http://2009.igem.org/wiki/index.php?title=Special:Contributions/Ivorugan&feed=atom&limit=50&target=Ivorugan&year=&month=2009.igem.org - User contributions [en]2024-03-29T07:59:16ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Brown/TeamTeam:Brown/Team2009-10-22T03:24:49Z<p>Ivorugan: </p>
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<div>{{Brown}}<br />
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[[Image:About-brown-igem-bar-1.png]]<br />
The Brown iGEM Lab is entirely student-run and consists of nine Brown University undergraduates. We began our training last spring with a lab course in Synthetic Biology. Under the gracious support of the Brown Undergraduate Teaching and Research Award (UTRA) Program and funding from various other academic departments, we were able to work throughout the summer on our project. Though the project's generation and implementation was an entirely student-directed process, the team owes much of its academic and research support to the faculty and graduate student advisers that helped make everything possible. Here at Brown, we pride ourselves in upholding the ideals set out by the iGEM competition, namely that the project we have set out to create is fully our own (from creation to completion) and that each student involved in the program be afforded his/her full opportunity to both learn and contribute in the lab. Therefore, in the true spirit of Synthetic Biology, our team's project this year works hard to reflect the many different research backgrounds contributed by its nine individual members; elements of electrical engineering, electrochemistry, genetics, and microbiology are incorporated. <br />
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'''Advisors:'''<br />
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*''' Faculty Advisor ''': Dr. Gary Wessel, Professor of Biology, Bio Med Molecular, Cellular Biology Biochemistry <br />
*'''Graduate Student Advisor ''': Adrian Reich, Graduate Student, Bio-Med (Bio) <br />
*'''Graduate Student Advisor ''': Diana Donovan, Graduate Student, Bio-Med (Bio)<br />
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'''Undergraduate Students:'''<br />
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*'''William Allen''': Will Allenquot<br />
*'''Michael Chang''': MC Mastermix<br />
*'''Stephanie Cheung''': Stephylococcus <br />
*'''Ashley Kim''': Ashley Kimwipe<br />
*'''Flora War War Ko''': Nasal Flora<br />
*'''Elias Scheer''': E.coli Scheer<br />
*'''Minoo Ramanathan''': Minoo Prep<br />
*'''Ahmad Rana''': Ahmad Ran-a-gel<br />
*'''Indu Voruganti''': The INDUcer<br />
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<gallery><br />
Image:Garywessel.jpg|Dr. Gary Wessel<br />
Image:Adrianreich.jpg|Adrian Reich<br />
Image:Diana D.JPG|Diana Donovan<br />
</gallery><br />
<gallery><br />
Image:Will.jpg|Will Allen<br />
Image:Michael.jpg|Michael Chang<br />
Image:Steph.jpg|Steph Cheung<br />
Image:Ashley.jpg|Ashley Kim<br />
Image:flora.jpg|Flora Ko<br />
Image:eli.jpg|Eli Scheer<br />
Image:ahmad.jpg|Ahmad Rana<br />
Image:minoo.jpg|Minoo Ramanathan<br />
Image:indu.jpg|Indu Voruganti<br />
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Team Brown is proud to collaborate with Team Valencia in raising awareness about '''human practices''' , '''safety''', '''ethics''', '''patents''', etc. in synthetic biology. Our team had 100% participation in the survey prepared by Team Valencia. <br />
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[[Image:V_Brown.jpg]]</div>Ivoruganhttp://2009.igem.org/Team:Brown/TeamTeam:Brown/Team2009-10-22T03:23:36Z<p>Ivorugan: </p>
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<div>{{Brown}}<br />
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<br />
[[Image:About-brown-igem-bar-1.png]]<br />
The Brown iGEM Lab is entirely student-run and consists of nine Brown University undergraduates. We began our training last spring with a lab course in Synthetic Biology. Under the gracious support of the Brown Undergraduate Teaching and Research Award (UTRA) Program and funding from various other academic departments, we were able to work throughout the summer on our project. Though the project's generation and implementation was an entirely student-directed process, the team owes much of its academic and research support to the faculty and graduate student advisers that helped make everything possible. Here at Brown, we pride ourselves in upholding the ideals set out by the iGEM competition, namely that the project we have set out to create is fully our own (from creation to completion) and that each student involved in the program be afforded his/her full opportunity to both learn and contribute in the lab. Therefore, in the true spirit of Synthetic Biology, our team's project this year works hard to reflect the many different research backgrounds contributed by its nine individual members; elements of electrical engineering, electrochemistry, genetics, and microbiology are incorporated. <br />
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'''Advisors:'''<br />
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*''' Faculty Advisor ''': Dr. Gary Wessel, Professor of Biology, Bio Med Molecular, Cellular Biology Biochemistry <br />
*'''Graduate Student Advisor ''': Adrian Reich, Graduate Student, Bio-Med (Bio) <br />
*'''Graduate Student Advisor ''': Diana Donovan, Graduate Student, Bio-Med (Bio)<br />
<br />
'''Undergraduate Students:'''<br />
<br />
*'''William Allen''': Will Allenquot<br />
*'''Michael Chang''': MC Mastermix<br />
*'''Stephanie Cheung''': Stephylococcus <br />
*'''Ashley Kim''': Ashley Kimwipe<br />
*'''Flora War War Ko''': Nasal Flora<br />
*'''Elias Scheer''': E.coli Scheer<br />
*'''Minoo Ramanathan''': Minoo Prep<br />
*'''Ahmad Rana''': Ahmad Ran-a-gel<br />
*'''Indu Voruganti''': The INDUcer<br />
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|<br />
<gallery><br />
Image:Garywessel.jpg|Dr. Gary Wessel<br />
Image:Adrianreich.jpg|Adrian Reich<br />
Image:Diana D.JPG|Diana Donovan<br />
</gallery><br />
<gallery><br />
Image:Will.jpg|Will Allen<br />
Image:Michael.jpg|Michael Chang<br />
Image:Steph.jpg|Steph Cheung<br />
Image:Ashley.jpg|Ashley Kim<br />
Image:flora.jpg|Flora Ko<br />
Image:eli.jpg|Eli Scheer<br />
Image:ahmad.jpg|Ahmad Rana<br />
Image:minoo.jpg|Minoo Ramanathan<br />
Image:indu.jpg|Indu Voruganti<br />
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<br />
<br />
Team Brown is proud to collaborate with Team Valencia in raising awareness about '''human practices''' , '''safety''', '''ethics''', '''patents''', etc. in synthetic biology. Our team had 100% participation in the survey prepared by Team Valencia. <br />
<br />
[[Image:Image:V_Brown.jpg]]</div>Ivoruganhttp://2009.igem.org/Team:Brown/TeamTeam:Brown/Team2009-10-22T03:18:40Z<p>Ivorugan: </p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:About-brown-igem-bar-1.png]]<br />
The Brown iGEM Lab is entirely student-run and consists of nine Brown University undergraduates. We began our training last spring with a lab course in Synthetic Biology. Under the gracious support of the Brown Undergraduate Teaching and Research Award (UTRA) Program and funding from various other academic departments, we were able to work throughout the summer on our project. Though the project's generation and implementation was an entirely student-directed process, the team owes much of its academic and research support to the faculty and graduate student advisers that helped make everything possible. Here at Brown, we pride ourselves in upholding the ideals set out by the iGEM competition, namely that the project we have set out to create is fully our own (from creation to completion) and that each student involved in the program be afforded his/her full opportunity to both learn and contribute in the lab. Therefore, in the true spirit of Synthetic Biology, our team's project this year works hard to reflect the many different research backgrounds contributed by its nine individual members; elements of electrical engineering, electrochemistry, genetics, and microbiology are incorporated. <br />
<br />
<br />
{|border = "0"<br />
|-<br />
|rowspan="0"|<br />
<br />
'''Advisors:'''<br />
<br />
*''' Faculty Advisor ''': Dr. Gary Wessel, Professor of Biology, Bio Med Molecular, Cellular Biology Biochemistry <br />
*'''Graduate Student Advisor ''': Adrian Reich, Graduate Student, Bio-Med (Bio) <br />
*'''Graduate Student Advisor ''': Diana Donovan, Graduate Student, Bio-Med (Bio)<br />
<br />
'''Undergraduate Students:'''<br />
<br />
*'''William Allen''': Will Allenquot<br />
*'''Michael Chang''': MC Mastermix<br />
*'''Stephanie Cheung''': Stephylococcus <br />
*'''Ashley Kim''': Ashley Kimwipe<br />
*'''Flora War War Ko''': Nasal Flora<br />
*'''Elias Scheer''': E.coli Scheer<br />
*'''Minoo Ramanathan''': Minoo Prep<br />
*'''Ahmad Rana''': Ahmad Ran-a-gel<br />
*'''Indu Voruganti''': The INDUcer<br />
<br />
|<br />
<gallery><br />
Image:Garywessel.jpg|Dr. Gary Wessel<br />
Image:Adrianreich.jpg|Adrian Reich<br />
Image:Diana D.JPG|Diana Donovan<br />
</gallery><br />
<gallery><br />
Image:Will.jpg|Will Allen<br />
Image:Michael.jpg|Michael Chang<br />
Image:Steph.jpg|Steph Cheung<br />
Image:Ashley.jpg|Ashley Kim<br />
Image:flora.jpg|Flora Ko<br />
Image:eli.jpg|Eli Scheer<br />
Image:ahmad.jpg|Ahmad Rana<br />
Image:minoo.jpg|Minoo Ramanathan<br />
Image:indu.jpg|Indu Voruganti<br />
<br />
<br />
<br />
Team Brown is proud to collaborate with Team Valencia in raising awareness about '''human practices''' , '''safety''', '''ethics''', '''patents''', etc. in synthetic biology. Our team had 100% participation in the survey prepared by Team Valencia. <br />
[[Image:Image:V_Brown.jpg]]</div>Ivoruganhttp://2009.igem.org/File:V_Brown.JPGFile:V Brown.JPG2009-10-22T03:18:21Z<p>Ivorugan: uploaded a new version of "Image:V Brown.JPG"</p>
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<div></div>Ivoruganhttp://2009.igem.org/Team:Brown/ProjectTeam:Brown/Project2009-10-22T03:07:26Z<p>Ivorugan: </p>
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<div>{{Brown}}<br />
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=A Synthetic Approach to Treating Allergic Rhinitis: Engineering Staphyloccocus Epidermidis to Secrete High-Affinity Histamine Binding Protein in Response to Elevated Levels of Histamine during an Allergic Attack=<br />
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<html><img src="http://img29.imageshack.us/img29/1341/abstractm.png"></html><br />
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Brown University’s 2009 iGEM Team presents an exciting new approach to treating nasal allergies through Allergene: a synthetically engineered, self-regulating drug factory in the nose. This revolutionary new product provides a much-needed alternative to current antihistamines by directly sequestering the histamine released in an allergic response. In order to do this, Allergene makes use of the unique histamine binding protein rEV131, native to the tick Rhipicephalus appendiculatus. By taking advantage of rEV131's high binding affinity for histamine, Allergene effectively eliminates both the symptoms and side effects associated with allergies and their treatments. <br />
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Rather than presenting a system that passively sequesters histamine; however, Allergene goes one step further to providing patients with much-needed allergy relief. Activation of the product only occurs in the explicit event of an actual allergic response. This ingenious mechanism is made possible through the novel engineering of a histamine receptor in prokaryotes, a grand undertaking that had never before been accomplished. By re-designing pre-existing prokaryotic chemoreceptors to bind histamine rather than their wild-type ligands, Allergene acquired its most impressive feature yet: the "Histamine Sensor". Site-directed mutagenesis on the ligand binding pockets of two particular prokaryotic chemoreceptors, Ribose Binding Protein and Tar (normally responsive to Aspartate)was performed to create this novel sensor. Through histamine detection, Allergene’s unique histamine sequestering system is engineered to function only when histamine levels are markedly high. This efficiently timed system is therefore completely self-regulating. <br />
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Allergene is introduced to its host patients by capitalizing on the endogenous existence of bacterium Staphylococcus epidermidis in human nasal flora. This naturally present organism is the perfect vehicle for delivery; its rapid production and secretion of Allergene’s genetic constructs effectively implement the system’s histamine sensing and sequestering capabilities in human hosts. Secretion is accomplished through the attachment of a signal peptide sequence specific to S. epidermidis, a clear and concise method by which to deliver the ready-to-use drug. In order to eliminate any safety concerns associated with the utilization of S. epidermidis, special care has additionally been taken to engineer the product’s accessory kill switch mechanism. Capitalizing on the cells' abilities to sense the growth of their own populations, a DNA gyrase poison responsible for triggering cell death (CCDB)is neatly placed under the quorum or population sensing promoter Agr, which normally functions in hazardous biofilm formation. Named the “Quorum Sensor”, this thoughtful, additional feature prompts Allergene’s swift response in the unfavorable event of over-cell-proliferation.<br />
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<li id="mainHome"><a href="https://2009.igem.org/Team:Brown">Home</a></li><br />
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<li><a href="https://2009.igem.org/Team:Brown/Team">About Us</a></li><br />
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<li><a href="https://2009.igem.org/Team:Brown/Project">Abstract</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Project_Introduction">The Allergic Response</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Project_Histamine_Sensor">Histamine Sensor</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Project_HBP">Histamine Binding Protein</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Project_S.epidermidis">S.epidermidis</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Project_All_Together">System Schematic</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Project_Implications">Human Practices</a></li><br />
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</body></html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_ImplicationsTeam:Brown/Project Implications2009-10-22T03:03:03Z<p>Ivorugan: </p>
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<html><img src="http://img11.imageshack.us/img11/1575/humanpractices.png"></html><br />
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= Safety Issues =<br />
[[Image:Staphylococcus_epidermidis_biofilm.jpg|200px|thumb|right|Staphylococcus epidermidis biofilm]]<br />
This project raises safety issues due to the use of Staphylococcus epidermidis for producing and secreting the histamine binding protein, EV131. Although S. epidermidis is one of the more benign species of Staphylococcus, it can form infectious biofilms that are impervious to antibiotic treatment if its cell density becomes too great. This could pose a grave risk to researchers involved and the public for whom Allergene aims to serve.<br />
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[[Image:Quorum sensing construct.jpg|195px|thumb|left|Quorum sensing construct with CCDb gene]] This change in S. epidermidis phenotype is accomplished by the S. epidermidis agr operon, which upregulates pathogenicity genes in response to a quorum. We reasoned that we could incorporate a safety mechanism into our bacteria by putting a death gene under the regulation of the agr promoter. This way, whenever the bacteria would reach a high enough density to become dangerous, they would simply begin to die until they reach a safer, lower density. In order to first test that our cassette works, we ligated the agr promoter upstream of GFP, so that whenever the cells reached a quorum, they would fluoresce green. Later, the GFP would be switched out for a CCDb, a death gene.<br />
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= Institutional Biosafety Regulation =<br />
[[Image:Brownlogo_biohazard.jpg|125px|left|]]<br />
Any member of the Brown University faculty, staff, post-doctoral, and student bodies who plans to initiate research involving human subjects must submit a protocol for review and approval by the Institutional Review Board (IRB) prior to beginning the project. The Research Protections Office (RPO) staff provides administrative support to researchers who are preparing protocols for presentation to the IRB. <br />
<br />
The Institutional Animal Care & Use Committee (IACUC) ensures the health, well-being, and humane use of animals used in research at Brown, following the regulations and guidance of the U.S. Department of Agriculture and the Department of Health and Human Services. IACUC staff is available to answer administrative questions, and campus veterinarians can provide consultation and assistance on animal care and questions regarding experimental protocols.<br />
For our project, however, we have not yet proceeded to preclinical testing stage. For in vivo research, the Office of Environmental Health and Safety (EHS) at Brown provides compliance tools to optimize laboratory and facility conditions across research projects, from safe handling of potentially hazardous materials to biosafety training. EHS also has created guidelines to describe its emergency response system, environmental strategies, and lab equipment rules.<br />
<br />
<br />
We presented and proposed our project to the Office of Environmental Health and Safety at Brown University. Initially, we ran into concerns about the Biosafety Level 2 classification of the proposed bacteria, Staphylococcus aureus, that we planned to use for expression of the histamine binding protein. With further consideration of the biohazard implications of S. aureus, we decided instead to utilize Staphylococcus epidermidis for the expression of the histamine binding protein. Using S. epidermidis provided us with two major benefits:<br />
<br />
<br />
'''''<big>1) S. epidermidis is endogenous to the nasal flora and thus, very well-suited for accomplishing the eventual goal of our project.</big>'''''<br />
<br />
'''''<big>2) S. epidermidis is Biosafety Level 1, eliminating the fears that would have been associated with a Biosafety Level 2 bacterium.</big>'''''<br />
<br />
<br />
<br />
= Ethics, Ethics, Ethics =<br />
[[Image:ugoku-themis.gif|225px|right|]]<br />
An important concern regarding many iGEM projects, including ours, is the introduction of genetically engineered organisms into a human body. In particular, for our project we are engineering a strain of a bacterium that normally lives in a human environment without any problems, by introducing an immune response suppressant function to that organism. This raises some potential ethical problems. For example, what if the engineered bacteria an allergy sufferer takes to relieve their symptoms causes an infection?<br />
<br />
Regarding the potential concern that synthetic biologists are "playing God", with potentially harmful effects, we on the Brown team feel that any technology can be used for "good" or "bad" purposes. Just as nuclear reactions can be used for generating electricity or destroying cities, so too can synthetic biology be used for both positive and negative ends. The same techniques that allow us to genetically engineer Staphylococcus epidermidis for the "good" purpose of treating allergies could allow one to create a "bad" super multi-antibiotic resistant strain of Staphylococcus aureus (a closely related species to Staphylococcus epidermidis that is highly pathogenic, and responsible for increasing numbers of fatal infections). Unlike nuclear energy, however, this technology is so cheap and relatively easy to use that it is accessible even to high school and college students. It would be extremely difficult to create a central body that controls what end people put synthetic biological technology towards. As such, we think that it is the responsibility of everyone in the synthetic biological community to use this technology only for "good" ends, and to think about the consequences of our manipulations. We on the Brown team have tried our best to be conscious of these implications, and to engineer our machine accordingly.<br />
<br />
<html><a href: "https://2009.igem.org/Team:Brown/Parts"><img src:"https://static.igem.org/mediawiki/2009/8/89/Part_submission.png"></html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_Protocols/sdspageTeam:Brown/Notebook Protocols/sdspage2009-10-22T03:01:45Z<p>Ivorugan: </p>
<hr />
<div>{{Brown}}<br />
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<br />
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<br />
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<br />
'''SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)'''<br />
<br />
The standard method to separate proteins in a complex mixture according to mass (formally, according to their ability to migrate through the gel). The proteins are denatured by SDS and boiling, and must be stained with dyes in order to see, yet the gels can also be blotted to identify specific proteins by antibodies, or lectins. “Bands” of individual proteins can be excised and analyzed by mass spectrometry or, eluted and used to make antibodies. <br />
<br />
Components: <br />
*1) Gel box, electrode buffer, power supply, pre-pored gels.<br />
*2) Protein molecular weight marker (not 1kb ladder), 2x sample buffer, samples<br />
<br />
*The protein MW marker is in sample buffer and ready to load. Simply thaw, and load 10 μl.<br />
<br />
*Electrode buffer: per 3 liters – 43.2 grams of Glycine, 9.09 grams of TRIS base, and 15 mls of 20% SDS, pH8.0<br />
<br />
*2x Sample Buffer: 5mM TRIS, pH 6.8; 2% SDS; 5mM EDTA; 20% sucrose; and a pinch of Bromphenol blue<br />
<br />
Sample preparation: <br />
*1) For protein in aqueous solution, add equal volume of 2x sample buffer and treat at 95oC for 5 minutes. Spin for 1 minute at high speed in the microfuge and load supernatant (being careful not to disturb pellet).<br />
<br />
*2) For bacteria: pellet (spin at high speed in the microfuge for 30 seconds) appropriate amount of cells (e.g. 100ul of liquid culture). Resuspend cells in dH2O (e.g. 20 ul) by vortexing vigorously. Add equal volume of 2x sample buffer and incubate at 95oC for 5 minutes with intermittent vigorous vortexing to lyse the cells. Spin for 1 minute at high speed in the microfuge and load supernatant (being careful not to disturb pellet).<br />
<br />
*Running gels: Assemble the gels in the gel box. For one gel use a blank to form a reservoir. Remove gel comb (and perhaps label wells for easier loading), add buffer to the two chambers, wash wells with pipette, and load samples (about 10-20 μl, being careful to not bump the gel box). Run 125V for 90 minutes. Turn off power supply before disassembling the gel box. <br />
<br />
*Staining gels: Carefully (‘cause it will stain you, your clothes, shoes, teeth, fingers etc) place the gel in Coomassie Blue (or similar choice) for >4 hours on a rotary shaker (not a rocker). Destain to taste.</div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_Protocols/sdspageTeam:Brown/Notebook Protocols/sdspage2009-10-22T03:01:11Z<p>Ivorugan: New page: {{Brown}} '''SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)''' The standard method to separate proteins in a complex mixture according to mass (formally, according to their ability...</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
'''SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)'''<br />
<br />
The standard method to separate proteins in a complex mixture according to mass (formally, according to their ability to migrate through the gel). The proteins are denatured by SDS and boiling, and must be stained with dyes in order to see, yet the gels can also be blotted to identify specific proteins by antibodies, or lectins. “Bands” of individual proteins can be excised and analyzed by mass spectrometry or, eluted and used to make antibodies. <br />
<br />
Components: <br />
*1) Gel box, electrode buffer, power supply, pre-pored gels.<br />
*2) Protein molecular weight marker (not 1kb ladder), 2x sample buffer, samples<br />
<br />
*The protein MW marker is in sample buffer and ready to load. Simply thaw, and load 10 μl.<br />
<br />
*Electrode buffer: per 3 liters – 43.2 grams of Glycine, 9.09 grams of TRIS base, and 15 mls of 20% SDS, pH8.0<br />
<br />
*2x Sample Buffer: 5mM TRIS, pH 6.8; 2% SDS; 5mM EDTA; 20% sucrose; and a pinch of Bromphenol blue<br />
<br />
Sample preparation: <br />
*1) For protein in aqueous solution, add equal volume of 2x sample buffer and treat at 95oC for 5 minutes. Spin for 1 minute at high speed in the microfuge and load supernatant (being careful not to disturb pellet).<br />
<br />
*2) For bacteria: pellet (spin at high speed in the microfuge for 30 seconds) appropriate amount of cells (e.g. 100ul of liquid culture). Resuspend cells in dH2O (e.g. 20 ul) by vortexing vigorously. Add equal volume of 2x sample buffer and incubate at 95oC for 5 minutes with intermittent vigorous vortexing to lyse the cells. Spin for 1 minute at high speed in the microfuge and load supernatant (being careful not to disturb pellet).<br />
<br />
*Running gels: Assemble the gels in the gel box. For one gel use a blank to form a reservoir. Remove gel comb (and perhaps label wells for easier loading), add buffer to the two chambers, wash wells with pipette, and load samples (about 10-20 μl, being careful to not bump the gel box). Run 125V for 90 minutes. Turn off power supply before disassembling the gel box. <br />
<br />
*Staining gels: Carefully (‘cause it will stain you, your clothes, shoes, teeth, fingers etc) place the gel in Coomassie Blue (or similar choice) for >4 hours on a rotary shaker (not a rocker). Destain to taste.</div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_ImplicationsTeam:Brown/Project Implications2009-10-22T02:58:14Z<p>Ivorugan: </p>
<hr />
<div>{{Brown}}<br />
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<br />
<br />
__NOTOC__<br />
<html><img src="http://img11.imageshack.us/img11/1575/humanpractices.png"></html><br />
<br />
= Safety Issues =<br />
[[Image:Staphylococcus_epidermidis_biofilm.jpg|200px|thumb|right|Staphylococcus epidermidis biofilm]]<br />
This project raises safety issues due to the use of Staphylococcus epidermidis for producing and secreting the histamine binding protein, EV131. Although S. epidermidis is one of the more benign species of Staphylococcus, it can form infectious biofilms that are impervious to antibiotic treatment if its cell density becomes too great. This could pose a grave risk to researchers involved and the public for whom Allergene aims to serve.<br />
<br />
<br />
[[Image:Quorum sensing construct.jpg|195px|thumb|left|Quorum sensing construct with CCDb gene]] This change in S. epidermidis phenotype is accomplished by the S. epidermidis agr operon, which upregulates pathogenicity genes in response to a quorum. We reasoned that we could incorporate a safety mechanism into our bacteria by putting a death gene under the regulation of the agr promoter. This way, whenever the bacteria would reach a high enough density to become dangerous, they would simply begin to die until they reach a safer, lower density. In order to first test that our cassette works, we ligated the agr promoter upstream of GFP, so that whenever the cells reached a quorum, they would fluoresce green. Later, the GFP would be switched out for a CCDb, a death gene.<br />
<br />
<br />
<br />
<br />
= Institutional Biosafety Regulation =<br />
[[Image:Brownlogo_biohazard.jpg|125px|left|]]<br />
Any member of the Brown University faculty, staff, post-doctoral, and student bodies who plans to initiate research involving human subjects must submit a protocol for review and approval by the Institutional Review Board (IRB) prior to beginning the project. The Research Protections Office (RPO) staff provides administrative support to researchers who are preparing protocols for presentation to the IRB. <br />
<br />
The Institutional Animal Care & Use Committee (IACUC) ensures the health, well-being, and humane use of animals used in research at Brown, following the regulations and guidance of the U.S. Department of Agriculture and the Department of Health and Human Services. IACUC staff is available to answer administrative questions, and campus veterinarians can provide consultation and assistance on animal care and questions regarding experimental protocols.<br />
For our project, however, we have not yet proceeded to preclinical testing stage. For in vivo research, the Office of Environmental Health and Safety (EHS) at Brown provides compliance tools to optimize laboratory and facility conditions across research projects, from safe handling of potentially hazardous materials to biosafety training. EHS also has created guidelines to describe its emergency response system, environmental strategies, and lab equipment rules.<br />
<br />
<br />
We presented and proposed our project to the Office of Environmental Health and Safety at Brown University. Initially, we ran into concerns about the Biosafety Level 2 classification of the proposed bacteria, Staphylococcus aureus, that we planned to use for expression of the histamine binding protein. With further consideration of the biohazard implications of S. aureus, we decided instead to utilize Staphylococcus epidermidis for the expression of the histamine binding protein. Using S. epidermidis provided us with two major benefits:<br />
<br />
<br />
'''''<big>1) S. epidermidis is endogenous to the nasal flora and thus, very well-suited for accomplishing the eventual goal of our project.</big>'''''<br />
<br />
'''''<big>2) S. epidermidis is Biosafety Level 1, eliminating the fears that would have been associated with a Biosafety Level 2 bacterium.</big>'''''<br />
<br />
<br />
<br />
= Ethics, Ethics, Ethics =<br />
[[Image:ugoku-themis.gif|225px|right|]]<br />
An important concern regarding many iGEM projects, including ours, is the introduction of genetically engineered organisms into a human body. In particular, for our project we are engineering a strain of a bacterium that normally lives in a human environment without any problems, by introducing an immune response suppressant function to that organism. This raises some potential ethical problems. For example, what if the engineered bacteria an allergy sufferer takes to relieve their symptoms causes an infection?<br />
<br />
Regarding the potential concern that synthetic biologists are "playing God", with potentially harmful effects, we on the Brown team feel that any technology can be used for "good" or "bad" purposes. Just as nuclear reactions can be used for generating electricity or destroying cities, so too can synthetic biology be used for both positive and negative ends. The same techniques that allow us to genetically engineer Staphylococcus epidermidis for the "good" purpose of treating allergies could allow one to create a "bad" super multi-antibiotic resistant strain of Staphylococcus aureus (a closely related species to Staphylococcus epidermidis that is highly pathogenic, and responsible for increasing numbers of fatal infections). Unlike nuclear energy, however, this technology is so cheap and relatively easy to use that it is accessible even to high school and college students. It would be extremely difficult to create a central body that controls what end people put synthetic biological technology towards. As such, we think that it is the responsibility of everyone in the synthetic biological community to use this technology only for "good" ends, and to think about the consequences of our manipulations. We on the Brown team have tried our best to be conscious of these implications, and to engineer our machine accordingly.<br />
<br />
[[Image:Part_submission.png]]</div>Ivoruganhttp://2009.igem.org/File:Part_submission.pngFile:Part submission.png2009-10-22T02:57:34Z<p>Ivorugan: </p>
<hr />
<div></div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_IntroductionTeam:Brown/Project Introduction2009-10-22T02:54:37Z<p>Ivorugan: </p>
<hr />
<div>{{Brown}}<br />
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<br />
<br />
<br />
<br />
<html><img src="http://img132.imageshack.us/img132/4105/theallergiceresponse.png"></html><br />
<br />
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. Histamine has been identified as a principal mediator of inflammatory responses. Upon first contact with an allergen, plasma cells release Immunoglobulin E (IgE) antibodies. These antibodies then activate mast cell degranulation and release of histamine. When histamine reaches histamine receptors on various target cells, vasodilation and inflammation occurs, resulting in allergic symptoms. <br />
<br />
Allergies are routinely treated with antihistamine drugs, which have many adverse effects. Antihistamines compete with histamine to bind and block these histamine receptors, preventing the initiation of the inflammatory response. However, antihistamines also block receptors of the nervous system, thereby causing drowsiness. For many people, sedation remains the primary concern when considering the adverse effects of the newer antihistamines, particularly since these drugs are given to patients with chronic disorders with treatment periods that often extend over several months or even years. Besides sedation, there also exists concern regarding the caridotoxicity of antihistamines and other adverse drug interactions. For patients suffering from chronic allergies and inflammation, there is a great need for an alternative strategy for combating allergic symptoms without causing significant side effects.<br />
<br />
<br />
<html><center><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/b4Bgia6_54c&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/b4Bgia6_54c&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object></center></html><br />
<br />
<br />
Allergene presents a favorable alternative because it utilizes the binding affinity of a histamine binding protein rEV131. Similar to antihistamines, this protein prevents histamine molecules from interacting with histamine. However, by directly sequestering the histamine molecules rather than blocking their receptors, the drowsiness side-effect is successfully avoided. <br />
<br />
<br />
<html><br />
<a href="https://2009.igem.org/Team:Brown/Project_Histamine_Sensor"><br />
<img src="https://static.igem.org/mediawiki/2009/5/51/Brown-button-2-histamine-sensor.png"><br />
</center></html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Links_SponsorsTeam:Brown/Links Sponsors2009-10-22T02:54:24Z<p>Ivorugan: /* Financial, logistical, and spatial sponsors: */</p>
<hr />
<div>{{Brown}}<br />
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<br />
<br />
<br />
<html><img src="http://img29.imageshack.us/img29/7569/teamsponsors.png"></html><br />
<br />
<br />
Thank you from the 2009 Brown iGEM Team!<br />
<br />
[[Image:Brown_mdl_logo.png]]<br />
<br />
[[Image:Brown mcb.png]]<br />
<br />
[[Image:Brown primo.png]]<br />
<br />
[[Image:Brown research logo.png]]<br />
<br />
[[Image:Brown comp bio.png]]<br />
<br />
[[Image:Brown div engineering.png]]<br />
<br />
[[Image:Brown biomed logo.png]]<br />
<br />
[[Image:Thermofisher.png]]</div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_IntroductionTeam:Brown/Project Introduction2009-10-22T02:53:46Z<p>Ivorugan: </p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<html><img src="http://img132.imageshack.us/img132/4105/theallergiceresponse.png"></html><br />
<br />
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. Histamine has been identified as a principal mediator of inflammatory responses. Upon first contact with an allergen, plasma cells release Immunoglobulin E (IgE) antibodies. These antibodies then activate mast cell degranulation and release of histamine. When histamine reaches histamine receptors on various target cells, vasodilation and inflammation occurs, resulting in allergic symptoms. <br />
<br />
Allergies are routinely treated with antihistamine drugs, which have many adverse effects. Antihistamines compete with histamine to bind and block these histamine receptors, preventing the initiation of the inflammatory response. However, antihistamines also block receptors of the nervous system, thereby causing drowsiness. For many people, sedation remains the primary concern when considering the adverse effects of the newer antihistamines, particularly since these drugs are given to patients with chronic disorders with treatment periods that often extend over several months or even years. Besides sedation, there also exists concern regarding the caridotoxicity of antihistamines and other adverse drug interactions. For patients suffering from chronic allergies and inflammation, there is a great need for an alternative strategy for combating allergic symptoms without causing significant side effects.<br />
<br />
<br />
<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/b4Bgia6_54c&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/b4Bgia6_54c&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object></html><br />
<br />
<br />
Allergene presents a favorable alternative because it utilizes the binding affinity of a histamine binding protein rEV131. Similar to antihistamines, this protein prevents histamine molecules from interacting with histamine. However, by directly sequestering the histamine molecules rather than blocking their receptors, the drowsiness side-effect is successfully avoided. <br />
<br />
<br />
<html><br />
<center><a href="https://2009.igem.org/Team:Brown/Project_Histamine_Sensor"><br />
<img src="https://static.igem.org/mediawiki/2009/5/51/Brown-button-2-histamine-sensor.png"><br />
</center></html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_IntroductionTeam:Brown/Project Introduction2009-10-22T02:51:23Z<p>Ivorugan: </p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<html><img src="http://img132.imageshack.us/img132/4105/theallergiceresponse.png"></html><br />
<br />
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. Histamine has been identified as a principal mediator of inflammatory responses. Upon first contact with an allergen, plasma cells release Immunoglobulin E (IgE) antibodies. These antibodies then activate mast cell degranulation and release of histamine. When histamine reaches histamine receptors on various target cells, vasodilation and inflammation occurs, resulting in allergic symptoms. <br />
<br />
Allergies are routinely treated with antihistamine drugs, which have many adverse effects. Antihistamines compete with histamine to bind and block these histamine receptors, preventing the initiation of the inflammatory response. However, antihistamines also block receptors of the nervous system, thereby causing drowsiness. For many people, sedation remains the primary concern when considering the adverse effects of the newer antihistamines, particularly since these drugs are given to patients with chronic disorders with treatment periods that often extend over several months or even years. Besides sedation, there also exists concern regarding the caridotoxicity of antihistamines and other adverse drug interactions. For patients suffering from chronic allergies and inflammation, there is a great need for an alternative strategy for combating allergic symptoms without causing significant side effects.<br />
<br />
<br />
<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/b4Bgia6_54c&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/b4Bgia6_54c&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object></html><br />
<br />
<br />
Allergene presents a favorable alternative because it utilizes the binding affinity of a histamine binding protein rEV131. Similar to antihistamines, this protein prevents histamine molecules from interacting with histamine. However, by directly sequestering the histamine molecules rather than blocking their receptors, the drowsiness side-effect is successfully avoided. <br />
<br />
<br />
<html><br />
<a href="https://2009.igem.org/Team:Brown/Project_Histamine_Sensor"><br />
<img src="https://static.igem.org/mediawiki/2009/5/51/Brown-button-2-histamine-sensor.png"><br />
</html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Links_AcknowledgementsTeam:Brown/Links Acknowledgements2009-10-22T02:51:10Z<p>Ivorugan: </p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<html><img src="http://img18.imageshack.us/img18/6854/acknow.png"></html><br />
<br />
'''We would like to thank the following individuals without whom this project would not have been made possible:'''<br />
<br />
<br />
*Dr. '''Gary Wessel''', for his invaluable advice, unremitting patience, continuous encouragement and most of all, tremendous enthusiasm for our project.<br />
<br />
*Graduate students '''Adrian Reich''', iGEM Adviser, and '''Diana Donovan''', for their continuous guidance and experimental support throughout the project. <br />
<br />
*'''John Szymanski''', iGEM alum, for his original competent cells protocol and generous assistance in the lab. <br />
<br />
<br />
<br />
*'''Adella Francis''', for her consistent and generous administrative assistance for Brown iGEM teams past and present.<br />
<br />
*'''John Cumbers''', Founder and hardcore supporter of Brown iGEM Teams.<br />
<br />
<br />
*The '''PRIMO Lab''', for their generous assistance in research protocols and laboratory facility use.<br />
<br />
*The '''Barnea Lab''', for the use of their laboratory facilities.<br />
<br />
*The Faculty Panel, for its invaluable support and advice.<br />
<br />
<br />
*The '''Brown UTRA Program''', for undergraduate summer research funding at Brown University. <br />
<br />
*Brown University Departments of Biology and Medicine, Engineering, Computational Biology, Molecular, Cell Biology, and Biochemistry.<br />
<br />
*'''Neil Parikh''', '''Kate Jacobs''', '''Rima Shah''', '''John Szymanski''' and '''Aaron Glieberman''': Former Brown iGEM Team Members who helped train and guide us, setting a high standard for all future iGEM Mentors. <br />
<br />
<br />
<br />
----<br />
<br />
<br />
*Dr. '''Guido Paesen''', Centre for Ecology & Hydrology, Oxford: for providing the initial inspiration to propose a project concerning the practical implications of a histamine binding protein, and also for generously sharing rEV131.<br />
<br />
*Dr. '''Loren Looger''', Howard Hughes Medical Institute: for the use of his computational protein design program to calculate mutations that would transform Tar’s aspartate binding pocket to one that selectively binds histamine. <br />
<br />
*Dr. '''Masayori Inouye''', Rutgers University: for his generous provision of Tar-EnvZ.<br />
<br />
*Dr. '''Luciano Marraffini''', Sontheimer Lab at Northwestern University, for the provision of plasmid pLM6, a shuttle vector between S.epidermidis and E.coli.<br />
<br />
*Dr. '''Reinholb Bruckner''', for the provision of shuttle vectors PRB474 and PRB473. <br />
<br />
<br />
----</div>Ivoruganhttp://2009.igem.org/Team:Brown/Links_AcknowledgementsTeam:Brown/Links Acknowledgements2009-10-22T02:50:42Z<p>Ivorugan: /* Acknowledgements */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<html><img src="http://img18.imageshack.us/img18/6854/acknow.png"></html><br />
<br />
<br />
<br />
'''We would like to thank the following individuals without whom this project would not have been made possible:'''<br />
<br />
<br />
*Dr. '''Gary Wessel''', for his invaluable advice, unremitting patience, continuous encouragement and most of all, tremendous enthusiasm for our project.<br />
<br />
*Graduate students '''Adrian Reich''', iGEM Adviser, and '''Diana Donovan''', for their continuous guidance and experimental support throughout the project. <br />
<br />
*'''John Szymanski''', iGEM alum, for his original competent cells protocol and generous assistance in the lab. <br />
<br />
<br />
<br />
*'''Adella Francis''', for her consistent and generous administrative assistance for Brown iGEM teams past and present.<br />
<br />
*'''John Cumbers''', Founder and hardcore supporter of Brown iGEM Teams.<br />
<br />
<br />
*The '''PRIMO Lab''', for their generous assistance in research protocols and laboratory facility use.<br />
<br />
*The '''Barnea Lab''', for the use of their laboratory facilities.<br />
<br />
*The Faculty Panel, for its invaluable support and advice.<br />
<br />
<br />
*The '''Brown UTRA Program''', for undergraduate summer research funding at Brown University. <br />
<br />
*Brown University Departments of Biology and Medicine, Engineering, Computational Biology, Molecular, Cell Biology, and Biochemistry.<br />
<br />
*'''Neil Parikh''', '''Kate Jacobs''', '''Rima Shah''', '''John Szymanski''' and '''Aaron Glieberman''': Former Brown iGEM Team Members who helped train and guide us, setting a high standard for all future iGEM Mentors. <br />
<br />
<br />
<br />
----<br />
<br />
<br />
*Dr. '''Guido Paesen''', Centre for Ecology & Hydrology, Oxford: for providing the initial inspiration to propose a project concerning the practical implications of a histamine binding protein, and also for generously sharing rEV131.<br />
<br />
*Dr. '''Loren Looger''', Howard Hughes Medical Institute: for the use of his computational protein design program to calculate mutations that would transform Tar’s aspartate binding pocket to one that selectively binds histamine. <br />
<br />
*Dr. '''Masayori Inouye''', Rutgers University: for his generous provision of Tar-EnvZ.<br />
<br />
*Dr. '''Luciano Marraffini''', Sontheimer Lab at Northwestern University, for the provision of plasmid pLM6, a shuttle vector between S.epidermidis and E.coli.<br />
<br />
*Dr. '''Reinholb Bruckner''', for the provision of shuttle vectors PRB474 and PRB473. <br />
<br />
<br />
----</div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_IntroductionTeam:Brown/Project Introduction2009-10-22T02:50:42Z<p>Ivorugan: </p>
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<div>{{Brown}}<br />
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<html><img src="http://img132.imageshack.us/img132/4105/theallergiceresponse.png"></html><br />
<br />
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. Histamine has been identified as a principal mediator of inflammatory responses. Upon first contact with an allergen, plasma cells release Immunoglobulin E (IgE) antibodies. These antibodies then activate mast cell degranulation and release of histamine. When histamine reaches histamine receptors on various target cells, vasodilation and inflammation occurs, resulting in allergic symptoms. <br />
<br />
Allergies are routinely treated with antihistamine drugs, which have many adverse effects. Antihistamines compete with histamine to bind and block these histamine receptors, preventing the initiation of the inflammatory response. However, antihistamines also block receptors of the nervous system, thereby causing drowsiness. For many people, sedation remains the primary concern when considering the adverse effects of the newer antihistamines, particularly since these drugs are given to patients with chronic disorders with treatment periods that often extend over several months or even years. Besides sedation, there also exists concern regarding the caridotoxicity of antihistamines and other adverse drug interactions. For patients suffering from chronic allergies and inflammation, there is a great need for an alternative strategy for combating allergic symptoms without causing significant side effects.<br />
<br />
<br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/b4Bgia6_54c&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/b4Bgia6_54c&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
<br />
<br />
Allergene presents a favorable alternative because it utilizes the binding affinity of a histamine binding protein rEV131. Similar to antihistamines, this protein prevents histamine molecules from interacting with histamine. However, by directly sequestering the histamine molecules rather than blocking their receptors, the drowsiness side-effect is successfully avoided. <br />
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<html><br />
<a href="https://2009.igem.org/Team:Brown/Project_Histamine_Sensor"><br />
<img src="https://static.igem.org/mediawiki/2009/5/51/Brown-button-2-histamine-sensor.png"><br />
</html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_meetingsTeam:Brown/Notebook meetings2009-10-22T02:49:48Z<p>Ivorugan: </p>
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<html><img src="http://img30.imageshack.us/img30/8074/teammeetingminutes.png"></html><br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/4-22-09 April 22, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-4-09 May 4, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-6-09 May 6, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-27-09 May 27, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-31-09 May 31, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-3-09 June 3, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-8-09 June 8, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-15-09 June 15, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-16-09 June 16, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-17-09 June 17, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-19-09 June 19, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-22-09 June 22, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-29-09 June 29, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/7-6-09 July 6, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/7-13-09 July 13, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/8-3-09 August 3, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/8-10-09 August 10, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/8-17-09 August 17, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/9-11-09 September 11, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/9-15-09 September 15, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/10-16-09 October 16, 2009]</div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_meetingsTeam:Brown/Notebook meetings2009-10-22T02:49:32Z<p>Ivorugan: </p>
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<html><img src="http://img30.imageshack.us/img30/8074/teammeetingminutes.png"></html><br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/4-22-09 April 22, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-4-09 May 4, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-6-09 May 6, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-27-09 May 27, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-31-09 May 31, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-3-09 June 3, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-8-09 June 8, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-15-09 June 15, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-16-09 June 16, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-17-09 June 17, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-19-09 June 19, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-22-09 June 22, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-29-09 June 29, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/7-6-09 July 6, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/7-13-09 July 13, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/8-3-09 August 3, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/8-10-09 August 10, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/8-17-09 August 17, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/9-11-09 September 11, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/9-15-09 September 15, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/10-16-09 October 16, 2009]</div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_meetingsTeam:Brown/Notebook meetings2009-10-22T02:49:12Z<p>Ivorugan: </p>
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<div>{{Brown}}<br />
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<html><img src="http://img30.imageshack.us/img30/8074/teammeetingminutes.png"></html><br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/4-22-09 April 22, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-4-09 May 4, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-6-09 May 6, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-27-09 May 27, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-31-09 May 31, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-3-09 June 3, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-8-09 June 8, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-15-09 June 15, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-16-09 June 16, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-17-09 June 17, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-19-09 June 19, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-22-09 June 22, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-29-09 June 29, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/7-6-09 July 6, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/7-13-09 July 13, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/8-3-09 August 3, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/8-10-09 August 10, 2009]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Meetings/8-17-09 August 17, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/9-11-09 September 11, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/9-15-09 September 15, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/10-16-09 October 16, 2009]</div>Ivoruganhttp://2009.igem.org/Team:BrownTeam:Brown2009-10-22T02:49:09Z<p>Ivorugan: </p>
<hr />
<div>{{Brown}}<br />
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<br />
[[Image:Brownbanner.gif|center|800px]]<br />
----<br />
<br />
<br />
Every year, over '''<big>fifty million people</big>''' in the US suffer from '''<big>allergic rhinitis</big>''',<br />
the most common type of allergy. Allergens such as pollen, dust, and dander result in nasal congestion, itching, burning, sneezing, and overall '''<big>discomfort</big>'''. Current treatments include over-the-counter '''<big>antihistamines</big>''', however, side effects of these drugs include drowsiness, restlessness, and poor concentration. For patients suffering from chronic allergies and inflammation, there is a great '''<big>need for an alternative strategy</big>''' for combating allergic symptoms '''<big>without</big>''' causing '''<big>significant side effects</big>'''.<br />
<br />
<br />
The '''<big>2009 Brown University iGEM</big>''' team worked to treat allergic rhinitis by '''<big>engineering Staphylococcus epidermidis</big>''', a microbe endogenous to the human nasal flora, to '''<big>secrete</big>''' a recombinant '''<big>histamine-binding protein</big>''' in response to the elevated histamine concentrations of an allergic response. The engineered strain of S. epidermidis will function as a '''<big>self-regulating drug factory in the nose</big>''', providing relief, '''<big>without</big>''' any negative '''<big>side effects</big>'''.<br />
<br />
<br />
----<br />
<html><head><META http-equiv="Content-Type" content="text/html; charset=utf-8"></head><body><br />
<center><br />
<div><br />
<table width="827" border="5"><br />
<br />
<font size="24"><br />
<tr><br />
<td width="165" height="107"><center><a href="https://2009.igem.org/Team:Brown/Team"><img src="https://static.igem.org/mediawiki/2009/8/8c/Teambutton.gif" width="168" height="100" align="absmiddle" longdesc="https://static.igem.org/mediawiki/2009/8/8c/Teambutton.gif"></td><br />
<br />
<td width="164" height="107"><center><a href="https://2009.igem.org/Team:Brown/Parts"><img src="https://static.igem.org/mediawiki/2009/3/33/Partsbutton.gif" alt="" width="161" height="100" longdesc="https://static.igem.org/mediawiki/2009/3/33/Partsbutton.gif"></td><br />
<br />
<td width="154" height="107"><center><a href="https://2009.igem.org/Team:Brown/Notebook_Weekly_Logs"><img src="https://static.igem.org/mediawiki/2009/c/ce/Notebookbutton.gif" width="157" height="100"></td><br />
<br />
<td width="154"><center><a href="https://2009.igem.org/Team:Brown/Project_Introduction"><img src="https://static.igem.org/mediawiki/2009/6/6e/Learnmoreallergiesbutton.gif" alt="" width="150" height="100" longdesc="https://static.igem.org/mediawiki/2009/6/6e/Learnmoreallergiesbutton.gif"></td><br />
<br />
<td width="156"><center><a href="https://2009.igem.org/Team:Brown/Project_Implications"><img src="https://static.igem.org/mediawiki/2009/9/97/Implicationsbutton.gif" alt="" width="157" height="100" longdesc="https://static.igem.org/mediawiki/2009/9/97/Implicationsbutton.gif"></td><br />
</tr><br />
<br />
<tr><br />
<td colspan="2"><a href="https://2009.igem.org/Team:Brown/Project"><img src="https://static.igem.org/mediawiki/2009/2/2a/Projectabstractbutton.gif" width="335" height="100"></td><br />
<br />
<td colspan="3">&nbsp;<i>Learn more about Allergene: a synthetically engineered, self-regulating drug factory in the &nbsp;nose.</i> </td><br />
</tr><br />
<tr><br />
<td colspan="2"><a href="https://2009.igem.org/Team:Brown/Project_Histamine_Sensor"><img src="https://static.igem.org/mediawiki/2009/d/d0/Histaminesensorbutton.gif" width="335" height="100"></td><br />
<td colspan="3">&nbsp;<i>When an allergic response occurs, our system senses a change in histamine levels above &nbsp;threshold and initiates an intracellular cascade, signaling cells to respond appropriately to &nbsp;the increase in histamine concentration.</i> </td><br />
</tr><br />
<tr><br />
<br />
<tr><br />
<td colspan="2"><a href="https://2009.igem.org/Team:Brown/Project_HBP"><img src="https://static.igem.org/mediawiki/2009/7/70/Hbpbutton.gif" alt="" width="335" height="100"></td><br />
<td colspan="3">&nbsp;<i>EV131 is a high-affinity histamine binding protein, that originates from the saliva of a &nbsp;female tick species.</i> </td><br />
</tr><br />
<td colspan="2"><a href="https://2009.igem.org/Team:Brown/Project_S.epidermidis"><img src="https://static.igem.org/mediawiki/2009/3/3c/S.epibutton.gif" alt="" width="335" height="100"></td><br />
<td colspan="3">&nbsp;<i>The chassis of choice for production and secretion of the Histamine Binding Protein is &nbsp;Staphyloccocus Epidermidis, an organism endogenous to the human nasal flora. A quorum &nbsp;sensing mechanism was also incorporated to regulate cell density.</i> </td><br />
</tr><br />
<tr><br />
<td colspan="2"><a href="https://2009.igem.org/Team:Brown/Project_All_Together"><img src="https://static.igem.org/mediawiki/2009/1/12/Alltogetherbutton.gif" alt="All Together" width="335" height="100"></a></td><br />
<td colspan="3">&nbsp;<i>What happens when an allergy attacks: Description of the overall construct mechanism in S.&nbsp;epidermidis incorporating the histamine sensor, intracellular signal cascade, and &nbsp;transcription of EV131 ligated to secretion peptide. </i> </td><br />
</tr><br />
</table><br />
</div><br />
</font><br />
</center><br />
</body></html><br />
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----<br />
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'''A special thank you to Team Heidelberg for inspiring our wiki design!'''</div>Ivoruganhttp://2009.igem.org/Team:BrownTeam:Brown2009-10-22T02:48:45Z<p>Ivorugan: </p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:Brownbanner.gif|center|800px]]<br />
----<br />
<br />
<br />
Every year, over '''<big>fifty million people</big>''' in the US suffer from '''<big>allergic rhinitis</big>''',<br />
the most common type of allergy. Allergens such as pollen, dust, and dander result in nasal congestion, itching, burning, sneezing, and overall '''<big>discomfort</big>'''. Current treatments include over-the-counter '''<big>antihistamines</big>''', however, side effects of these drugs include drowsiness, restlessness, and poor concentration. For patients suffering from chronic allergies and inflammation, there is a great '''<big>need for an alternative strategy</big>''' for combating allergic symptoms '''<big>without</big>''' causing '''<big>significant side effects</big>'''.<br />
<br />
<br />
The '''<big>2009 Brown University iGEM</big>''' team worked to treat allergic rhinitis by '''<big>engineering Staphylococcus epidermidis</big>''', a microbe endogenous to the human nasal flora, to '''<big>secrete</big>''' a recombinant '''<big>histamine-binding protein</big>''' in response to the elevated histamine concentrations of an allergic response. The engineered strain of S. epidermidis will function as a '''<big>self-regulating drug factory in the nose</big>''', providing relief, '''<big>without</big>''' any negative '''<big>side effects</big>'''.<br />
<br />
<br />
----<br />
<html><head><META http-equiv="Content-Type" content="text/html; charset=utf-8"></head><body><br />
<center><br />
<div><br />
<table width="827" border="5"><br />
<br />
<font size="24"><br />
<tr><br />
<td width="165" height="107"><center><a href="https://2009.igem.org/Team:Brown/Team"><img src="https://static.igem.org/mediawiki/2009/8/8c/Teambutton.gif" width="168" height="100" align="absmiddle" longdesc="https://static.igem.org/mediawiki/2009/8/8c/Teambutton.gif"></td><br />
<br />
<td width="164" height="107"><center><a href="https://2009.igem.org/Team:Brown/Parts"><img src="https://static.igem.org/mediawiki/2009/3/33/Partsbutton.gif" alt="" width="161" height="100" longdesc="https://static.igem.org/mediawiki/2009/3/33/Partsbutton.gif"></td><br />
<br />
<td width="154" height="107"><center><a href="https://2009.igem.org/Team:Brown/Notebook_Weekly_Logs"><img src="https://static.igem.org/mediawiki/2009/c/ce/Notebookbutton.gif" width="157" height="100"></td><br />
<br />
<td width="154"><center><a href="https://2009.igem.org/Team:Brown/Project_Introduction"><img src="https://static.igem.org/mediawiki/2009/6/6e/Learnmoreallergiesbutton.gif" alt="" width="150" height="100" longdesc="https://static.igem.org/mediawiki/2009/6/6e/Learnmoreallergiesbutton.gif"></td><br />
<br />
<td width="156"><center><a href="https://2009.igem.org/Team:Brown/Project_Implications"><img src="https://static.igem.org/mediawiki/2009/9/97/Implicationsbutton.gif" alt="" width="157" height="100" longdesc="https://static.igem.org/mediawiki/2009/9/97/Implicationsbutton.gif"></td><br />
</tr><br />
<br />
<tr><br />
<td colspan="2"><a href="https://2009.igem.org/Team:Brown/Project"><img src="https://static.igem.org/mediawiki/2009/2/2a/Projectabstractbutton.gif" width="335" height="100"></td><br />
<br />
<td colspan="3">&nbsp;<i>Learn more about Allergene: a synthetically engineered, self-regulating drug factory in the &nbsp;nose.</i> </td><br />
</tr><br />
<tr><br />
<td colspan="2"><a href="https://2009.igem.org/Team:Brown/Project_Histamine_Sensor"><img src="https://static.igem.org/mediawiki/2009/d/d0/Histaminesensorbutton.gif" width="335" height="100"></td><br />
<td colspan="3">&nbsp;<i>When an allergic response occurs, our system senses a change in histamine levels above &nbsp;threshold and initiates an intracellular cascade, signaling cells to respond appropriately to &nbsp;the increase in histamine concentration.</i> </td><br />
</tr><br />
<tr><br />
<br />
<tr><br />
<td colspan="2"><a href="https://2009.igem.org/Team:Brown/Project_HBP"><img src="https://static.igem.org/mediawiki/2009/7/70/Hbpbutton.gif" alt="" width="335" height="100"></td><br />
<td colspan="3">&nbsp;<i>EV131 is a high-affinity histamine binding protein, that originates from the saliva of a &nbsp;female tick species.</i> </td><br />
</tr><br />
<td colspan="2"><a href="https://2009.igem.org/Team:Brown/Project_S.epidermidis"><img src="https://static.igem.org/mediawiki/2009/3/3c/S.epibutton.gif" alt="" width="335" height="100"></td><br />
<td colspan="3">&nbsp;<i>The chassis of choice for production and secretion of the Histamine Binding Protein is &nbsp;Staphyloccocus Epidermidis, an organism endogenous to the human nasal flora. A quorum &nbsp;sensing mechanism was also incorporated to regulate cell density.</i> </td><br />
</tr><br />
<tr><br />
<td colspan="2"><a href="https://2009.igem.org/Team:Brown/Project_All_Together"><img src="https://static.igem.org/mediawiki/2009/1/12/Alltogetherbutton.gif" alt="All Together" width="335" height="100"></a></td><br />
<td colspan="3">&nbsp;<i>What happens when an allergy attacks: Description of the overall construct mechanism in S. &nbsp;epidermidis incorporating the histamine sensor, intracellular signal cascade, and &nbsp;transcription of EV131 ligated to secretion peptide. </i> </td><br />
</tr><br />
</table><br />
</div><br />
</font><br />
</center><br />
</body></html><br />
<br />
----<br />
<br />
<br />
'''A special thank you to Team Heidelberg for inspiring our wiki design!'''</div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_Meetings/9-15-09Team:Brown/Notebook Meetings/9-15-092009-10-22T02:47:52Z<p>Ivorugan: New page: {{Brown}} '''iGEM Meeting''' '''B25''' '''9/15/09, 8AM''' *Team 1 update: **Need pNoTat to express EV131! **Binding assay by next week **Will try pBlueScript *Team 2 update...</p>
<hr />
<div>{{Brown}}<br />
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<br />
<br />
'''iGEM Meeting'''<br />
<br />
'''B25'''<br />
<br />
'''9/15/09, 8AM'''<br />
<br />
*Team 1 update:<br />
**Need pNoTat to express EV131! <br />
**Binding assay by next week<br />
**Will try pBlueScript <br />
<br />
*Team 2 update:<br />
**Talking to Diana to electroporate <br />
**Vector in B. subtilis<br />
**Growing B. subtilis <br />
<br />
*Team 3 update:<br />
**Mutagenesis is not working <br />
**Trg-EnvZ test too?</div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_RecipesTeam:Brown/Notebook Recipes2009-10-22T02:47:37Z<p>Ivorugan: </p>
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<html><img src="http://img132.imageshack.us/img132/2905/recipese.png"></html><br />
<br />
Making Medium<br />
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<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/LB Preparing Luria Broth]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/TSB Preparing Trypticase Soy Broth]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/LBPlate Preparing Luria Broth Plates]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/TSBPlates Preparing Trypticase Soy Broth Plates]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/SOB Preparing SOB Medium]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/SMMP Preparing SMMP Medium]<br />
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<br />
Making Antibiotic<br />
----<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/Ampicillin Preparing Ampicillin (Amp) Stock = 100x]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/Tetracycline Preparing Tetracyclin (Tet) Stock = 100x]<br />
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Making Laboratory Stocks<br />
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[https://2009.igem.org/Team:Brown/Notebook_Recipes/Glycerolstocks Preparing 15% Glycerol stock for E.Coli]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/TAE Making 1x TAE Buffer]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/Detergent Making 1% Lab Detergent]</div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_RecipesTeam:Brown/Notebook Recipes2009-10-22T02:47:27Z<p>Ivorugan: </p>
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<html><img src="http://img132.imageshack.us/img132/2905/recipese.png"></html><br />
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Making Medium<br />
----<br />
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[https://2009.igem.org/Team:Brown/Notebook_Recipes/LB Preparing Luria Broth]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/TSB Preparing Trypticase Soy Broth]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/LBPlate Preparing Luria Broth Plates]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/TSBPlates Preparing Trypticase Soy Broth Plates]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/SOB Preparing SOB Medium]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Recipes/SMMP Preparing SMMP Medium]<br />
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<br />
Making Antibiotic<br />
----<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/Ampicillin Preparing Ampicillin (Amp) Stock = 100x]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/Tetracycline Preparing Tetracyclin (Tet) Stock = 100x]<br />
<br />
<br />
<br />
Making Laboratory Stocks<br />
----<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/Glycerolstocks Preparing 15% Glycerol stock for E.Coli]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/TAE Making 1x TAE Buffer]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Recipes/Detergent Making 1% Lab Detergent]</div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_ProtocolsTeam:Brown/Notebook Protocols2009-10-22T02:46:40Z<p>Ivorugan: /* Protocols */</p>
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[https://2009.igem.org/Team:Brown/Notebook_Protocols/bacterialbasics Bacterial Basics]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Protocols/movingdna Moving DNA]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Protocols/primerresusp Primer Resuspension]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Protocols/PCR Polymerase Chain Reaction]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Protocols/glycerolstocks Making Glycerol Stocks & Plating Cells]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Protocols/redigest DNA Restriction Enzyme Digest]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Protocols/sdspage SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Protocols/immunoblotting Immunoblotting (Western Blotting)]<br />
<br />
[https://2009.igem.org/Team:Brown/Notebook_Protocols/CFU CFU Calculation for Competent Cells]</div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_Meetings/8-17-09Team:Brown/Notebook Meetings/8-17-092009-10-22T02:46:03Z<p>Ivorugan: New page: {{Brown}} '''iGEM Meeting''' '''8-17-09''' '''SFH 218''' '''9 AM''' *Team 1 update: **Today they are making the Blotto solution with 6-His **Purchased Qiagen nickel column puri...</p>
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'''iGEM Meeting'''<br />
<br />
'''8-17-09'''<br />
<br />
'''SFH 218'''<br />
<br />
'''9 AM'''<br />
<br />
*Team 1 update:<br />
**Today they are making the Blotto solution with 6-His<br />
**Purchased Qiagen nickel column purification kit<br />
**After protein comes down in a nickel column <br />
**Tests for histamine binding; selective binding?<br />
<br />
*Possible ideas for after binding test; Will suggests Barnea lab competitive binding assay involving GPCR => fluorescence<br />
*Adrian suggestion: Look at crystal structure, and binding pocket; see if you can cut the rest of the protein off and make minimized histamine binding domain<br />
*Degradation mechanism?<br />
<br />
*Team 2 update:<br />
**“Chugging right along” all this before Thursday<br />
**Signal peptide construct finished in expression vector for S. epi<br />
**Agr promoter construct in S. epi <br />
**Checking Trg-EnvZ ligations tonight <br />
**S. epi is resistance to Tet<br />
**Looger sent us designs to modify Taz to a histidine receptor; then will move to histamine receptor<br />
<br />
*Team 3 update:<br />
**Mutagenic PCR….<br />
**Administrative<br />
**Bio 1950 - Fill out proposal form on bio website<br />
**Planning in advance<br />
**Practice presentation in front of people. <br />
**Google calendar? Weekly lab meetings <br />
**Wiki has to be done a week before the week its due<br />
**Cleaner lab book<br />
**Clean wiki<br />
<br />
**Mutagenic PCR</div>Ivoruganhttp://2009.igem.org/Team:Brown/PartsTeam:Brown/Parts2009-10-22T02:45:50Z<p>Ivorugan: </p>
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<html><img src="http://img29.imageshack.us/img29/9726/biobricksparts.png"></html><br />
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'''rEV131''' (<partinfo>BBa_K212000</partinfo>)<br />
----<br />
<br />
<br />
[[Image:Ev131structure.png|350px]]<br />
<br />
'''Paraphrased description from “Tick Histamine-Binding Proteins: Isolation, Cloning, and Three-Dimensional Structure” by Paesen et al. (1999)'''<br />
<br />
<br />
EV131 (also known as rRa-HBP2) is a histamine binding protein, and one of three discovered in the salivary gland extracts of Rhipicephalus appendiculatus ticks. Two major proteins (Ra-HBP1 and Ra-HBP2) were found in the saliva of female ticks, and a third protein (Ra-HBP3) was retained from male salivary gland extracts. On SDS gels, the proteins have apparent molecular masses between 20 and 25 kDa.<br />
<br />
The crystallographic structure and biological activity of these HBPs (Histamine binding proteins) indicate that they sequester histamine at wound site, outcompeting histamine receptors for the ligand, thereby overcoming their hosts’ inflammatory (and related immune) responses and feeding successfully. Acting independently of the membrane-bound H1, H2, and H3 receptors, HBPs offer a new approach to the control of histamine-based diseases, such as allergic rhinitis.<br />
<br />
Binding of histamine to the three rHBPs appears to be saturable. Scatchard plots show high affinities for rRa-HBP3 (equilibrium dissociation constant [KD] 1.2X10^-9 M; SD=0.4; three measurements) and for rRa-HBP2 (KD 1.7x10^-9 M; SD=0.9), but a lower affinity for rRa-HBP1 (KD 1.8x10^-8 M; SD=1.2). <br />
<br />
A series of histamine-like compounds were tested for their ability to compete with 3H-histamine for binding to the proteins. Depending on the protein tested, 100–240 times more 1-methylhistamine than cold histamine, and 600–1000 times more 3-methylhistamine, were needed for a 50% reduction of bound radioactivity. No significant competition was observed with other related compounds (histidine, imidazole, serotonin, dopamine, the H1 receptor agonist betahistine, the H1 antagonists chlorpheniramine and pyrilamine, the H2 agonist dimaprit, the H2 antagonists ranitidine and cimetidine, and the polyamines putrescine, spermine, and spermidine). This indicates highly specific histamine binding, different from that of the mammalian H1 and H2 receptors (Gantz et al., 1992). <br />
<br />
<br />
<br />
'''Paraphrased description from “Arthropod-Derived Histamine-Binding Protein Prevents Murine Allergic Asthma” by Couillin et al. (2004)'''<br />
<br />
<br />
EV131 has a distinctive feature because it presents a second specific binding site for histamine with lower affinity than the high affinity binding site, as revealed by its crystal structure. <br />
<br />
Allergen challenge enhances histamine release upon OVA-specific IgE cross-linking on mast cell and subsequent degranulation with release of histamine in sensitized mice, leading to bronchoconstriction, eosinophilia, and mucus hypersecretion. Administration of EV131 to BP2 strain of mice reduced significantly the peribronchial eosinophilia, mucus hypersecretion, and hyperplasia of bronchial smooth muscles. Therefore, complete in vivo neutralization of histamine with the high affinity histamine-binding protein EV131 inhibited the inflammatory cell recruitment and suppressed the characteristic allergic inflammation of the airways.<br />
<br />
<br />
[[https://2009.igem.org/Team:Brown/Parts/EV131 DNA Sequence]]<br />
<br />
<br />
<br />
'''Tar-EnvZ<br />
''' (<partinfo>BBa_K212001</partinfo>)<br />
----<br />
<br />
<br />
[[Image:Taz.png|450px]]<br />
<br />
<br />
In our system, the Tar-EnvZ (or Taz) chimera protein is used to indicate and signal the presence of a chemical ligand. Endogenous to E. coli cells, Tar has three domains: a periplasmic ligand binding receptor domain, a transmembrane domain, and an intracellular kinase domain. When aspartate binds to the Tar receptor domain, the kinase domain subsequently propagates a message by modifying intracellular components, ultimately resulting in regulation of flagella rotation. <br />
Also endogenous to E. Coli is the EnvZ protein, an inner membrane kinase which responds to changes in osmolarity. When activated, the EnvZ kinase phosphorylates transcription factor OmpR, which subsequently activates transcription of the OmpC gene. <br />
Tar-EnvZ (Taz) is a chimera protein, manufactured by Inouye, et al. We obtained the gene from his lab and Biobricked it. Taz comprises of the aspartate chemoreceptor region of Tar, the transmembrane region of Tar, and the intracellular kinase region of EnvZ. The genes were fused by digesting both with NdeI and ligating the overlapping ends together. The cut site is between amino acids H256 and M257. <br />
<br />
[[https://2009.igem.org/Team:Brown/Parts/Tar-EnvZ DNA Sequence]]<br />
<br />
<br />
<br />
<br />
'''OmpC promoter-RFP<br />
''' (<partinfo>BBa_K212002</partinfo>)<br />
----<br />
<br />
<br />
This construct is a reporter for EnvZ activation. Once activated, EnvZ phosphorylates transcription factor OmpR, which in turn activates transcription of the OmpC gene. This cassette contains the promoter region of OmpC (BBa_R0082), placed over a red fluorescence protein gene (BBa_E1010). The ribosome binding site is . There is also a double terminator sequence. <br />
[[https://2009.igem.org/Team:Brown/Parts/OmpC DNA Sequence]]<br />
<br />
<br />
<br />
<br />
'''pAgr''' (<partinfo>BBa_K212003</partinfo>)<br />
----<br />
<br />
This part is the promoter region + RBS of the Agr operon in S. epidermidis. <br />
The agr operon transduces the signal that a quorum of S. epidermidis has been reached, based on the extracellular concentration of signalling oligopeptides. When the bacteria reaches a quorum, the genes under the agr operon are usually turned on (this includes virulence factors that can create a biofilm).<br />
<br />
[[Image:pagr.png|The pAgr Part is the P3 promoter in this diagram, along with a native RBS. Diagram is from Novick and Geisinger, "Quorum Sensing in Staphylococci", Annual Review of Genetics, Vol. 42: 541-564 (2008).]]<br />
<br />
NOTE: This part only works in S. epidermidis.<br />
<br />
<br />
<br />
<br />
'''secretion signal peptide + GFPmut3b''' (<partinfo>BBa_K212004</partinfo>)<br />
----<br />
<br />
This is a composite signalling part. The secretion signal peptide for beta-lactamase in S. epidermidis is ligated N-terminally to registry part BBa_E0040 (GFPmut3b). The signal peptide motif is used by the bacteria to target the protein for secretion through the Sec pathway. Any part may be put in place of GFP, but in its current state, the amount of secretion of any protein may be studied using fluorescent microscopy.<br />
<br />
NOTE: This part only works for S. epidermidis.<br />
<br />
<br />
<br />
<br />
'''TrgEnvZ''' (<partinfo>BBa_K212005</partinfo>)<br />
----<br />
<br />
Transduces ribose-binding protein signal to OmpC promoter.<br />
This chimeric protein is composed of a trg domain joined to an EnvZ histidine kinase domain. <br />
<br />
<br />
<br />
<br />
'''mRBP (modified ribose binding protein - now binds histamine)''' (<partinfo>BBa_K212006</partinfo>)<br />
----<br />
This is a mutated version of RBP, an E. coli periplasmic ribose-binding protein. Natively, ribose binds as a ligand, transducing a message to the trg domain of a transmembrane histidine kinase. Here we have computationally redesigned RBP's ligand binding pocket to bind histamine preferentially over ribose, yielding mRBP. <br />
<br />
There are sixteen locations (S9K, N13D, F15E, F16H, N64H, D89G, R90D, S103T, I132D, A137T, R141L, F164D, N190H, F214V, D215G, Q235T) at which RBP is mutated to create mRBP. These mutations redesigned the protein's ligand-binding pocket to bind to histamine instead of ribose, while still allowing the protein to fold properly and maintain its shape. These mutations were introduced based on predictions made by the Rosetta 3 macromolecular modeling program, which probabilistically searches through mutations to RBP's ligand binding pocket, then simulating the interaction between the mutated protein and histamine (similar to the method in Looger, et al., "Computational design of receptor and sensor proteins with novel functions", Nature (2003)). The predicted design with the lowest energy was selected and the DNA that coded for it was synthesized by GeneArt (see Brown 2009 wiki>Projects>Histamine Binding for more details).<br />
<br />
The mechanism of mRBP's binding may be thought of like that of a venus fly-trap. There are two domains that form the top and bottom of its "mouth", and the ligand binding pocket is between these two domains. The two domains are connected by a hinge that allows the protein to snap shut on a ligand when it goes into its bound conformation. <br />
[[Image:Ompc_envz_pathway.png|Adapted from The TrgEnvZ-OmpR pathway. From Fig. 3 of Looger, et al.]]<br />
<br />
<br />
In its bound conformation, mRBP interacts with the Trg domain of the chimeric TrgEnvZ histidine kinase, which then phosphorylates OmpR, which causes expression of genes under the OmpC promoter. The more mRBPs exist in the periplasmic domain bound to histamine, the more it interacts with TrgEnvZ, and the more gene expression there is in a cell containing all three genes and the promoter.</div>Ivoruganhttp://2009.igem.org/Team:Brown/PartsTeam:Brown/Parts2009-10-22T02:45:28Z<p>Ivorugan: </p>
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<div>{{Brown}}<br />
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<html><img src="http://img29.imageshack.us/img29/9726/biobricksparts.png"></html><br />
'''rEV131''' (<partinfo>BBa_K212000</partinfo>)<br />
----<br />
<br />
<br />
[[Image:Ev131structure.png|350px]]<br />
<br />
'''Paraphrased description from “Tick Histamine-Binding Proteins: Isolation, Cloning, and Three-Dimensional Structure” by Paesen et al. (1999)'''<br />
<br />
<br />
EV131 (also known as rRa-HBP2) is a histamine binding protein, and one of three discovered in the salivary gland extracts of Rhipicephalus appendiculatus ticks. Two major proteins (Ra-HBP1 and Ra-HBP2) were found in the saliva of female ticks, and a third protein (Ra-HBP3) was retained from male salivary gland extracts. On SDS gels, the proteins have apparent molecular masses between 20 and 25 kDa.<br />
<br />
The crystallographic structure and biological activity of these HBPs (Histamine binding proteins) indicate that they sequester histamine at wound site, outcompeting histamine receptors for the ligand, thereby overcoming their hosts’ inflammatory (and related immune) responses and feeding successfully. Acting independently of the membrane-bound H1, H2, and H3 receptors, HBPs offer a new approach to the control of histamine-based diseases, such as allergic rhinitis.<br />
<br />
Binding of histamine to the three rHBPs appears to be saturable. Scatchard plots show high affinities for rRa-HBP3 (equilibrium dissociation constant [KD] 1.2X10^-9 M; SD=0.4; three measurements) and for rRa-HBP2 (KD 1.7x10^-9 M; SD=0.9), but a lower affinity for rRa-HBP1 (KD 1.8x10^-8 M; SD=1.2). <br />
<br />
A series of histamine-like compounds were tested for their ability to compete with 3H-histamine for binding to the proteins. Depending on the protein tested, 100–240 times more 1-methylhistamine than cold histamine, and 600–1000 times more 3-methylhistamine, were needed for a 50% reduction of bound radioactivity. No significant competition was observed with other related compounds (histidine, imidazole, serotonin, dopamine, the H1 receptor agonist betahistine, the H1 antagonists chlorpheniramine and pyrilamine, the H2 agonist dimaprit, the H2 antagonists ranitidine and cimetidine, and the polyamines putrescine, spermine, and spermidine). This indicates highly specific histamine binding, different from that of the mammalian H1 and H2 receptors (Gantz et al., 1992). <br />
<br />
<br />
<br />
'''Paraphrased description from “Arthropod-Derived Histamine-Binding Protein Prevents Murine Allergic Asthma” by Couillin et al. (2004)'''<br />
<br />
<br />
EV131 has a distinctive feature because it presents a second specific binding site for histamine with lower affinity than the high affinity binding site, as revealed by its crystal structure. <br />
<br />
Allergen challenge enhances histamine release upon OVA-specific IgE cross-linking on mast cell and subsequent degranulation with release of histamine in sensitized mice, leading to bronchoconstriction, eosinophilia, and mucus hypersecretion. Administration of EV131 to BP2 strain of mice reduced significantly the peribronchial eosinophilia, mucus hypersecretion, and hyperplasia of bronchial smooth muscles. Therefore, complete in vivo neutralization of histamine with the high affinity histamine-binding protein EV131 inhibited the inflammatory cell recruitment and suppressed the characteristic allergic inflammation of the airways.<br />
<br />
<br />
[[https://2009.igem.org/Team:Brown/Parts/EV131 DNA Sequence]]<br />
<br />
<br />
<br />
'''Tar-EnvZ<br />
''' (<partinfo>BBa_K212001</partinfo>)<br />
----<br />
<br />
<br />
[[Image:Taz.png|450px]]<br />
<br />
<br />
In our system, the Tar-EnvZ (or Taz) chimera protein is used to indicate and signal the presence of a chemical ligand. Endogenous to E. coli cells, Tar has three domains: a periplasmic ligand binding receptor domain, a transmembrane domain, and an intracellular kinase domain. When aspartate binds to the Tar receptor domain, the kinase domain subsequently propagates a message by modifying intracellular components, ultimately resulting in regulation of flagella rotation. <br />
Also endogenous to E. Coli is the EnvZ protein, an inner membrane kinase which responds to changes in osmolarity. When activated, the EnvZ kinase phosphorylates transcription factor OmpR, which subsequently activates transcription of the OmpC gene. <br />
Tar-EnvZ (Taz) is a chimera protein, manufactured by Inouye, et al. We obtained the gene from his lab and Biobricked it. Taz comprises of the aspartate chemoreceptor region of Tar, the transmembrane region of Tar, and the intracellular kinase region of EnvZ. The genes were fused by digesting both with NdeI and ligating the overlapping ends together. The cut site is between amino acids H256 and M257. <br />
<br />
[[https://2009.igem.org/Team:Brown/Parts/Tar-EnvZ DNA Sequence]]<br />
<br />
<br />
<br />
<br />
'''OmpC promoter-RFP<br />
''' (<partinfo>BBa_K212002</partinfo>)<br />
----<br />
<br />
<br />
This construct is a reporter for EnvZ activation. Once activated, EnvZ phosphorylates transcription factor OmpR, which in turn activates transcription of the OmpC gene. This cassette contains the promoter region of OmpC (BBa_R0082), placed over a red fluorescence protein gene (BBa_E1010). The ribosome binding site is . There is also a double terminator sequence. <br />
[[https://2009.igem.org/Team:Brown/Parts/OmpC DNA Sequence]]<br />
<br />
<br />
<br />
<br />
'''pAgr''' (<partinfo>BBa_K212003</partinfo>)<br />
----<br />
<br />
This part is the promoter region + RBS of the Agr operon in S. epidermidis. <br />
The agr operon transduces the signal that a quorum of S. epidermidis has been reached, based on the extracellular concentration of signalling oligopeptides. When the bacteria reaches a quorum, the genes under the agr operon are usually turned on (this includes virulence factors that can create a biofilm).<br />
<br />
[[Image:pagr.png|The pAgr Part is the P3 promoter in this diagram, along with a native RBS. Diagram is from Novick and Geisinger, "Quorum Sensing in Staphylococci", Annual Review of Genetics, Vol. 42: 541-564 (2008).]]<br />
<br />
NOTE: This part only works in S. epidermidis.<br />
<br />
<br />
<br />
<br />
'''secretion signal peptide + GFPmut3b''' (<partinfo>BBa_K212004</partinfo>)<br />
----<br />
<br />
This is a composite signalling part. The secretion signal peptide for beta-lactamase in S. epidermidis is ligated N-terminally to registry part BBa_E0040 (GFPmut3b). The signal peptide motif is used by the bacteria to target the protein for secretion through the Sec pathway. Any part may be put in place of GFP, but in its current state, the amount of secretion of any protein may be studied using fluorescent microscopy.<br />
<br />
NOTE: This part only works for S. epidermidis.<br />
<br />
<br />
<br />
<br />
'''TrgEnvZ''' (<partinfo>BBa_K212005</partinfo>)<br />
----<br />
<br />
Transduces ribose-binding protein signal to OmpC promoter.<br />
This chimeric protein is composed of a trg domain joined to an EnvZ histidine kinase domain. <br />
<br />
<br />
<br />
<br />
'''mRBP (modified ribose binding protein - now binds histamine)''' (<partinfo>BBa_K212006</partinfo>)<br />
----<br />
This is a mutated version of RBP, an E. coli periplasmic ribose-binding protein. Natively, ribose binds as a ligand, transducing a message to the trg domain of a transmembrane histidine kinase. Here we have computationally redesigned RBP's ligand binding pocket to bind histamine preferentially over ribose, yielding mRBP. <br />
<br />
There are sixteen locations (S9K, N13D, F15E, F16H, N64H, D89G, R90D, S103T, I132D, A137T, R141L, F164D, N190H, F214V, D215G, Q235T) at which RBP is mutated to create mRBP. These mutations redesigned the protein's ligand-binding pocket to bind to histamine instead of ribose, while still allowing the protein to fold properly and maintain its shape. These mutations were introduced based on predictions made by the Rosetta 3 macromolecular modeling program, which probabilistically searches through mutations to RBP's ligand binding pocket, then simulating the interaction between the mutated protein and histamine (similar to the method in Looger, et al., "Computational design of receptor and sensor proteins with novel functions", Nature (2003)). The predicted design with the lowest energy was selected and the DNA that coded for it was synthesized by GeneArt (see Brown 2009 wiki>Projects>Histamine Binding for more details).<br />
<br />
The mechanism of mRBP's binding may be thought of like that of a venus fly-trap. There are two domains that form the top and bottom of its "mouth", and the ligand binding pocket is between these two domains. The two domains are connected by a hinge that allows the protein to snap shut on a ligand when it goes into its bound conformation. <br />
[[Image:Ompc_envz_pathway.png|Adapted from The TrgEnvZ-OmpR pathway. From Fig. 3 of Looger, et al.]]<br />
<br />
<br />
In its bound conformation, mRBP interacts with the Trg domain of the chimeric TrgEnvZ histidine kinase, which then phosphorylates OmpR, which causes expression of genes under the OmpC promoter. The more mRBPs exist in the periplasmic domain bound to histamine, the more it interacts with TrgEnvZ, and the more gene expression there is in a cell containing all three genes and the promoter.</div>Ivoruganhttp://2009.igem.org/Team:Brown/PartsTeam:Brown/Parts2009-10-22T02:44:26Z<p>Ivorugan: /* Biobricks...saving one sneeze at a time! */</p>
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'''rEV131''' (<partinfo>BBa_K212000</partinfo>)<br />
----<br />
<br />
<br />
[[Image:Ev131structure.png|350px]]<br />
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'''Paraphrased description from “Tick Histamine-Binding Proteins: Isolation, Cloning, and Three-Dimensional Structure” by Paesen et al. (1999)'''<br />
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EV131 (also known as rRa-HBP2) is a histamine binding protein, and one of three discovered in the salivary gland extracts of Rhipicephalus appendiculatus ticks. Two major proteins (Ra-HBP1 and Ra-HBP2) were found in the saliva of female ticks, and a third protein (Ra-HBP3) was retained from male salivary gland extracts. On SDS gels, the proteins have apparent molecular masses between 20 and 25 kDa.<br />
<br />
The crystallographic structure and biological activity of these HBPs (Histamine binding proteins) indicate that they sequester histamine at wound site, outcompeting histamine receptors for the ligand, thereby overcoming their hosts’ inflammatory (and related immune) responses and feeding successfully. Acting independently of the membrane-bound H1, H2, and H3 receptors, HBPs offer a new approach to the control of histamine-based diseases, such as allergic rhinitis.<br />
<br />
Binding of histamine to the three rHBPs appears to be saturable. Scatchard plots show high affinities for rRa-HBP3 (equilibrium dissociation constant [KD] 1.2X10^-9 M; SD=0.4; three measurements) and for rRa-HBP2 (KD 1.7x10^-9 M; SD=0.9), but a lower affinity for rRa-HBP1 (KD 1.8x10^-8 M; SD=1.2). <br />
<br />
A series of histamine-like compounds were tested for their ability to compete with 3H-histamine for binding to the proteins. Depending on the protein tested, 100–240 times more 1-methylhistamine than cold histamine, and 600–1000 times more 3-methylhistamine, were needed for a 50% reduction of bound radioactivity. No significant competition was observed with other related compounds (histidine, imidazole, serotonin, dopamine, the H1 receptor agonist betahistine, the H1 antagonists chlorpheniramine and pyrilamine, the H2 agonist dimaprit, the H2 antagonists ranitidine and cimetidine, and the polyamines putrescine, spermine, and spermidine). This indicates highly specific histamine binding, different from that of the mammalian H1 and H2 receptors (Gantz et al., 1992). <br />
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'''Paraphrased description from “Arthropod-Derived Histamine-Binding Protein Prevents Murine Allergic Asthma” by Couillin et al. (2004)'''<br />
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EV131 has a distinctive feature because it presents a second specific binding site for histamine with lower affinity than the high affinity binding site, as revealed by its crystal structure. <br />
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Allergen challenge enhances histamine release upon OVA-specific IgE cross-linking on mast cell and subsequent degranulation with release of histamine in sensitized mice, leading to bronchoconstriction, eosinophilia, and mucus hypersecretion. Administration of EV131 to BP2 strain of mice reduced significantly the peribronchial eosinophilia, mucus hypersecretion, and hyperplasia of bronchial smooth muscles. Therefore, complete in vivo neutralization of histamine with the high affinity histamine-binding protein EV131 inhibited the inflammatory cell recruitment and suppressed the characteristic allergic inflammation of the airways.<br />
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[[https://2009.igem.org/Team:Brown/Parts/EV131 DNA Sequence]]<br />
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'''Tar-EnvZ<br />
''' (<partinfo>BBa_K212001</partinfo>)<br />
----<br />
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[[Image:Taz.png|450px]]<br />
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In our system, the Tar-EnvZ (or Taz) chimera protein is used to indicate and signal the presence of a chemical ligand. Endogenous to E. coli cells, Tar has three domains: a periplasmic ligand binding receptor domain, a transmembrane domain, and an intracellular kinase domain. When aspartate binds to the Tar receptor domain, the kinase domain subsequently propagates a message by modifying intracellular components, ultimately resulting in regulation of flagella rotation. <br />
Also endogenous to E. Coli is the EnvZ protein, an inner membrane kinase which responds to changes in osmolarity. When activated, the EnvZ kinase phosphorylates transcription factor OmpR, which subsequently activates transcription of the OmpC gene. <br />
Tar-EnvZ (Taz) is a chimera protein, manufactured by Inouye, et al. We obtained the gene from his lab and Biobricked it. Taz comprises of the aspartate chemoreceptor region of Tar, the transmembrane region of Tar, and the intracellular kinase region of EnvZ. The genes were fused by digesting both with NdeI and ligating the overlapping ends together. The cut site is between amino acids H256 and M257. <br />
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[[https://2009.igem.org/Team:Brown/Parts/Tar-EnvZ DNA Sequence]]<br />
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'''OmpC promoter-RFP<br />
''' (<partinfo>BBa_K212002</partinfo>)<br />
----<br />
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This construct is a reporter for EnvZ activation. Once activated, EnvZ phosphorylates transcription factor OmpR, which in turn activates transcription of the OmpC gene. This cassette contains the promoter region of OmpC (BBa_R0082), placed over a red fluorescence protein gene (BBa_E1010). The ribosome binding site is . There is also a double terminator sequence. <br />
[[https://2009.igem.org/Team:Brown/Parts/OmpC DNA Sequence]]<br />
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'''pAgr''' (<partinfo>BBa_K212003</partinfo>)<br />
----<br />
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This part is the promoter region + RBS of the Agr operon in S. epidermidis. <br />
The agr operon transduces the signal that a quorum of S. epidermidis has been reached, based on the extracellular concentration of signalling oligopeptides. When the bacteria reaches a quorum, the genes under the agr operon are usually turned on (this includes virulence factors that can create a biofilm).<br />
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[[Image:pagr.png|The pAgr Part is the P3 promoter in this diagram, along with a native RBS. Diagram is from Novick and Geisinger, "Quorum Sensing in Staphylococci", Annual Review of Genetics, Vol. 42: 541-564 (2008).]]<br />
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NOTE: This part only works in S. epidermidis.<br />
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'''secretion signal peptide + GFPmut3b''' (<partinfo>BBa_K212004</partinfo>)<br />
----<br />
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This is a composite signalling part. The secretion signal peptide for beta-lactamase in S. epidermidis is ligated N-terminally to registry part BBa_E0040 (GFPmut3b). The signal peptide motif is used by the bacteria to target the protein for secretion through the Sec pathway. Any part may be put in place of GFP, but in its current state, the amount of secretion of any protein may be studied using fluorescent microscopy.<br />
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NOTE: This part only works for S. epidermidis.<br />
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'''TrgEnvZ''' (<partinfo>BBa_K212005</partinfo>)<br />
----<br />
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Transduces ribose-binding protein signal to OmpC promoter.<br />
This chimeric protein is composed of a trg domain joined to an EnvZ histidine kinase domain. <br />
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'''mRBP (modified ribose binding protein - now binds histamine)''' (<partinfo>BBa_K212006</partinfo>)<br />
----<br />
This is a mutated version of RBP, an E. coli periplasmic ribose-binding protein. Natively, ribose binds as a ligand, transducing a message to the trg domain of a transmembrane histidine kinase. Here we have computationally redesigned RBP's ligand binding pocket to bind histamine preferentially over ribose, yielding mRBP. <br />
<br />
There are sixteen locations (S9K, N13D, F15E, F16H, N64H, D89G, R90D, S103T, I132D, A137T, R141L, F164D, N190H, F214V, D215G, Q235T) at which RBP is mutated to create mRBP. These mutations redesigned the protein's ligand-binding pocket to bind to histamine instead of ribose, while still allowing the protein to fold properly and maintain its shape. These mutations were introduced based on predictions made by the Rosetta 3 macromolecular modeling program, which probabilistically searches through mutations to RBP's ligand binding pocket, then simulating the interaction between the mutated protein and histamine (similar to the method in Looger, et al., "Computational design of receptor and sensor proteins with novel functions", Nature (2003)). The predicted design with the lowest energy was selected and the DNA that coded for it was synthesized by GeneArt (see Brown 2009 wiki>Projects>Histamine Binding for more details).<br />
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The mechanism of mRBP's binding may be thought of like that of a venus fly-trap. There are two domains that form the top and bottom of its "mouth", and the ligand binding pocket is between these two domains. The two domains are connected by a hinge that allows the protein to snap shut on a ligand when it goes into its bound conformation. <br />
[[Image:Ompc_envz_pathway.png|Adapted from The TrgEnvZ-OmpR pathway. From Fig. 3 of Looger, et al.]]<br />
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In its bound conformation, mRBP interacts with the Trg domain of the chimeric TrgEnvZ histidine kinase, which then phosphorylates OmpR, which causes expression of genes under the OmpC promoter. The more mRBPs exist in the periplasmic domain bound to histamine, the more it interacts with TrgEnvZ, and the more gene expression there is in a cell containing all three genes and the promoter.</div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_ImplicationsTeam:Brown/Project Implications2009-10-22T02:43:13Z<p>Ivorugan: /* Human Practices */</p>
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= Safety Issues =<br />
[[Image:Staphylococcus_epidermidis_biofilm.jpg|200px|thumb|right|Staphylococcus epidermidis biofilm]]<br />
This project raises safety issues due to the use of Staphylococcus epidermidis for producing and secreting the histamine binding protein, EV131. Although S. epidermidis is one of the more benign species of Staphylococcus, it can form infectious biofilms that are impervious to antibiotic treatment if its cell density becomes too great. This could pose a grave risk to researchers involved and the public for whom Allergene aims to serve.<br />
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[[Image:Quorum sensing construct.jpg|195px|thumb|left|Quorum sensing construct with CCDb gene]] This change in S. epidermidis phenotype is accomplished by the S. epidermidis agr operon, which upregulates pathogenicity genes in response to a quorum. We reasoned that we could incorporate a safety mechanism into our bacteria by putting a death gene under the regulation of the agr promoter. This way, whenever the bacteria would reach a high enough density to become dangerous, they would simply begin to die until they reach a safer, lower density. In order to first test that our cassette works, we ligated the agr promoter upstream of GFP, so that whenever the cells reached a quorum, they would fluoresce green. Later, the GFP would be switched out for a CCDb, a death gene.<br />
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= Institutional Biosafety Regulation =<br />
[[Image:Brownlogo_biohazard.jpg|125px|left|]]<br />
Any member of the Brown University faculty, staff, post-doctoral, and student bodies who plans to initiate research involving human subjects must submit a protocol for review and approval by the Institutional Review Board (IRB) prior to beginning the project. The Research Protections Office (RPO) staff provides administrative support to researchers who are preparing protocols for presentation to the IRB. <br />
<br />
The Institutional Animal Care & Use Committee (IACUC) ensures the health, well-being, and humane use of animals used in research at Brown, following the regulations and guidance of the U.S. Department of Agriculture and the Department of Health and Human Services. IACUC staff is available to answer administrative questions, and campus veterinarians can provide consultation and assistance on animal care and questions regarding experimental protocols.<br />
For our project, however, we have not yet proceeded to preclinical testing stage. For in vivo research, the Office of Environmental Health and Safety (EHS) at Brown provides compliance tools to optimize laboratory and facility conditions across research projects, from safe handling of potentially hazardous materials to biosafety training. EHS also has created guidelines to describe its emergency response system, environmental strategies, and lab equipment rules.<br />
<br />
<br />
We presented and proposed our project to the Office of Environmental Health and Safety at Brown University. Initially, we ran into concerns about the Biosafety Level 2 classification of the proposed bacteria, Staphylococcus aureus, that we planned to use for expression of the histamine binding protein. With further consideration of the biohazard implications of S. aureus, we decided instead to utilize Staphylococcus epidermidis for the expression of the histamine binding protein. Using S. epidermidis provided us with two major benefits:<br />
<br />
<br />
'''''<big>1) S. epidermidis is endogenous to the nasal flora and thus, very well-suited for accomplishing the eventual goal of our project.</big>'''''<br />
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'''''<big>2) S. epidermidis is Biosafety Level 1, eliminating the fears that would have been associated with a Biosafety Level 2 bacterium.</big>'''''<br />
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= Ethics, Ethics, Ethics =<br />
[[Image:ugoku-themis.gif|225px|right|]]<br />
An important concern regarding many iGEM projects, including ours, is the introduction of genetically engineered organisms into a human body. In particular, for our project we are engineering a strain of a bacterium that normally lives in a human environment without any problems, by introducing an immune response suppressant function to that organism. This raises some potential ethical problems. For example, what if the engineered bacteria an allergy sufferer takes to relieve their symptoms causes an infection?<br />
<br />
Regarding the potential concern that synthetic biologists are "playing God", with potentially harmful effects, we on the Brown team feel that any technology can be used for "good" or "bad" purposes. Just as nuclear reactions can be used for generating electricity or destroying cities, so too can synthetic biology be used for both positive and negative ends. The same techniques that allow us to genetically engineer Staphylococcus epidermidis for the "good" purpose of treating allergies could allow one to create a "bad" super multi-antibiotic resistant strain of Staphylococcus aureus (a closely related species to Staphylococcus epidermidis that is highly pathogenic, and responsible for increasing numbers of fatal infections). Unlike nuclear energy, however, this technology is so cheap and relatively easy to use that it is accessible even to high school and college students. It would be extremely difficult to create a central body that controls what end people put synthetic biological technology towards. As such, we think that it is the responsibility of everyone in the synthetic biological community to use this technology only for "good" ends, and to think about the consequences of our manipulations. We on the Brown team have tried our best to be conscious of these implications, and to engineer our machine accordingly.</div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_Meetings/8-10-09Team:Brown/Notebook Meetings/8-10-092009-10-22T02:42:56Z<p>Ivorugan: New page: {{Brown}} '''iGEM Meeting 8-10-09''' '''SFH 218''' '''Attendees: Diana, Adrian''' *Team 1 update **Got EV131 into pNoTat **Growing in competent BL21 **IPTG induction **Last year...</p>
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'''iGEM Meeting 8-10-09'''<br />
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'''SFH 218'''<br />
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'''Attendees: Diana, Adrian'''<br />
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*Team 1 update<br />
**Got EV131 into pNoTat<br />
**Growing in competent BL21<br />
**IPTG induction <br />
**Last year Team Resistance did an IPTG induction<br />
**Will do Western blot (Diana has 6-His antibody)<br />
<br />
*Team 2 update<br />
**Successfully cloned Agr operon promoter, moved into pGEM<br />
**Digested and ligated into Biobrick vector<br />
**Agr promoter + RFP growing on plates, will test quorum sensor<br />
**Electroporation failed; might be due to Tet plates, or faulty electrocompetent cells<br />
**Made Trg-EnvZ. Doing blue-white test<br />
**However, maybe did not distribute X-gal and IPTG evenly<br />
**Looger said he will get back this week about receptor<br />
**Running out of S. epi expression vector <br />
<br />
*Team 3 update<br />
**Ran SDS PAGE on Taz1, too much noise on protein gel<br />
**Tested mutagenic primers <br />
**Will send off for sequencing <br />
<br />
*Administrative stuff<br />
**Booked hotels for the Jamboree<br />
**We have 2 time slots at the Jamboree, may split up??</div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_All_TogetherTeam:Brown/Project All Together2009-10-22T02:42:37Z<p>Ivorugan: </p>
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<html><img src="http://img18.imageshack.us/img18/1602/constructinaction.png"></html><br />
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[[Image:Cascade.gif|center]]1) Mast cells release histamine during the allergic response. <br />
<br />
2) Histamine binds to our re-engineered histamine receptor.<br />
<br />
3) This receptor’s intracellular kinase domain EnvZ phosphorylates transcription factor OmpR.<br />
<br />
4) OmpR turns on transcription of DNA under the OmpC promoter.<br />
<br />
5) The genes for rEV131 and its attached secretion signal are transcribed.<br />
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6) After translation, the secretion peptide allows rEV131 to be released into the extracellular fluid.<br />
<br />
7) rEV131 sequesters histamine<br />
<br />
8) The transcription and secretion of rEV131 continues as long as histamine is present in the extracellular fluid. When histamine concentration returns to its pre-allergic response state, production of rEV131 stops because the initiating ligand histamine is no longer present.<br />
<br />
<html><br />
<a href="https://2009.igem.org/Team:Brown/Project_Implications"><br />
<img src="https://static.igem.org/mediawiki/2009/b/b2/Human_practices_brown_button.png"><br />
</html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_All_TogetherTeam:Brown/Project All Together2009-10-22T02:42:27Z<p>Ivorugan: /* Allergene: The Genetic Construct in Action */</p>
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<div>{{Brown}}<br />
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<html><img src="http://img18.imageshack.us/img18/1602/constructinaction.png"></html><br />
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[[Image:Cascade.gif|center]]1) Mast cells release histamine during the allergic response. <br />
<br />
2) Histamine binds to our re-engineered histamine receptor.<br />
<br />
3) This receptor’s intracellular kinase domain EnvZ phosphorylates transcription factor OmpR.<br />
<br />
4) OmpR turns on transcription of DNA under the OmpC promoter.<br />
<br />
5) The genes for rEV131 and its attached secretion signal are transcribed.<br />
<br />
6) After translation, the secretion peptide allows rEV131 to be released into the extracellular fluid.<br />
<br />
7) rEV131 sequesters histamine<br />
<br />
8) The transcription and secretion of rEV131 continues as long as histamine is present in the extracellular fluid. When histamine concentration returns to its pre-allergic response state, production of rEV131 stops because the initiating ligand histamine is no longer present.<br />
<br />
<html><br />
<a href="https://2009.igem.org/Team:Brown/Project_Implications"><br />
<img src="https://static.igem.org/mediawiki/2009/b/b2/Human_practices_brown_button.png"><br />
</html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_S.epidermidisTeam:Brown/Project S.epidermidis2009-10-22T02:41:50Z<p>Ivorugan: </p>
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<html><img src="http://img26.imageshack.us/img26/6156/thechassis.png"></html><br />
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[[Image:Staphylococcus epidermidis.jpg|250 px|thumb|right|Staphylococcus epidermidis biofilm]]Staphylococcus epidermidis is an ideal vehicle for our genetic machine for several reasons: it is a native organism in the human nasal flora, it is generally non-pathogenic, and its sequenced genome is used with some frequency in lab work.<br />
<br />
<br />
[[Image:Plasmid_with_HBP+secretion_tag.jpg|200 px|thumb|left|Final construct with secretion tag attached to HBP]]<br />
Since our gene of interest, rEV131, can only bind histamine if it is outside the cell, we have to engineer its secretion from S. epidermidis. We decided to use a signal peptide motif normally found on ß-lactamase, the S. epidermidis gene for ampicillin resistance, which uses the native Sec pathway for its secretion.<br />
We had the DNA sequence of this peptide synthesized by GeneArt AG and ligated it N-terminally to GFP as a test construct (<partinfo>BBa_K212004</partinfo>). This reporter would allow us to visually ascertain<br />
whether the produced protein was secreted using differential centrifugation. An additional advantage is that GFP is the approximately the same size as rEV131, increasing the likelihood that the two proteins behave similarly in terms of secretion. If the secretion tag works properly, the GFP will be secreted through the Sec pathway into the surrounding environment. If we grow S. epi in solution, the secreted GFP will remain in the solution. We can then use differential centrifugation to separate the cells from whatever proteins are in the solution. Once we have obtained the cell-free supernatant, we will use our fluorospectrophotometer to determine the fluorescence of GFP in the supernatant, which can be compared to the fluorescence of the supernatant from similarly prepared cells expressing GFP without a secretion tag to determine whether the secretion tag is functional. <br />
<br />
In our final genetic construct, the GFP reporter is replaced by rEV131.<br />
<br />
=Quorum Sensor=<br />
<br />
<br />
Although Staphylococcus epidermidis is one of the more benign species of Staphylococcus, it can form infectious biofilms that are impervious to antibiotic treatment if its cell<br />
density becomes too great. This change in phenotype is accomplished by the S. epidermidis agr operon, which upregulates pathogenicity genes in response to a quorum.<br />
We reasoned that we could incorporate a safety mechanism into our bacteria by putting a death gene under the regulation of the agr promoter. This way, whenever the bacteria would reach a high enough density to become dangerous, they would simply begin to die until they reach a safer, lower density. In order to first test that our cassette works, we ligated the agr promoter upstream of GFP, so that whenever the cells reached a quorum, they would fluoresce green. Later, the GFP would be switched out for a CCDB, a DNA gyrase poison <partinfo>BBa_P1010</partinfo>.<br />
<br />
Staphylococcus epidermidis is not readily made chemically competent due to its thick cell wall. Most researchers use electroporation to induce cells to take up their target DNA. Throughout the course of our research, we have tweaked the electroporation protocol many times but there are always a very small number of transformants.<br />
<br />
[https://2009.igem.org/Team:Brown/Project_Implications Read more about human practices and safety issues here]<br />
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<html><br />
<a href="https://2009.igem.org/Team:Brown/Project_All_Together"><br />
<img src="https://static.igem.org/mediawiki/2009/9/91/Brown_system_schematic_button.png"><br />
</html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_S.epidermidisTeam:Brown/Project S.epidermidis2009-10-22T02:41:37Z<p>Ivorugan: </p>
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<div>{{Brown}}<br />
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<html><img src="http://img26.imageshack.us/img26/6156/thechassis.png"></html><br />
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[[Image:Staphylococcus epidermidis.jpg|250 px|thumb|right|Staphylococcus epidermidis biofilm]]Staphylococcus epidermidis is an ideal vehicle for our genetic machine for several reasons: it is a native organism in the human nasal flora, it is generally non-pathogenic, and its sequenced genome is used with some frequency in lab work.<br />
<br />
<br />
[[Image:Plasmid_with_HBP+secretion_tag.jpg|200 px|thumb|left|Final construct with secretion tag attached to HBP]]<br />
Since our gene of interest, rEV131, can only bind histamine if it is outside the cell, we have to engineer its secretion from S. epidermidis. We decided to use a signal peptide motif normally found on ß-lactamase, the S. epidermidis gene for ampicillin resistance, which uses the native Sec pathway for its secretion.<br />
We had the DNA sequence of this peptide synthesized by GeneArt AG and ligated it N-terminally to GFP as a test construct (<partinfo>BBa_K212004</partinfo>). This reporter would allow us to visually ascertain<br />
whether the produced protein was secreted using differential centrifugation. An additional advantage is that GFP is the approximately the same size as rEV131, increasing the likelihood that the two proteins behave similarly in terms of secretion. If the secretion tag works properly, the GFP will be secreted through the Sec pathway into the surrounding environment. If we grow S. epi in solution, the secreted GFP will remain in the solution. We can then use differential centrifugation to separate the cells from whatever proteins are in the solution. Once we have obtained the cell-free supernatant, we will use our fluorospectrophotometer to determine the fluorescence of GFP in the supernatant, which can be compared to the fluorescence of the supernatant from similarly prepared cells expressing GFP without a secretion tag to determine whether the secretion tag is functional. <br />
<br />
In our final genetic construct, the GFP reporter is replaced by rEV131.<br />
<br />
=Quorum Sensor=<br />
<br />
<br />
Although Staphylococcus epidermidis is one of the more benign species of Staphylococcus, it can form infectious biofilms that are impervious to antibiotic treatment if its cell<br />
density becomes too great. This change in phenotype is accomplished by the S. epidermidis agr operon, which upregulates pathogenicity genes in response to a quorum.<br />
We reasoned that we could incorporate a safety mechanism into our bacteria by putting a death gene under the regulation of the agr promoter. This way, whenever the bacteria would reach a high enough density to become dangerous, they would simply begin to die until they reach a safer, lower density. In order to first test that our cassette works, we ligated the agr promoter upstream of GFP, so that whenever the cells reached a quorum, they would fluoresce green. Later, the GFP would be switched out for a CCDB, a DNA gyrase poison <partinfo>BBa_P1010</partinfo>.<br />
<br />
Staphylococcus epidermidis is not readily made chemically competent due to its thick cell wall. Most researchers use electroporation to induce cells to take up their target DNA. Throughout the course of our research, we have tweaked the electroporation protocol many times but there are always a very small number of transformants.<br />
<br />
[https://2009.igem.org/Team:Brown/Project_Implications Read more about human practices and safety issues here]<br />
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<html><br />
<a href="https://2009.igem.org/Team:Brown/Project_All_Together"><br />
<img src="https://static.igem.org/mediawiki/2009/9/91/Brown_system_schematic_button.png"><br />
</html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_S.epidermidisTeam:Brown/Project S.epidermidis2009-10-22T02:41:11Z<p>Ivorugan: /* The Chassis: Staphyloccocus Epidermidis */</p>
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<br />
<br />
<br />
[[Image:Staphylococcus epidermidis.jpg|250 px|thumb|right|Staphylococcus epidermidis biofilm]]Staphylococcus epidermidis is an ideal vehicle for our genetic machine for several reasons: it is a native organism in the human nasal flora, it is generally non-pathogenic, and its sequenced genome is used with some frequency in lab work.<br />
<br />
<br />
[[Image:Plasmid_with_HBP+secretion_tag.jpg|200 px|thumb|left|Final construct with secretion tag attached to HBP]]<br />
Since our gene of interest, rEV131, can only bind histamine if it is outside the cell, we have to engineer its secretion from S. epidermidis. We decided to use a signal peptide motif normally found on ß-lactamase, the S. epidermidis gene for ampicillin resistance, which uses the native Sec pathway for its secretion.<br />
We had the DNA sequence of this peptide synthesized by GeneArt AG and ligated it N-terminally to GFP as a test construct (<partinfo>BBa_K212004</partinfo>). This reporter would allow us to visually ascertain<br />
whether the produced protein was secreted using differential centrifugation. An additional advantage is that GFP is the approximately the same size as rEV131, increasing the likelihood that the two proteins behave similarly in terms of secretion. If the secretion tag works properly, the GFP will be secreted through the Sec pathway into the surrounding environment. If we grow S. epi in solution, the secreted GFP will remain in the solution. We can then use differential centrifugation to separate the cells from whatever proteins are in the solution. Once we have obtained the cell-free supernatant, we will use our fluorospectrophotometer to determine the fluorescence of GFP in the supernatant, which can be compared to the fluorescence of the supernatant from similarly prepared cells expressing GFP without a secretion tag to determine whether the secretion tag is functional. <br />
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In our final genetic construct, the GFP reporter is replaced by rEV131.<br />
<br />
=Quorum Sensor=<br />
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Although Staphylococcus epidermidis is one of the more benign species of Staphylococcus, it can form infectious biofilms that are impervious to antibiotic treatment if its cell<br />
density becomes too great. This change in phenotype is accomplished by the S. epidermidis agr operon, which upregulates pathogenicity genes in response to a quorum.<br />
We reasoned that we could incorporate a safety mechanism into our bacteria by putting a death gene under the regulation of the agr promoter. This way, whenever the bacteria would reach a high enough density to become dangerous, they would simply begin to die until they reach a safer, lower density. In order to first test that our cassette works, we ligated the agr promoter upstream of GFP, so that whenever the cells reached a quorum, they would fluoresce green. Later, the GFP would be switched out for a CCDB, a DNA gyrase poison <partinfo>BBa_P1010</partinfo>.<br />
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Staphylococcus epidermidis is not readily made chemically competent due to its thick cell wall. Most researchers use electroporation to induce cells to take up their target DNA. Throughout the course of our research, we have tweaked the electroporation protocol many times but there are always a very small number of transformants.<br />
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[https://2009.igem.org/Team:Brown/Project_Implications Read more about human practices and safety issues here]<br />
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</html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_HBPTeam:Brown/Project HBP2009-10-22T02:40:25Z<p>Ivorugan: /* rEV131: High-Affinity Histamine Binding Protein */</p>
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[[Image:Mmdbimage.fcgi.png|300px|thumb|right|]] rEV131 effectively sequesters histamine without disrupting histamine receptors, hence this treatment may prove to be a viable alternative to current antihistamines in combating allergic symptoms without causing negative side effects. 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, outcompeting the host's histamine receptors for the ligand, thereby effectively combating the allergic response. rEV131 has the highest affinity (lowest dissociation constant) among all histamine binding proteins secreted by R. appendiculatus (Paesen et al, 1999). <br />
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In our genetic construct, rEV131 will be placed under the OmpC promoter so that histamine molecule binding to our engineered receptor triggers the transcription of rEV131. <br />
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Though the protein has been purified, cloned, expressed and well characterized (Paesen et al, 1999) (Couillin et al, 2004) our future plans include further characterization of the protein by conducting binding assays in which we will vary levels of free histamine, above and below allergic threshold, to mimic the extracellular fluid in the nasal cavity. We will also 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<br />
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'''<big>Cloning the EV131 Sequence into an Expression Plasmid, Transformation of E. coli, and Cell Selection</big>'''<br />
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[[Image:ev131cloning.jpg|250px|thumb|left|Cloning of EV131 into pNOTAT expression plasmid]]The insert of interest coding for EV131 is 572 base pairs (bp) in length. After synthesizing primers with the restriction sites EcoRI and BamHI engineered onto the ends, we performed a polymerase chain reaction (PCR) in order to amplify the insert from the pBluescript SK II plasmid in which it was sent. In order to express the recombinant protein, the coding DNA sequence was cloned into the pGEM easy-T expression vector for more efficient excision before cloning into the pNOTAT expression vector. <br />
<br />
The pNOTAT expression vector is important because it adds a 6-His tag to the protein which allows for selective detection (SDS page) and purification (nickel column chromatography) of our particular protein EV131. Once the protein’s coding sequence was cloned into an expression vector, the vector was introduced into competent BL21 E. coli cells by way of heat shock transformation. The BL21 competent cell strain allows for high-efficiency protein expression of any gene that is under the control of a T7 promoter and has a ribosome binding site. BL21 cells contain the T7 bacteriophage gene I, encoding T7 RNA polymerase under the control of the lac UV5 promoter, which is induced by isopropyl-β-D-thiogalactoside (IPTG). After transformation, an aliquot of the transformed cells was spread onto an agar plate to allow for isolation of E. coli colonies containing the expression plasmid ligated to EV131. pNOTAT expression vectors carry a gene for ampicillin resistance, which allows E. coli containing the expression plasmid to be selected for on agar plates containing ampicillin. <br />
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<br />
'''AR-any gels that you have of EV131, put them in.'''<br />
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'''<big>Expression of rEV131 Protein in BL21 E.coli</big>'''<br />
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Efficient and controlled expression of protein in E. coli cells is regulated by the presence of the lac repressor protein. During normal cell growth, this protein binds to the operator sequences in the plasmid and prevents recombinant protein expression. Expression of recombinant proteins encoded by the pNOTAT expression vector is induced by the addition of IPTG, which binds to the lac repressor protein and inactivates it. Once the lac repressor is inactivated, the host cell’s RNA polymerase can transcribe the sequences downstream from the promoter. The transcripts produced are then translated into the recombinant protein. Inducing protein expression with IPTG means that the cellular metabolism concentrates almost exclusively on the production of recombinant protein under tight control. 5-mL liquid cultures of cell colonies isolated from ampicillin plates were grown up and then induced with IPTG. The amount of time allowed for protein expression was varied in a range from two to five hours for different cultures. Thereafter, we began sample preparations for SDS-PAGE and Nickel Column Chromatography.<br />
<br />
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'''<big>Protein Purification Under Native or Denaturing Conditions</big>'''<br />
<br />
Protein purification at this stage of our project is still in process. In order to retain the biological activity of the protein, we wish to purify 6-His-tagged proteins under native conditions. For this, the 6-His-tagged protein must be soluble. In this case, however, there is greater potential for nonspecific proteins to interact with the Nickel-NTA resin. Also, since there is a chance of the 6-His tag being hidden by the tertiary structure of the native protein, the soluble proteins require denaturation before they can be purified on the Ni- NTA column. As a control, a parallel purification under denaturing conditions will be carried out. If purification is only possible under denaturing conditions, the tag can be made generally accessible by moving it to the opposite terminus of the protein. The QIAgen QIAexpress® Ni-NTA Fast Start Kit is an appropriate kit for the purification and detection of recombinant 6-His-tagged proteins in high yield.<br />
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'''<big>Histamine and Competing Molecule Binding Assays</big>'''<br />
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''Test 1: Testing for Histamine Binding Affinity''<br />
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As a model of the free histamine-EV131 interaction, we will be testing the binding affinity strength of a solution of EV131 being poured into a column that contains histamine beads. The threshold level for eliciting a significant allergic response is 12 ng/mL (Proud et al, 1992). The binding capacity of EV131 is two molecules of histamine for one molecule of protein (Paesen et al, 1999) (Couillin et al, 2004). We will test the amount of protein necessary to sequester the threshold level of histamine by running protein solutions of varying concentrations along the histamine bead column. We will measure the initial amount of protein added to the column and then measure the remaining solution eluted from the column. This will determine the amount of protein needed to sequester histamine at threshold levels. <br />
<br />
Another component of the binding affinity test includes the running of free histamine along the histamine-protein-bound column. We will be able to determine the rate of dissociation of the EV131 based upon the interaction of which histamine the EV131 will competitively bind to: the stationary histamine column or the poured free histamine. We will observe the time the protein takes to dissociate from the column and confirm the dissociation constant for the protein as described in the literature.<br />
<br />
<br />
''Test 2: Testing EV131 with Histamine Competitor Molecules'' <br />
<br />
After having investigated the histamine-EV131 interaction, we will test the binding affinity that molecules similar in structure to histamine will have with EV131. Such molecules include histadine, imidazole, and aspartate. The procedure will be similar to that of testing free histamine versus stationary histamine binding with the recombinant EV131 protein. We will pour solutions of free histadine, imidazole, or aspartate into a stationary histamine bead column with EV131 protein bound to it. Thereafter, we will observe how much histamine, imidazole, or aspartate elutes with the protein bound to it. Ideally, the results of this experiment will suggest that EV131 has a much higher binding affinity for histamine than do other molecules of a similar structure.<br />
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'''<big>Further Improvements and Insights Upon EV131 Recombinant Protein</big>'''<br />
<br />
EV131 is a novel protein because it has a high affinity for histamine and it outcompetes histamine receptors located throughout the body for histamine. For these reasons, EV131 has tremendous potential as a therapeutic drug for allergies and other inflammatory diseases. We hope to improve the existing functions of EV131 and to broaden the scope of protein functions by modifying the protein. We could potentially improve the binding affinity of the protein for histamine by increasing the number of binding pockets available, which would optimize the protein to histamine ratio. We could also look at the possibility of constructing a fusion protein that includes cell receptor signals, such as ApoE, that may signal faster uptake and degradation of saturated histamine binding proteins or histamine sensors. This can be accomplished by structurally changing the protein, which will involve single amino acid modifications via site directed mutagenesis. Forthcoming research into these methods will help determine their feasibility and practicality.<br />
<br />
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''<big>References</big>''<br />
<br />
Paesen, G.C., Adams, P.L., Harlos, K., Nuttall, P.A., and Stuart, D.I. "Tick Histamine-Binding Proteins: Isolation,<br />
Cloning, and Three-Dimensional Structure," ''Molecular Cell'', '''1999''' , ''3'', 661-671.<br />
<br />
Couillin, I., Maillet, I., Vargaftig, B.B., Jacobs, M., Paesen, G.C., Nuttall, P.A., Lefort, J., Moser, R., Weston-Davies, W., and Bernhard, R. "Arthropod-Derived Histamine-Binding Protein Prevents Murine Allergic Asthma," ''Journal of Immunology'', '''2004''', ''173'' , 3281-3286.<br />
<br />
Proud, D., Bailey, G.S., Naclerio, R.M., Reynolds, C.J., Cruz A.A., Eggleston, P.A., Lichtenstein, L.M., and Togias, A.G. "Tryptase and histamine as markers to evaluate mast cell activation during the responses to nasal challenge with allergen, cold, dry air, and hyperosmolar solutions," ''Journal of Allergy and Clinical Immunology'', '''1992''' , ''89'' , 1098-1110.<br />
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</html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_Meetings/8-3-09Team:Brown/Notebook Meetings/8-3-092009-10-22T02:38:57Z<p>Ivorugan: New page: {{Brown}} '''iGEM Meeting 8-3-09''' '''SFH 218''' *9:06: UTRA poster presentation. Call Kinko’s and let them know. Or contact Judy Nathanson for printing next door for much c...</p>
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'''iGEM Meeting 8-3-09'''<br />
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'''SFH 218'''<br />
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*9:06: UTRA poster presentation. Call Kinko’s and let them know. Or contact Judy Nathanson for printing next door for much cheaper.<br />
*9:08: Gary will be out Thursday to Thursday. Poster practice on Wednesday. <br />
*9:13: Team 1 update<br />
**EV131 is in pNoTat<br />
**Making BL21 competent – need T7 polymerase; greater control over gene expression <br />
**When running protein gel, run control cell w/o bacteria. To purify protein, will get 6-His antibodies and nickel columns.<br />
**Possible binding assays – run against stationary histamine. Running free histamine. <br />
**Threshold level of allergic rhinitis discussed. <br />
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**WE SHOULD INCLUDE ON POSTER the affinity information about EV131 <br />
**Also include considerations. <br />
**What next after we get EV131 binding? Gary belabors. <br />
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*9:40: Team 2 update<br />
**Assembled Sec-GFP. Waiting on primers to amplify and drop into expression vector<br />
**Also building Trg-EnvZ <br />
**Working on making S. epi electrocompetent<br />
**Timeline: make electrocompetent S. epi. Transform with expression vector. By end of week have expression in S. epi. <br />
**Get a picture of S. epi on the microscope. Screen for contaminants. <br />
**Also ordering primers for ccdB. <br />
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*9:46: Gary’s advice about gel extractions<br />
**Either run multiple lanes and combine, or run a big well<br />
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*9:55: Team 3 update<br />
**Now working with RFP as a selection marker<br />
**Now using assembly service to build OmpC-RBS-RFP-DT. We need to focus on Taz1 mutation.<br />
**Testing mutagenic primers this week. <br />
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*10:06: Wednesday at 3PM. Poster practice.</div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_meetingsTeam:Brown/Notebook meetings2009-10-22T02:36:50Z<p>Ivorugan: </p>
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Team Meetings- Minutes<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/4-22-09 April 22, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-4-09 May 4, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-6-09 May 6, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-27-09 May 27, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/5-31-09 May 31, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-3-09 June 3, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-8-09 June 8, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-15-09 June 15, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-16-09 June 16, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-17-09 June 17, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-19-09 June 19, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-22-09 June 22, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/6-29-09 June 29, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/7-6-09 July 6, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/7-13-09 July 13, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/8-3-09 August 3, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/8-10-09 August 10, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/8-17-09 August 17, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/9-11-09 September 11, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/9-15-09 September 15, 2009]<br />
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[https://2009.igem.org/Team:Brown/Notebook_Meetings/10-16-09 October 16, 2009]</div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-22T02:36:42Z<p>Ivorugan: </p>
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<html><img src="http://img10.imageshack.us/img10/8299/thehistaminesensor.png"></html><br />
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During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
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Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
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To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands.<br />
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= Re-engineering Chemoreceptor #1: Ribose Binding Protein =<br />
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We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger, et al., "Computational design of receptor and sensor proteins with novel functions", Nature (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger, et al "Kemp elimination catalysts by computational enzyme design", Nature (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. [[Image:Rbp img.png|400px|thumb|The native E. coli ribose-binding protein]]<br />
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'''<big>Protein design:</big>'''<br />
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1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose). [[Image:polyala_rbp_img.png|400px|thumb|The polyala RBP.]]<br />
<br />
4) Used Rosetta's Ligand Docking mode on 100 of Brown's Center for Computational Molecular Biology (http://www.brown.edu/Research/CCMB/) clustered servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. The scores for the top few final mRBP designs (the selected design highlighted in bold):<br />
<br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
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6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500. <br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode, run on the same cluster. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. Filtered the final designs to only those with a total score better than or equal to that of the scaffold RBP. Then set cutoffs for several of the relevant criteria, found the sets of the best designs in each relevant category, and found the designs that were in the intersection of those sets. The final design (in bold) selected had the best overall scores in each category.<br />
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[[Image:Final_sel.png|800px|center|thumb|The final selection list of mRBP.]]<br />
[[Image:native_score.png|800px|center|thumb|The score of the native RBP]]<br />
<br />
{|<br />
!total_score<br />
!tot_pstat_pm<br />
!tot_nlpstat_pm<br />
!tot_burunsat_pm<br />
!tot_hbond<br />
!SR_1_interf_E_1_2<br />
!SR_1_dasa_1_2<br />
|-<br />
|Weighted sum of the other terms. (Lower better)<br />
|How well packed the protein is. (0-1)<br />
|How well packed the protein is without the ligand. (0-1)<br />
|Number buried unsatisfied polars. (Lower better)<br />
|Number of H-bonds. (Higher better)<br />
|How well the ligand binds to protein. (Lower better)<br />
|How exposed the ligand is. (0-1)<br />
|}<br />
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[[Image:MRBP.png|400px| thumb|The mutated Ribose Binding Protein (mRBP)]][[Image:ligandbindingprotein.png|400px|thumb|Histamine in the mRBP ligand binding pocket]] <br />
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The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
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In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. [[Image:ompc_envz_pathway.png|400px|thumb|The TrgEnvZ-OmpR pathway. From Fig. 3 of Looger, et al., "Computational design of receptor and sensor proteins with novel functions", Nature (2003).]]<br />
<br />
= Re-engineering Chemoreceptor #2: Tar Receptor =<br />
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In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
In normal E. coli cells, aspartate binds to the Tar receptor. The Tar-EnvZ chimera protein (created by Utsumi et. al in "Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate" 1989) allowed the Taz protein to be linked to the EnvZ cascade in the same manner as Trg-EnvZ. Just as in that system, ligand binding to its receptor leads to an intracellular signaling cascade promoting gene transcription.<br />
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= Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription =<br />
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The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering [https://2009.igem.org/Team:Brown/Project_HBP gene transcription]. The successful mutation of the binding pockets on both RBP and Tar was therefore followed by the complete construction of each receptor as it would function in the cell membrane. For both RBP and Tar, this meant replicating the work done by Masayori Inouye et. al to create the chimeric proteins Trg-EnvZ (Trz) and Tar-EnvZ (Taz) (respectively). Chimeric proteins were created by fusing the intracellular domains of Tar and Trg (Trg is the membrane receptor associated with ligand-bound RBP) to the kinase domain of EnvZ. Activation of the functional receptor domains by histamine binding is thus able to initiate an intracellular cascade that phosphorylates the transcription factor ompR thereby activating gene transcription under the ompC promoter. By replacing the gene normally present under this promoter with our gene of interest, we successfully manipulated the cascade to produce an appropriate cellular response when allergens are present.<br />
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'''<big>Assay to Test Signaling Cascade Functionality</big>'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription under the OmpC promoter, we have constructed a cassette that places the OmpC promoter over the RFP gene for red fluorescence. Testing for functionality of the cascade then simply involved observing whether RFP expression occurred under ompC, a simple fluorescence assay conducted on an epifluorescence microscope. Qualitative visualization of red-fluorescing colonies transformed with both the receptor and cascade components indicated a functional intracellular signaling system. <br />
<br />
We have tested this signaling cascade by performing the following series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. As efforts to construct a novel histamine receptor were being conducted in parallel to these assays, testing of the cascade was conducted with the original chimeric chemoreceptor Tar-EnvZ. The binding of the wild-type ligand aspartate to Tar and the intracellular transcription of ompC-RFP it initiated was thus used as a proof-of-concept of our future histamine-initiated system. <br />
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1. RU1012 with no plasmid<br />
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2. RU1012 with OmpC-RFP <br />
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3. RU1012 with Tar-EnvZ<br />
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4. RU1012 with Trg-EnvZ<br />
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5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
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6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
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'''Results: Testing the Cascade'''<br />
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Our fluorescence assays of these transformed colonies indicate that signal transduction is indeed effective. <br />
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All photographs were taken on an epi-fluorescent microscope: Olympus SZX16, excitation source X-cite Series 120. The first image in each series is under bright light, the second under a fluorescent filter for RFP. <br />
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1. RU1012 with no plasmid<br />
<br />
[[Image:RU1012 BL.jpg|300px]] [[Image:RU1012 RFP.jpg|300px]]<br />
<br />
<br />
2. RU1012 with OmpC-RFP <br />
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[[Image:RU1012 OmpC BL.jpg|300px]] [[Image:RU1012 OmpC RFP.jpg|300px]]<br />
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<br />
3. RU1012 with Tar-EnvZ<br />
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[[Image:RU1012 Taz BL.jpg|300px]] [[Image:RU1012 Taz RFP.jpg|300px]]<br />
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4. RU1012 with Trg-EnvZ<br />
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[[Image:RU1012 Trg BL.jpg|300px]][[Image:RU1012 Trg RFP.jpg|300px]]<br />
<br />
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5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
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[[Image:RU1012 Taz OmpC BL.jpg|300px]] [[Image:RU1012 Taz OmpC RFP.jpg|300px]]<br />
<br />
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6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
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[[Image:RU1012 Trg OmpC BL.jpg|300px]] [[Image:RU1012 Trg OmpC RFP.jpg|300px]]<br />
<br />
----<br />
<br />
<br />
''<big>References</big>''<br />
<br />
Baumgartner, James W., Changhoon Kim, Renée E. Brisette, Masoyuri Inouye, Changkyu Park, and Gerald L. Hazelbauer. "Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ." Journal of Bacteriology 176: 1157-163. Journal of Bacteriology. Web. 25 July 2009. <http://www.ncbi.nlm.nih.gov:80/pmc/articles/PMC205168/>.<br />
<br />
Looger, Loren L., Mary A. Dwyer, James J. Smith, and Homme H. Hellinga. "Computational design of receptor and sensor proteins with novel functions." Nature 423 (2003): 185-90. PubMed. Web. 15 July 2009. <http://www.ncbi.nlm.nih.gov/pubmed/12736688>.<br />
<br />
Meiler, Jens, and David Baker. "ROSETTALIGAND: protein-small molecule docking with full side-chain flexibility." Protein 65.3 (2006): 538-48. PubMed. Web. 12 Aug. 2009. <http://www.ncbi.nlm.nih.gov/pubmed/16972285>.<br />
<br />
Rhiju, Das, and David Baker. "Macromolecular modeling with rosetta." Annual Review of Biochemistry 77: 368-82. PubMed. Web. 12 July 2009. <http://www.ncbi.nlm.nih.gov/pubmed/18410248>.<br />
<br />
Rothlisberger, Daniela, Olga Kersonsky, Andrew M. Wollacott, Lin Jiang, Jason Dechancie, Jamie Betger, Jasmine L. Gallaher, Eric A. Althoff, Alexandre Zanghellini, Orly Dym, Shira Albeck, Kendall N. Houk, Dan S. Tawfik, and David Baker. "Kemp elimination catalysts by computational enzyme design." Nature 453 (2008): 190-95. Nature. 19 Mar. 2008. Web. 5 July 2009. <http://www.nature.com/nature/journal/v453/n7192/abs/nature06879.html>.<br />
<br />
Utsumi, R., R. E. Brisette, A. Rampersaud, S. A. Forst, K. Oosawa, and M. Inouye. "Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate." Science 245.4923 (1989): 1246-249. Science. Web. 27 June 2009. <http://www.sciencemag.org/cgi/content/abstract/245/4923/1246>.<br />
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<html><br />
<a href="https://2009.igem.org/Team:Brown/Project_HBP"><br />
<img src="https://static.igem.org/mediawiki/2009/d/d8/Brown_hbp_bottom_3.png"><br />
</html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-22T02:36:18Z<p>Ivorugan: /* Histamine Sensor */</p>
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<html><img src="http://img10.imageshack.us/img10/8299/thehistaminesensor.png"></html><br />
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During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
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Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
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To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands.<br />
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= Re-engineering Chemoreceptor #1: Ribose Binding Protein =<br />
<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger, et al., "Computational design of receptor and sensor proteins with novel functions", Nature (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger, et al "Kemp elimination catalysts by computational enzyme design", Nature (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. [[Image:Rbp img.png|400px|thumb|The native E. coli ribose-binding protein]]<br />
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'''<big>Protein design:</big>'''<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose). [[Image:polyala_rbp_img.png|400px|thumb|The polyala RBP.]]<br />
<br />
4) Used Rosetta's Ligand Docking mode on 100 of Brown's Center for Computational Molecular Biology (http://www.brown.edu/Research/CCMB/) clustered servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. The scores for the top few final mRBP designs (the selected design highlighted in bold):<br />
<br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500. <br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode, run on the same cluster. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. Filtered the final designs to only those with a total score better than or equal to that of the scaffold RBP. Then set cutoffs for several of the relevant criteria, found the sets of the best designs in each relevant category, and found the designs that were in the intersection of those sets. The final design (in bold) selected had the best overall scores in each category.<br />
<br />
[[Image:Final_sel.png|800px|center|thumb|The final selection list of mRBP.]]<br />
[[Image:native_score.png|800px|center|thumb|The score of the native RBP]]<br />
<br />
{|<br />
!total_score<br />
!tot_pstat_pm<br />
!tot_nlpstat_pm<br />
!tot_burunsat_pm<br />
!tot_hbond<br />
!SR_1_interf_E_1_2<br />
!SR_1_dasa_1_2<br />
|-<br />
|Weighted sum of the other terms. (Lower better)<br />
|How well packed the protein is. (0-1)<br />
|How well packed the protein is without the ligand. (0-1)<br />
|Number buried unsatisfied polars. (Lower better)<br />
|Number of H-bonds. (Higher better)<br />
|How well the ligand binds to protein. (Lower better)<br />
|How exposed the ligand is. (0-1)<br />
|}<br />
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[[Image:MRBP.png|400px| thumb|The mutated Ribose Binding Protein (mRBP)]][[Image:ligandbindingprotein.png|400px|thumb|Histamine in the mRBP ligand binding pocket]] <br />
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The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
<br />
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In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. [[Image:ompc_envz_pathway.png|400px|thumb|The TrgEnvZ-OmpR pathway. From Fig. 3 of Looger, et al., "Computational design of receptor and sensor proteins with novel functions", Nature (2003).]]<br />
<br />
= Re-engineering Chemoreceptor #2: Tar Receptor =<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
In normal E. coli cells, aspartate binds to the Tar receptor. The Tar-EnvZ chimera protein (created by Utsumi et. al in "Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate" 1989) allowed the Taz protein to be linked to the EnvZ cascade in the same manner as Trg-EnvZ. Just as in that system, ligand binding to its receptor leads to an intracellular signaling cascade promoting gene transcription.<br />
<br />
= Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription =<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering [https://2009.igem.org/Team:Brown/Project_HBP gene transcription]. The successful mutation of the binding pockets on both RBP and Tar was therefore followed by the complete construction of each receptor as it would function in the cell membrane. For both RBP and Tar, this meant replicating the work done by Masayori Inouye et. al to create the chimeric proteins Trg-EnvZ (Trz) and Tar-EnvZ (Taz) (respectively). Chimeric proteins were created by fusing the intracellular domains of Tar and Trg (Trg is the membrane receptor associated with ligand-bound RBP) to the kinase domain of EnvZ. Activation of the functional receptor domains by histamine binding is thus able to initiate an intracellular cascade that phosphorylates the transcription factor ompR thereby activating gene transcription under the ompC promoter. By replacing the gene normally present under this promoter with our gene of interest, we successfully manipulated the cascade to produce an appropriate cellular response when allergens are present.<br />
<br />
'''<big>Assay to Test Signaling Cascade Functionality</big>'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription under the OmpC promoter, we have constructed a cassette that places the OmpC promoter over the RFP gene for red fluorescence. Testing for functionality of the cascade then simply involved observing whether RFP expression occurred under ompC, a simple fluorescence assay conducted on an epifluorescence microscope. Qualitative visualization of red-fluorescing colonies transformed with both the receptor and cascade components indicated a functional intracellular signaling system. <br />
<br />
We have tested this signaling cascade by performing the following series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. As efforts to construct a novel histamine receptor were being conducted in parallel to these assays, testing of the cascade was conducted with the original chimeric chemoreceptor Tar-EnvZ. The binding of the wild-type ligand aspartate to Tar and the intracellular transcription of ompC-RFP it initiated was thus used as a proof-of-concept of our future histamine-initiated system. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
2. RU1012 with OmpC-RFP <br />
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3. RU1012 with Tar-EnvZ<br />
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4. RU1012 with Trg-EnvZ<br />
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5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
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6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
'''Results: Testing the Cascade'''<br />
<br />
Our fluorescence assays of these transformed colonies indicate that signal transduction is indeed effective. <br />
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All photographs were taken on an epi-fluorescent microscope: Olympus SZX16, excitation source X-cite Series 120. The first image in each series is under bright light, the second under a fluorescent filter for RFP. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
[[Image:RU1012 BL.jpg|300px]] [[Image:RU1012 RFP.jpg|300px]]<br />
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2. RU1012 with OmpC-RFP <br />
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[[Image:RU1012 OmpC BL.jpg|300px]] [[Image:RU1012 OmpC RFP.jpg|300px]]<br />
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3. RU1012 with Tar-EnvZ<br />
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[[Image:RU1012 Taz BL.jpg|300px]] [[Image:RU1012 Taz RFP.jpg|300px]]<br />
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4. RU1012 with Trg-EnvZ<br />
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[[Image:RU1012 Trg BL.jpg|300px]][[Image:RU1012 Trg RFP.jpg|300px]]<br />
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5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
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[[Image:RU1012 Taz OmpC BL.jpg|300px]] [[Image:RU1012 Taz OmpC RFP.jpg|300px]]<br />
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6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
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[[Image:RU1012 Trg OmpC BL.jpg|300px]] [[Image:RU1012 Trg OmpC RFP.jpg|300px]]<br />
<br />
----<br />
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''<big>References</big>''<br />
<br />
Baumgartner, James W., Changhoon Kim, Renée E. Brisette, Masoyuri Inouye, Changkyu Park, and Gerald L. Hazelbauer. "Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ." Journal of Bacteriology 176: 1157-163. Journal of Bacteriology. Web. 25 July 2009. <http://www.ncbi.nlm.nih.gov:80/pmc/articles/PMC205168/>.<br />
<br />
Looger, Loren L., Mary A. Dwyer, James J. Smith, and Homme H. Hellinga. "Computational design of receptor and sensor proteins with novel functions." Nature 423 (2003): 185-90. PubMed. Web. 15 July 2009. <http://www.ncbi.nlm.nih.gov/pubmed/12736688>.<br />
<br />
Meiler, Jens, and David Baker. "ROSETTALIGAND: protein-small molecule docking with full side-chain flexibility." Protein 65.3 (2006): 538-48. PubMed. Web. 12 Aug. 2009. <http://www.ncbi.nlm.nih.gov/pubmed/16972285>.<br />
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Rhiju, Das, and David Baker. "Macromolecular modeling with rosetta." Annual Review of Biochemistry 77: 368-82. PubMed. Web. 12 July 2009. <http://www.ncbi.nlm.nih.gov/pubmed/18410248>.<br />
<br />
Rothlisberger, Daniela, Olga Kersonsky, Andrew M. Wollacott, Lin Jiang, Jason Dechancie, Jamie Betger, Jasmine L. Gallaher, Eric A. Althoff, Alexandre Zanghellini, Orly Dym, Shira Albeck, Kendall N. Houk, Dan S. Tawfik, and David Baker. "Kemp elimination catalysts by computational enzyme design." Nature 453 (2008): 190-95. Nature. 19 Mar. 2008. Web. 5 July 2009. <http://www.nature.com/nature/journal/v453/n7192/abs/nature06879.html>.<br />
<br />
Utsumi, R., R. E. Brisette, A. Rampersaud, S. A. Forst, K. Oosawa, and M. Inouye. "Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate." Science 245.4923 (1989): 1246-249. Science. Web. 27 June 2009. <http://www.sciencemag.org/cgi/content/abstract/245/4923/1246>.<br />
<br />
<html><br />
<a href="https://2009.igem.org/Team:Brown/Project_HBP"><br />
<img src="https://static.igem.org/mediawiki/2009/d/d8/Brown_hbp_bottom_3.png"><br />
</html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Project_IntroductionTeam:Brown/Project Introduction2009-10-22T02:35:15Z<p>Ivorugan: /* The Allergic Response */</p>
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<html><img src="http://img132.imageshack.us/img132/4105/theallergiceresponse.png"></html><br />
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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. Histamine has been identified as a principal mediator of inflammatory responses. Upon first contact with an allergen, plasma cells release Immunoglobulin E (IgE) antibodies. These antibodies then activate mast cell degranulation and release of histamine. When histamine reaches histamine receptors on various target cells, vasodilation and inflammation occurs, resulting in allergic symptoms. <br />
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Allergies are routinely treated with antihistamine drugs, which have many adverse effects. Antihistamines compete with histamine to bind and block these histamine receptors, preventing the initiation of the inflammatory response. However, antihistamines also block receptors of the nervous system, thereby causing drowsiness. For many people, sedation remains the primary concern when considering the adverse effects of the newer antihistamines, particularly since these drugs are given to patients with chronic disorders with treatment periods that often extend over several months or even years. Besides sedation, there also exists concern regarding the caridotoxicity of antihistamines and other adverse drug interactions. For patients suffering from chronic allergies and inflammation, there is a great need for an alternative strategy for combating allergic symptoms without causing significant side effects.<br />
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<Video coming soon!><br />
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Allergene presents a favorable alternative because it utilizes the binding affinity of a histamine binding protein rEV131. Similar to antihistamines, this protein prevents histamine molecules from interacting with histamine. However, by directly sequestering the histamine molecules rather than blocking their receptors, the drowsiness side-effect is successfully avoided. <br />
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<html><br />
<a href="https://2009.igem.org/Team:Brown/Project_Histamine_Sensor"><br />
<img src="https://static.igem.org/mediawiki/2009/5/51/Brown-button-2-histamine-sensor.png"><br />
</html></div>Ivoruganhttp://2009.igem.org/Team:Brown/ProjectTeam:Brown/Project2009-10-22T02:34:42Z<p>Ivorugan: /* Project Abstract */</p>
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== A Synthetic Approach to Treating Allergic Rhinitis: Engineering Staphyloccocus Epidermidis to Secrete High-Affinity Histamine Binding Protein in Response to Elevated Levels of Histamine during an Allergic Attack ==<br />
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<html><img src="http://img29.imageshack.us/img29/1341/abstractm.png"></html><br />
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Brown University’s 2009 iGEM Team presents an exciting new approach to treating nasal allergies through Allergene: a synthetically engineered, self-regulating drug factory in the nose. This revolutionary new product provides a much-needed alternative to current antihistamines by directly sequestering the histamine released in an allergic response. In order to do this, Allergene makes use of the unique histamine binding protein rEV131, native to the tick Rhipicephalus appendiculatus. By taking advantage of rEV131's high binding affinity for histamine, Allergene effectively eliminates both the symptoms and side effects associated with allergies and their treatments. <br />
<br />
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Rather than presenting a system that passively sequesters histamine; however, Allergene goes one step further to providing patients with much-needed allergy relief. Activation of the product only occurs in the explicit event of an actual allergic response. This ingenious mechanism is made possible through the novel engineering of a histamine receptor in prokaryotes, a grand undertaking that had never before been accomplished. By re-designing pre-existing prokaryotic chemoreceptors to bind histamine rather than their wild-type ligands, Allergene acquired its most impressive feature yet: the "Histamine Sensor". Site-directed mutagenesis on the ligand binding pockets of two particular prokaryotic chemoreceptors, Ribose Binding Protein and Tar (normally responsive to Aspartate)was performed to create this novel sensor. Through histamine detection, Allergene’s unique histamine sequestering system is engineered to function only when histamine levels are markedly high. This efficiently timed system is therefore completely self-regulating. <br />
<br />
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Allergene is introduced to its host patients by capitalizing on the endogenous existence of bacterium Staphylococcus epidermidis in human nasal flora. This naturally present organism is the perfect vehicle for delivery; its rapid production and secretion of Allergene’s genetic constructs effectively implement the system’s histamine sensing and sequestering capabilities in human hosts. Secretion is accomplished through the attachment of a signal peptide sequence specific to S. epidermidis, a clear and concise method by which to deliver the ready-to-use drug. In order to eliminate any safety concerns associated with the utilization of S. epidermidis, special care has additionally been taken to engineer the product’s accessory kill switch mechanism. Capitalizing on the cells' abilities to sense the growth of their own populations, a DNA gyrase poison responsible for triggering cell death (CCDB)is neatly placed under the quorum or population sensing promoter Agr, which normally functions in hazardous biofilm formation. Named the “Quorum Sensor”, this thoughtful, additional feature prompts Allergene’s swift response in the unfavorable event of over-cell-proliferation.<br />
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</html></div>Ivoruganhttp://2009.igem.org/Template:BrownTemplate:Brown2009-10-22T02:30:08Z<p>Ivorugan: </p>
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<li id="mainHome"><a href="https://2009.igem.org/Team:Brown">Home</a></li><br />
<li id="mainTeam"><a class="aTeam" href="https://2009.igem.org/Team:Brown/Team">Team</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Brown/Team">About Us</a></li><br />
<li><a href="http://www.brown.edu">Brown University</a></li><br />
</ul><br />
</li><br />
<li id="mainProject"><a class="aProject" href="https://2009.igem.org/Team:Brown/Project">Project</a><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Brown/Project">Abstract</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Project_Introduction">The Allergic Response</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Project_Histamine_Sensor">Histamine Sensor</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Project_HBP">Histamine Binding Protein</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Project_S.epidermidis">S.epidermidis</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Project_All_Together">System Schematic</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Project_Implications">Human Practices</a></li><br />
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<ul><br />
<li><a href="https://2009.igem.org/Team:Brown/Notebook_Weekly_Logs">Weekly Logs </a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Notebook_Protocols">Protocols</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Notebook_Recipes">Recipes</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Notebook_meetings">Team Meetings</a></li><br />
</ul><br />
</li><br />
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<ul><br />
<li><a href="https://2009.igem.org/Team:Brown/Links_Acknowledgements">Acknowledgements</a></li><br />
<li><a href="https://2009.igem.org/Team:Brown/Links_Sponsors">Sponsors</a></li><br />
</ul><br />
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</body></html></div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_Meetings/6-8-09Team:Brown/Notebook Meetings/6-8-092009-10-22T02:27:06Z<p>Ivorugan: </p>
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<div>{{Brown}}<br />
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'''iGEM Meeting'''<br />
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'''June 8, 2009'''<br />
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*6:00 PM PST<br />
*6:19: We discovered we had video chat capabilities. It made the meeting AWESOME.<br />
*6:23: Possible 3rd kill switch: SulA stops FtsZ gene which is responsible for forming a contractile ring in binary fission<br />
**Another possibility: having a cell synthesize a colicin under certain conditions. Colicin is a toxin that kills bacteria. Check out this Pubmed article. **http://www.ncbi.nlm.nih.gov/pubmed/17347522 <br />
*6:28: Will discusses the EV131 failed clinical trials. The Histamine Binding Protein needs to not be in the bloodstream because it works outside of the bloodstream to cause vasodilation. and the IGE Antibody binding protein needs to be in the bloodstream to bind. This defeats our bifunctional protein idea. <br />
**Will’s new tactic: we could create a safe strain of S. aureus that secretes EV131 in the nose, and focus on establishing a secretion protocol. We can make biobricks for secretion. We can create new parts for the bacteria. Will is making a list of other things we can focus on. It’s on the google group. <br />
<br />
*Something like this: <br />
<br />
*Going from: <br />
**Cambridge's project to make a gram-positive chassis: https://2007.igem.org/Cambridge/Gram-positive_chassis_project<br />
*The Parts page about B. subtilis: http://partsregistry.org/Chassis/B.subtilis_Strains<br />
<br />
*Use Bacillus subtilis as our chassis.<br />
*Figure out how to turn off the genes that allow for mobility.<br />
*Add genes from S. aureus that allow B. subtilis to cling to mucus.<br />
*Add new parts for protein secretion in gram-positive bacteria (in addition to the one already there)<br />
*Come up with a new technical standard regarding gram-positive bacteria as a factory for proteins (this hasn't been done, I think)<br />
*Biobricks for uptake into blood<br />
**Indu will email professor Wessel about the bifunctional issue. We will go from there.<br />
**New issue, the bifunctional protein is that we are targeting to very different things, with different properties, quantities, and location. The IGE Antibody binding protein may be too large compared to the histamine binding protein, and this protein may be too large to be taken into the bloodstream. This can be solved by finding the single important region of each receptor and bridging them together. Protocols and biobricks for secretion, production, protein bridge can be our focus.<br />
*7:02: We need faculty input on these issues! Michael will follow up with Wessel and Adrian about the June 12 meeting. <br />
*7:08: Indu and Steph are researching parts of the IGE Antibody that we can focus on to minimize the size of the binding protein<br />
*7:20: We need to order / obtain UCP, Holin, colicin, and SulA. There are many different holins to choose from; Ahmad wants us to pick a couple! Ahmad: randomly order a couple from the Texas A&M professor, just so we have them on hand. Get the sequences.<br />
**We can characterize the one Holin in the registry and see if it works for our project. We can do the same for UCP. SulA Promoter (?) and colicin are in the registry…<br />
*7:25: For the first meeting at 8:30 AM on Monday, we will be talking about project plans and splitting up into teams. <br />
*7:38: We still need a repressor for the entire Safe Cell system!! We should consult Wessel about repressors. Gotta be tight, not endogenous to the organism, and uncommon in the world. Michael will send him an email. <br />
*7:43: Time and money about protocols are a concern. We can ask Adrian to take a look at the protocols and advise us step by step.<br />
*7:47: Everyone send out your emails! And forward the replies to the entire group. See you Monday at 9 AM Eastern Time.</div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_Meetings/6-3-09Team:Brown/Notebook Meetings/6-3-092009-10-22T02:23:48Z<p>Ivorugan: New page: {{Brown}} '''Brown iGEM Meeting''' '''June 2, 2009''' '''WebEx Conference Call''' *Attendees: Steph, Will, Indu, Eli, Michael *8:20: Steph will be researching S. Aureus express...</p>
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<div>{{Brown}}<br />
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'''Brown iGEM Meeting'''<br />
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'''June 2, 2009'''<br />
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'''WebEx Conference Call'''<br />
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*Attendees: Steph, Will, Indu, Eli, Michael<br />
<br />
*8:20: Steph will be researching S. Aureus expression vectors, and the natural levels of Staph in the nasal colony<br />
*8:25: We are looking into nanoparticle repressor system for Safe Cell<br />
*8:31: For Histamine – We are going to focus on making a bifunctional protein – EV131 and IGE antibody binding protein<br />
**The protein will be made by a Staph strain endogenous to the nasal colony<br />
**We are targeting specifically the symptoms of Hay fever<br />
**Secretion and uptake into the bloodstream needs to be researched. <br />
*8:36: We should look at past iGEM projects to see if any other teams have done projects with similar aspects as our current 2<br />
**Have any teams worked with S. epidermidis, or any gram positive bacteria?<br />
*8:42: Since S. aureus might be dangerous to work with in the lab, we can do proof of concept with another well-studied gram positive bacteria<br />
*8:44: Indu will put up the Histamine step by step project design<br />
*8:47: For Safe Cell, we need to research the additional novel kill switch for the 3rd way to kill a cell. <br />
**We also need to find a solid repressor system. <br />
*8:55: S. epidermidis is endogenous to the nose!<br />
**Can we order colonies of S. epidermidis? Eli is looking at that.<br />
*9:00: Will is researching everything about S. epidermidis<br />
**Eli is looking into secretion / uptake<br />
**Indu and Michael are looking into creating a bifunctional proteins<br />
*9:02: Flora, can you start looking up nanoparticles in a repressor system?<br />
**Ahmad, we need you to look into a 3rd killing mechanism for the cell.<br />
**Everyone should start fleshing out potential project designs. <br />
*9:04: Indu will be making a list of “materials” we need<br />
**Can we get Histamine plates? Or IGE Antibody plates? Where can we get these?<br />
**How can we test binding affinity? We will having our binding proteins on a stationary phase. Wash over with fluorescent protein. (?)<br />
*9:11: As you can see there are a lot of things to be researched! There is a new sign up sheet on the Google group. <br />
*9:14: Next meeting is Monday 6PM Pacific Time.</div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_Meetings/5-31-09Team:Brown/Notebook Meetings/5-31-092009-10-22T02:21:23Z<p>Ivorugan: </p>
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<div>{{Brown}}<br />
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'''Brown iGEM 2009'''<br />
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'''WebEx Conference Call'''<br />
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'''May 31, 2009 8:00 PM'''<br />
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'''Attendees: Will, Eli, Indu, Michael'''<br />
<br />
*8:00: Will introduces new idea to deal with localization problem using S. aureus. See google group for details.<br />
*8:12: We are going to send the email out to the professors. Ask Adrian for the password for igem@brown.edu so we can also check up on which professors are coming to the conference on June 12.<br />
*8:19: Indu could not get a hold of Harvard but will try again tomorrow. Indu will check issues with patents.<br />
*8:37: For Safe Cell, we would like to come up with another unique killing mechanism (instead of ccdb) that we can call our own in the project. Possibilities: interrupt cell cycle / fission<br />
*8:43: Eli mentions something in the media that facilitates transport of the repressor<br />
*8:47: We should ask some other people about the repressor. <br />
*8:49: For next meeting: we need Safe Cell repressor candidates, a 3rd novel killing mechanism, details from Steph about S. aureus. <br />
*8:53: We should look into gram positive protein secretion. Look up natural levels of staph in the nose.<br />
*9:00: Next Meeting: Wed June 3. 8 AM PST. If your trial is expired before then just make a new account with different info.</div>Ivoruganhttp://2009.igem.org/Team:Brown/Notebook_Meetings/5-27-09Team:Brown/Notebook Meetings/5-27-092009-10-22T02:19:55Z<p>Ivorugan: </p>
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<div>{{Brown}}<br />
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'''Brown iGEM 2009'''<br />
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'''Phone Conference'''<br />
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'''5/27/09'''<br />
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'''(Pacific Time)'''<br />
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*8:20PM: Meeting starts after a lot of technical difficulties<br />
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*8:22: Brad Lowell at Harvard does not have UCP2, Invitrogen may have it looked up UCPs on invitrogen<br />
**What expression vectors has the past iGEM teams used?<br />
**We need E. coli expression vectors <br />
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*8:32 Ahmad, ask them for the UCP<br />
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*8:29: Indu wrote a nicely worded email.<br />
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*8:31: Picking holins (there’s 3000!)<br />
**There are some in the registry<br />
**UC Berkeley from last year used Holin<br />
**Ahmad will ask them for a couple Holins<br />
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*8:49: CCDB – stops cell growth – can be another redundant mechanism<br />
**It is in the registry<br />
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*8:56: All of the parts so far are in the registry, we need a unique part to submit to the registry<br />
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*9:01: We need to schedule a meeting with the panel for when we get back!<br />
**June 17th<br />
**Email the professors<br />
**It would be nice to have Gary Wessel email them.<br />
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*9:08: Michael write an email out to the professors inviting them<br />
**Have the draft proofread by the group<br />
**Have Wessel forward it to everybody<br />
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*9:15: Somebody else – research a tight repressor!!<br />
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*9:17: Indu will follow up phone call the lab for the tick protein<br />
**Second idea to the histamine binding protein – have something that also binds IgE antibodies<br />
**This provides redundancy – both binding current histamine and stopping further secretion of histamine<br />
**Link them with glycine bridge<br />
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*9:26: Probiotic system: natural bacteria that secretes protein into bloodstream<br />
**We have getting it into the system from MIT yogurt<br />
**We have intestinal implantation BactoKidney<br />
**How do we get the bifunctional protein secreted?<br />
<br />
*9:44: There is a lady named Edith that Will will email – she can help us on probiotic drug transport across organs<br />
**Ashley, Eli, Will, Minoo can research probiotic <br />
<br />
*9:45: Indu found a paper on protein that binds IGE antibody – she will put it on the google group<br />
**Somebody needs to research IGE<br />
**Eli will put up a paper about packaging<br />
**Will is emailing about drug delivery systems<br />
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*9:46: Any paper that may be useful to either project will be put on the google groups<br />
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*9:56: Before 8pm PST – everyone go on the google group and build on the research that’s already been done.<br />
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*10:00: Secretion: Eli, Will, Ashley<br />
**OK everyone just decide what to research on the google group. <br />
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*10:01: We are meeting on SUNDAY MAY 31 SAME TIME (Monday for Minoo)<br />
<br />
*10:02: Put up minutes.<br />
<br />
*10:16: bye everybody</div>Ivoruganhttp://2009.igem.org/Team:Brown/TeamTeam:Brown/Team2009-10-22T02:17:11Z<p>Ivorugan: </p>
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<div>{{Brown}}<br />
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[[Image:About-brown-igem-bar-1.png]]<br />
The Brown iGEM Lab is entirely student-run and consists of nine Brown University undergraduates. We began our training last spring with a lab course in Synthetic Biology. Under the gracious support of the Brown Undergraduate Teaching and Research Award (UTRA) Program and funding from various other academic departments, we were able to work throughout the summer on our project. Though the project's generation and implementation was an entirely student-directed process, the team owes much of its academic and research support to the faculty and graduate student advisers that helped make everything possible. Here at Brown, we pride ourselves in upholding the ideals set out by the iGEM competition, namely that the project we have set out to create is fully our own (from creation to completion) and that each student involved in the program be afforded his/her full opportunity to both learn and contribute in the lab. Therefore, in the true spirit of Synthetic Biology, our team's project this year works hard to reflect the many different research backgrounds contributed by its nine individual members; elements of electrical engineering, electrochemistry, genetics, and microbiology are incorporated. <br />
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{|border = "0"<br />
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'''Advisors:'''<br />
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*''' Faculty Advisor ''': Dr. Gary Wessel, Professor of Biology, Bio Med Molecular, Cellular Biology Biochemistry <br />
*'''Graduate Student Advisor ''': Adrian Reich, Graduate Student, Bio-Med (Bio) <br />
*'''Graduate Student Advisor ''': Diana Donovan, Graduate Student, Bio-Med (Bio)<br />
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'''Undergraduate Students:'''<br />
<br />
*'''William Allen''': Will Allenquot<br />
*'''Michael Chang''': MC Mastermix<br />
*'''Stephanie Cheung''': Stephylococcus <br />
*'''Ashley Kim''': Ashley Kimwipe<br />
*'''Flora War War Ko''': Nasal Flora<br />
*'''Elias Scheer''': E.coli Scheer<br />
*'''Minoo Ramanathan''': Minoo Prep<br />
*'''Ahmad Rana''': Ahmad Ran-a-gel<br />
*'''Indu Voruganti''': The INDUcer<br />
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|<br />
<gallery><br />
Image:Garywessel.jpg|Dr. Gary Wessel<br />
Image:Adrianreich.jpg|Adrian Reich<br />
Image:Diana D.JPG|Diana Donovan<br />
</gallery><br />
<gallery><br />
Image:Will.jpg|Will Allen<br />
Image:Michael.jpg|Michael Chang<br />
Image:Steph.jpg|Steph Cheung<br />
Image:Ashley.jpg|Ashley Kim<br />
Image:flora.jpg|Flora Ko<br />
Image:eli.jpg|Eli Scheer<br />
Image:ahmad.jpg|Ahmad Rana<br />
Image:minoo.jpg|Minoo Ramanthan<br />
Image:indu.jpg|Indu Voruganti</div>Ivorugan