain relief and pleasure. The delivery of opiorphin by The E.ncapsulator, marks the iGEM first entry to tackle psychological problems such as chronic pain and depression.

<img style="vertical-align:bottom;" width=90px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png"></a>  About opiorphin.

Enzyme Delivery
To carry out their functions, enzymes must maintain their precise three dimensional conformations. Many enzymes denature in the acidic conditions of the stomach rendering them inactive. Even enzymes that survive stomach acid must face an assalt from stomach proteases. For these two reasons, enzymes are well suited for encapsulation. The E.ncapsulator has been showcased with two important enzymes: phenylalanine hydroxylase (PAH) and cellulase.

Phenylalanine Hydroxylase (PAH)
<img style="border:3px solid #000;" src="http://upload.wikimedia.org/wikipedia/commons/1/16/Phenylketonuria_testing.jpg" width="250">

PAH converts one amino acid (phenylalanine) into another (tyrosine). PAH is normally found in the liver, however individuals lacking this important enzyme suffer from the genetic disorder Phenylketonuria (PKU). Individuals with PKU must limit their consumption of phenylalanine otherwise its accumulation can result in problems with brain development, leading to progressive mental retardation, brain damage and seizures.

PKU is currently tested for by genetic screening newborn babies. There is no cure for this disease, and current treatments revolve around following a strict low protein diet.

<img style="vertical-align:bottom;" width=90px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png"></a>  About PKU and current treatments.

Since PAH is usually found in the liver, it unsurprisingly lacks any natural resistance to proteases found in the intestine. In order to overcome this problem we introduced a mutation into the structure of PAH to increase its resistance to proteolytic degradation. The delivery of PAH by The Encapsulator is particularly relevant on two accounts. Firstly, it represents a landmark in metabolic subcontraction and secondly, it offers a treatment for a genetic disease.

<img style="vertical-align:bottom;" width=90px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png"></a>  About PAH and how we modified it.

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Cellulose is a compound that is abundant in the human diet - it is what is commonly referred to as 'dietary fibre.' However, the human body is unable to degrade cellulose into glucose naturally, as it lacks the enzymes required to break down this fibrous material. Cellulase is an enzyme that breaks down cellulose to glucose. By engineering The E.ncapsulator to produce this enzyme and delivering it to the small intestine, we would give the consumer the ability to digest dietary fibre. This would allow more energy and nutrients to be extracted from food; that which was previously unavailable.

More information on cellulase can be found here

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Phenylalanine Hydroxylase
Phenylketonuria is a digestive disorder caused by an inability of the body to break down the amino acid phenylalanine. The amino acid accumulates in the blood, and can cause serious health problems for afflicted individuals. Current treatments for the condition revolve around following a strict very low protein diet.

Phenylalanine Hydroxylase (PAH) is an enzyme that breaks down the amino acid phenylalanine into tyrosine. By introducing this enzyme into the digestive system with The E.ncapsulator's unique drug delivery mechanism, we hope to treat the sufferers of PKU.

To find out more about phenylketonuria and PAH, click here.

=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.

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:


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 (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 (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. -->