"
Team:Brown/Project Introduction
From 2009.igem.org
(New page: {{Brown}}) |
|||
| Line 1: | Line 1: | ||
{{Brown}} | {{Brown}} | ||
| + | |||
| + | |||
| + | The Problem | ||
| + | ---- | ||
| + | |||
| + | The prevalence of food, seasonal, and other allergies has been rapidly increasing in recent times. In particular, over 50 million people in the United States suffer from allergic rhinitis, more commonly known as hay fever. This allergy is caused by allergens such as pollen or dust and causes the mucous membranes of the eyes and nose to become itchy and inflamed, resulting in irritating symptoms such as runny nose and watery eyes. Upon first contact with an allergen, plasma cells release IgE antibodies. These antibodies then activate mast cell release of histamine. When histamine reaches histamine receptors on various target cells, vasodilation and inflammation occurs, resulting in allergic symptoms. Allergies and related inflammatory disorders are common and disabling; and current treatments such as histamine-receptor blocking drugs are not satisfactory, offering only symptomatic relief rather than long term benefits. For patients suffering from chronic allergies, there is a great need for an alternative strategy for combating allergic symptoms without causing significant side effects. | ||
| + | |||
| + | The Objective | ||
| + | ---- | ||
| + | |||
| + | The 2009 Brown iGEM team aims to treat allergic rhinitis by engineering Staphylococcus epidermidis, a specimen endogenous to the nasal flora, to secrete a recombinant histamine-binding protein in response to the elevated histamine concentrations of an allergic response. The engineered strain of S. epidermidis will function as a self-regulating drug factory in the nose. Our novel strain of S. epidermidis will contain three distinct plasmids, responsible for each of the following functions: | ||
| + | |||
| + | 1. Histamine Binding and Secretion | ||
| + | 2. Histamine Sensing | ||
| + | 3. Safety Kill Switch | ||
| + | |||
| + | |||
| + | |||
| + | ---- | ||
| + | |||
| + | |||
| + | |||
| + | Part 1 | ||
| + | a) Producing Recombinant EV131 Tick Histamine Binding Protein and Testing its Competitive Binding Affinity | ||
| + | rEV131 is a high affinity histamine binding protein secreted by the tick Rhipicephalus appendiculatus. This protein allows the tick to overcome the host’s inflammatory response by sequestering histamine at the site of feeding. rEV131 has the ability to outcompete host histamine receptors for the ligand, effectively combating the allergic response. rEV131 has the highest affinity (lowest Kd) among all histamine binding proteins secreted by R. appendiculatus (Paesen, 1999). rEV131 histamine binding protein may prove to be an effective alternative to current histamine antagonists in combating allergic symptoms without causing negative side effects. EV131 has been purified, cloned, expressed and well characterized. We hope to further characterize the protein by conducting histamine-binding assays. We will vary levels of free histamine to illustrate protein-binding capacity and demonstrate protein-ligand specificity by exposing the protein to molecules similar to histamine, such as histidine and imidazole. Further research goals include investigating modifications to the EV131 protein that could enhance histamine-binding affinity, including modifying or introducing novel binding pockets and honing protein residues. | ||
| + | |||
| + | b) Secretion of rEV131 by S.epidermis, our Biological Chassis | ||
| + | Staphyloccocus epidermidis, a gram-positive bacterium endogenous to the human nasal flora, is an attractive candidate in which to implement our system. In regulated levels, it is safe and non-pathogenic. In order to secrete rEV131 from S. epidermidis, a signal peptide that can be recognized by the cell's Sec secretion pathway must be attached to target the protein to its appropriate membrane translocation sites. This secretion tag will be tested first with GFP and later attached to rEV131. | ||
| + | |||
| + | |||
| + | ---- | ||
| + | |||
| + | Part 2 | ||
| + | Creating a Novel Histamine Receptor in S.epidermidis | ||
| + | To synchronize rEV131 production with the event of an allergic attack, a histamine sensor is necessary. This sensor will respond to changing levels of histamine, so that histamine binding will be triggered only as histamine levels dramatically increase in the presence of an allergen. Because S. epidermidis does not contain histamine receptors, a novel receptor that both binds to extracellular histamine but also leads to the intracellular transcription of rEV131 must be engineered. | ||
| + | Chimeric protein Taz1 contains an aspartate-binding periplasmic domain (Tar) and a cytoplasmic kinase domain (EnvZ). EnvZ phosphorylates transcription factor OmpR, which subsequently binds and activates the OmpC promoter, leading to transcription of the downstream gene. | ||
| + | Using mutagenic PCR, specific amino acid residues of the Tar binding pocket will be randomly mutated. Testing mutants for histamine binding involves screening for expression of the reporter gene placed downstream of OmpC (RFP). The novel histamine receptor that is isolated can then serve as an effective sensor to link histamine binding to rEV131 production. | ||
| + | |||
| + | |||
| + | ---- | ||
| + | |||
| + | Part 3 | ||
| + | S.epidermidis Kill Switch: Safety Mechanism | ||
| + | Although S. epidermidis is a safe, nonpathogenic bacterium, a sufficient number of cells existing together can result in the formation of a biofilm, facilitating harmful proliferation. Each cell contributes to biofilm formation through its ability to detect neighboring cells of the same species. The promoter involved in this self-detection, Agr, normally activates biofilm formation. However, in an attempt to ensure safe handling of the strain, activation of the Agr promoter will be engineered to initiate instead the transcription of ccdB, a DNA gyrase inhibitor gene. Such a construct would result in the controlled extinction (suicide) of S. epidermidis cells when conditions for biofilm formation are favorable, ensuring that safe populations of cells can continue producing rEV131. | ||
| + | |||
| + | ---- | ||
Revision as of 02:15, 19 October 2009
The Problem
The prevalence of food, seasonal, and other allergies has been rapidly increasing in recent times. In particular, over 50 million people in the United States suffer from allergic rhinitis, more commonly known as hay fever. This allergy is caused by allergens such as pollen or dust and causes the mucous membranes of the eyes and nose to become itchy and inflamed, resulting in irritating symptoms such as runny nose and watery eyes. Upon first contact with an allergen, plasma cells release IgE antibodies. These antibodies then activate mast cell release of histamine. When histamine reaches histamine receptors on various target cells, vasodilation and inflammation occurs, resulting in allergic symptoms. Allergies and related inflammatory disorders are common and disabling; and current treatments such as histamine-receptor blocking drugs are not satisfactory, offering only symptomatic relief rather than long term benefits. For patients suffering from chronic allergies, there is a great need for an alternative strategy for combating allergic symptoms without causing significant side effects.
The Objective
The 2009 Brown iGEM team aims to treat allergic rhinitis by engineering Staphylococcus epidermidis, a specimen endogenous to the nasal flora, to secrete a recombinant histamine-binding protein in response to the elevated histamine concentrations of an allergic response. The engineered strain of S. epidermidis will function as a self-regulating drug factory in the nose. Our novel strain of S. epidermidis will contain three distinct plasmids, responsible for each of the following functions:
1. Histamine Binding and Secretion 2. Histamine Sensing 3. Safety Kill Switch
Part 1 a) Producing Recombinant EV131 Tick Histamine Binding Protein and Testing its Competitive Binding Affinity rEV131 is a high affinity histamine binding protein secreted by the tick Rhipicephalus appendiculatus. This protein allows the tick to overcome the host’s inflammatory response by sequestering histamine at the site of feeding. rEV131 has the ability to outcompete host histamine receptors for the ligand, effectively combating the allergic response. rEV131 has the highest affinity (lowest Kd) among all histamine binding proteins secreted by R. appendiculatus (Paesen, 1999). rEV131 histamine binding protein may prove to be an effective alternative to current histamine antagonists in combating allergic symptoms without causing negative side effects. EV131 has been purified, cloned, expressed and well characterized. We hope to further characterize the protein by conducting histamine-binding assays. We will vary levels of free histamine to illustrate protein-binding capacity and demonstrate protein-ligand specificity by exposing the protein to molecules similar to histamine, such as histidine and imidazole. Further research goals include investigating modifications to the EV131 protein that could enhance histamine-binding affinity, including modifying or introducing novel binding pockets and honing protein residues.
b) Secretion of rEV131 by S.epidermis, our Biological Chassis Staphyloccocus epidermidis, a gram-positive bacterium endogenous to the human nasal flora, is an attractive candidate in which to implement our system. In regulated levels, it is safe and non-pathogenic. In order to secrete rEV131 from S. epidermidis, a signal peptide that can be recognized by the cell's Sec secretion pathway must be attached to target the protein to its appropriate membrane translocation sites. This secretion tag will be tested first with GFP and later attached to rEV131.
Part 2 Creating a Novel Histamine Receptor in S.epidermidis To synchronize rEV131 production with the event of an allergic attack, a histamine sensor is necessary. This sensor will respond to changing levels of histamine, so that histamine binding will be triggered only as histamine levels dramatically increase in the presence of an allergen. Because S. epidermidis does not contain histamine receptors, a novel receptor that both binds to extracellular histamine but also leads to the intracellular transcription of rEV131 must be engineered. Chimeric protein Taz1 contains an aspartate-binding periplasmic domain (Tar) and a cytoplasmic kinase domain (EnvZ). EnvZ phosphorylates transcription factor OmpR, which subsequently binds and activates the OmpC promoter, leading to transcription of the downstream gene. Using mutagenic PCR, specific amino acid residues of the Tar binding pocket will be randomly mutated. Testing mutants for histamine binding involves screening for expression of the reporter gene placed downstream of OmpC (RFP). The novel histamine receptor that is isolated can then serve as an effective sensor to link histamine binding to rEV131 production.
Part 3 S.epidermidis Kill Switch: Safety Mechanism Although S. epidermidis is a safe, nonpathogenic bacterium, a sufficient number of cells existing together can result in the formation of a biofilm, facilitating harmful proliferation. Each cell contributes to biofilm formation through its ability to detect neighboring cells of the same species. The promoter involved in this self-detection, Agr, normally activates biofilm formation. However, in an attempt to ensure safe handling of the strain, activation of the Agr promoter will be engineered to initiate instead the transcription of ccdB, a DNA gyrase inhibitor gene. Such a construct would result in the controlled extinction (suicide) of S. epidermidis cells when conditions for biofilm formation are favorable, ensuring that safe populations of cells can continue producing rEV131.
