http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=100&target=Gcromar&year=&month=2010.igem.org - User contributions [en]2024-03-28T09:36:32ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/File:Toronto_EncapsulinExpression.pngFile:Toronto EncapsulinExpression.png2010-10-27T08:28:58Z<p>Gcromar: </p>
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<div></div>Gcromarhttp://2010.igem.org/File:Toronto_EncapsulinConstruction.pngFile:Toronto EncapsulinConstruction.png2010-10-27T08:28:10Z<p>Gcromar: </p>
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<div></div>Gcromarhttp://2010.igem.org/File:Fba.pngFile:Fba.png2010-10-27T08:26:28Z<p>Gcromar: </p>
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<div></div>Gcromarhttp://2010.igem.org/Team:Toronto/ResultsTeam:Toronto/Results2010-10-27T08:22:23Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
|}<br />
<br />
<br />
<h1>'''Results'''</h1><br />
<br />
<h2>'''''E. coli'' DH5a is sensitive to Catechol exposure'''</h2><br />
<br />
To measure the effect of our pathway manipulations on the viability of ''E.coli'' exposed to catechol we plan to perform a tolerance test. In preliminary experiments we established the baseline sensitivity of ''E.coli'' (DH5a) exposed to 50mM catechol in liquid media over a six hour time course. Aliquots of this mixture were plated on LB media at various dilutions and colonies were counted and compared with untreated controls (see "Protocols"). There was no measurable difference between control and treated cells when bacteria were exposed to catechol in a growth media (data not shown). However, in minimal media (MM2) growth of control cells was moderate while exposed cells exhibited a marked decrease in survival (Figure 1). Although the effects of Catechol exposure in ''E.coli'' and ''P. putida'' have previously been investigated [Park et al. 2001] completion of these baseline experiments will enable us to determine the effect of pathway manipulations on the proposed engineered ''E.coli'' strains containing various configurations of the pathway.<br />
<br />
<p><br />
'''A'''<br />
[[Image:Toronto-LB-Plate_1.jpg|400px]] [[Image:Toronto-LB-Plate_2.jpg|400px]]<br />
</p><br />
<p><br />
'''B'''[[Image:Toronto-results-catechol_tolerance.png|center]]<br />
</p><br />
<blockquote>'''Figure 1:''' A. Growth of ''E.coli'' DH5a on LB plates (1x10<sup>-5</sup> dilution) following exposure to minimal media and minimal media with 50mM catechol in solution. Plates shown are for the last time points plotted. B. Survival curves of ''E. coli'' DH5a exposed to catechol over a six hour time course.</blockquote><br />
<br />
<H2>'''Synthetic construction of a functioning catechol ortho degradation pathway in E.coli is predicted to increase cell growth rate'''</H2><br />
To predict the effects of adding the catechol ortho-cleavage pathway on ''E.coli'' metabolism we used a previously developed genome-scale reconstruction of E. coli (iJR904) and applied flux balance analysis (FBA) to compare likely growth outcomes for normal and optimized pathways. We found that optimization of this pathway in the presence of glucose and sufficient oxygen leads to a prediction of increased growth rate. This result bodes well for the success of engineered bacteria introduced into mixed culture within the tailings ponds as they might be expected to have a survival advantage over their native counterparts. However, it should be pointed out that a corresponding genome-scale reconstruction of the target organism ''P. putida'' is not available at this time. These methods and results are discussed at length in the Modeling section.<br />
<br />
[[image:fba.png|500px|center]]<br />
Figure 1. Predicted growth of E. coli at various uptake rates of glucose and catechol. The points specified on the surface are considered further in simulations. Points are named according to the uptake rate of glucose and catechol (eg. Glc6/cat0 corresponds to a glucose uptake of 6 mmol/gDW/h and catechol uptake of zero). Glc6/cat0 corresponds to a “wildtype” (WT) condition, while glc6/cat2.52 corresponds to the “optimal” (OPT) ratio predicted for by FBA to maximize biomass production. Glc0/cat6 and glc6/cat20 represent additional hypothetical situtions where catechol replaces glucose as an energy source, and where both are used at their maximum rates, respectively.<br />
<br />
<br />
<h2>'''Colocalization of muconolactone D-isomerase (EC:5.3.3.4) and 3-oxoadipate enol-lactone hydrolase (EC:3.1.1.24) are predicted to lead to increased catechol degradation through metabolic channeling'''</h2><br />
In order to determine which enzymes would most benefit from metabolic channeling in the catechol degradation pathway, we used a cellular simulation tool, developed in our lab, called Cell++. It allows us to place enzymes of choice in a compartment within a cellular environment and calculates the effect of localization on metabolite concentrations in a user-defined biochemical pathway (Sanford et al. 2006). We gathered kinetic data of the five enzymes in the degradation pathway and investigated the effects of localizing pairs of sequential enzymes (i.e. catechol 1, 2-dioxygenase and muconate cycloisomeras) on metabolite concentrations. Four co-localization simulations were performed in Cell++ along with a control simulation where no enzymes were co-localized. The initial metabolite in the simulation environment, catechol, was added into the simulation environment and the simulation environment was run for 10,000 iterations to observe the effect on catechol degradation in each simulation. The results are displayed as a table indicating the concentration of all metabolites at 1000 equidistant time points during the simulation. <br />
<p>To make use of this information, we extracted the maximum metabolite concentration and the maximum rate of metabolite formation for all metabolites during each simulation. These values are normalized from a scale of 0 to 1, where 0 indicates the lowest value of the concentration/rate of formation (C/RoF) in the simulations and 1 is the highest. These values were further evaluated against the control simulation to the find the percentage change of the C/RoF values of each metabolite in the four co-localization simulations. In order to classify a particular enzyme co-localization as beneficial, we need to see an increase in the maximum concentration and rate of formation of the final metabolite in the pathway (meta6). These increases would represent an increased flux through the pathway. Of the four co-localization simulations, simulation 3 (which co-localizes the enzymes muconolactone D-isomerase and 3-oxodipate-enol lactone hydrolase) was the only simulation that matched the criteria for a beneficial co-localization. From these simulations, we decided to co-localize these two enzymes for our wet lab experiments to see if Cell++ accurately depicted the effects on catechol degradation.</p><br />
<br />
''' '''[[Image:Cellppheatmap.jpg|center]]<br />
<br />
<blockquote>'''Figure 2.''' Simulating the effects of enzyme co-localization on catechol degradation efficiency.<br />
Red indicates increase in the value of the kinetic parameter relative to the simulation where no enzymes are co-localized (0.0), while green indicates a decrease in the kinetic parameter. 1.0, 2.0, 3.0 and 4.0 represent pair-wise fusions of the 1st and 2nd, 2nd and 3rd, 3rd and 4th, and 4th and 5th enzymes respectively. “Max[]” and “Vmax” represent the maximum concentration and the maximum rate of formation of a particular metabolite in a particular simulation. The fusion of the 3rd and 4th enzymes (3.0) resulted in an increase of the production of the final pathway product, while the other fusion pairs demonstrate no increase or even reductions in the production of the final metabolite in the simulated pathway.</blockquote><br />
<br />
<h2>'''Encapsulin derived from ''T. maritima'' can be stably expressed in ''E.coli'''''</h2><br />
As part of our 2009 igem project we described a new bacterial microcompartment based on encapsulin which we intend to use for metabolic channeling. We obtained cloned dna from ''T. maritima'' and created a biobrick corresponding to this unique protein. Here we have shown that this part can be stably expressed in ''E.coli''.<br />
<br />
[[image:Toronto_EncapsulinConstruction.png]]<br />
[[image:Toronto_EncapsulinExpression.png]]</div>Gcromarhttp://2010.igem.org/Team:Toronto/ResultsTeam:Toronto/Results2010-10-27T08:19:30Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
|}<br />
<br />
<br />
<h1>'''Results'''</h1><br />
<br />
<h2>'''''E. coli'' DH5a is sensitive to Catechol exposure'''</h2><br />
<br />
To measure the effect of our pathway manipulations on the viability of ''E.coli'' exposed to catechol we plan to perform a tolerance test. In preliminary experiments we established the baseline sensitivity of ''E.coli'' (DH5a) exposed to 50mM catechol in liquid media over a six hour time course. Aliquots of this mixture were plated on LB media at various dilutions and colonies were counted and compared with untreated controls (see "Protocols"). There was no measurable difference between control and treated cells when bacteria were exposed to catechol in a growth media (data not shown). However, in minimal media (MM2) growth of control cells was moderate while exposed cells exhibited a marked decrease in survival (Figure 1). Although the effects of Catechol exposure in ''E.coli'' and ''P. putida'' have previously been investigated [Park et al. 2001] completion of these baseline experiments will enable us to determine the effect of pathway manipulations on the proposed engineered ''E.coli'' strains containing various configurations of the pathway.<br />
<br />
<p><br />
'''A'''<br />
[[Image:Toronto-LB-Plate_1.jpg|400px]] [[Image:Toronto-LB-Plate_2.jpg|400px]]<br />
</p><br />
<p><br />
'''B'''[[Image:Toronto-results-catechol_tolerance.png|center]]<br />
</p><br />
<blockquote>'''Figure 1:''' A. Growth of ''E.coli'' DH5a on LB plates (1x10<sup>-5</sup> dilution) following exposure to minimal media and minimal media with 50mM catechol in solution. Plates shown are for the last time points plotted. B. Survival curves of ''E. coli'' DH5a exposed to catechol over a six hour time course.</blockquote><br />
<br />
<H2>'''Synthetic construction of a functioning catechol ortho degradation pathway in E.coli is predicted to increase cell growth rate'''</H2><br />
To predict the effects of adding the catechol ortho-cleavage pathway on ''E.coli'' metabolism we used a previously developed genome-scale reconstruction of E. coli (iJR904) and applied flux balance analysis (FBA) to compare likely growth outcomes for normal and optimized pathways. We found that optimization of this pathway in the presence of glucose and sufficient oxygen leads to a prediction of increased growth rate. This result bodes well for the success of engineered bacteria introduced into mixed culture within the tailings ponds as they might be expected to have a survival advantage over their native counterparts. However, it should be pointed out that a corresponding genome-scale reconstruction of the target organism ''P. putida'' is not available at this time. These methods and results are discussed at length in the Modeling section.<br />
<br />
<h2>'''Colocalization of muconolactone D-isomerase (EC:5.3.3.4) and 3-oxoadipate enol-lactone hydrolase (EC:3.1.1.24) are predicted to lead to increased catechol degradation through metabolic channeling'''</h2><br />
In order to determine which enzymes would most benefit from metabolic channeling in the catechol degradation pathway, we used a cellular simulation tool, developed in our lab, called Cell++. It allows us to place enzymes of choice in a compartment within a cellular environment and calculates the effect of localization on metabolite concentrations in a user-defined biochemical pathway (Sanford et al. 2006). We gathered kinetic data of the five enzymes in the degradation pathway and investigated the effects of localizing pairs of sequential enzymes (i.e. catechol 1, 2-dioxygenase and muconate cycloisomeras) on metabolite concentrations. Four co-localization simulations were performed in Cell++ along with a control simulation where no enzymes were co-localized. The initial metabolite in the simulation environment, catechol, was added into the simulation environment and the simulation environment was run for 10,000 iterations to observe the effect on catechol degradation in each simulation. The results are displayed as a table indicating the concentration of all metabolites at 1000 equidistant time points during the simulation. <br />
<p>To make use of this information, we extracted the maximum metabolite concentration and the maximum rate of metabolite formation for all metabolites during each simulation. These values are normalized from a scale of 0 to 1, where 0 indicates the lowest value of the concentration/rate of formation (C/RoF) in the simulations and 1 is the highest. These values were further evaluated against the control simulation to the find the percentage change of the C/RoF values of each metabolite in the four co-localization simulations. In order to classify a particular enzyme co-localization as beneficial, we need to see an increase in the maximum concentration and rate of formation of the final metabolite in the pathway (meta6). These increases would represent an increased flux through the pathway. Of the four co-localization simulations, simulation 3 (which co-localizes the enzymes muconolactone D-isomerase and 3-oxodipate-enol lactone hydrolase) was the only simulation that matched the criteria for a beneficial co-localization. From these simulations, we decided to co-localize these two enzymes for our wet lab experiments to see if Cell++ accurately depicted the effects on catechol degradation.</p><br />
<br />
''' '''[[Image:Cellppheatmap.jpg|center]]<br />
<br />
<blockquote>'''Figure 2.''' Simulating the effects of enzyme co-localization on catechol degradation efficiency.<br />
Red indicates increase in the value of the kinetic parameter relative to the simulation where no enzymes are co-localized (0.0), while green indicates a decrease in the kinetic parameter. 1.0, 2.0, 3.0 and 4.0 represent pair-wise fusions of the 1st and 2nd, 2nd and 3rd, 3rd and 4th, and 4th and 5th enzymes respectively. “Max[]” and “Vmax” represent the maximum concentration and the maximum rate of formation of a particular metabolite in a particular simulation. The fusion of the 3rd and 4th enzymes (3.0) resulted in an increase of the production of the final pathway product, while the other fusion pairs demonstrate no increase or even reductions in the production of the final metabolite in the simulated pathway.</blockquote><br />
<br />
<h2>'''Encapsulin derived from ''T. maritima'' can be stably expressed in ''E.coli'''''</h2><br />
As part of our 2009 igem project we described a new bacterial microcompartment based on encapsulin which we intend to use for metabolic channeling. We obtained cloned dna from ''T. maritima'' and created a biobrick corresponding to this unique protein. Here we have shown that this part can be stably expressed in ''E.coli''.<br />
<br />
[[image:Toronto_EncapsulinConstruction.png]]<br />
[[image:Toronto_EncapsulinExpression.png]]</div>Gcromarhttp://2010.igem.org/File:Toronto_UTACCEL_Systems_Biology.pptFile:Toronto UTACCEL Systems Biology.ppt2010-10-27T08:06:22Z<p>Gcromar: </p>
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<div></div>Gcromarhttp://2010.igem.org/Team:Toronto/Human_PracticesTeam:Toronto/Human Practices2010-10-27T08:04:32Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
|}<br />
<br />
<br />
<br />
<h1>'''Human Practices'''</h1><br />
<br />
This year, the Toronto iGEM team partnered with UTACCEL to bring synthetic biology to high school students in China! We designed a six-hour course entitled "Adventures in Biotechnology" which was delivered to two dozen high school students over the course of a 10-day academic conference. Our goals were threefold:<br />
<br />
<ol><br />
<li>Spread the word about synthetic biology.<br />
<li>Experience a different perspective on the demand for energy.<br />
<li>Have fun!<br />
</ol><br />
<br />
[[image:Toronto_China_1.jpg|600px|center]]<br />
<center>Friend Justin and iGEM team members Graham, Kenny and Mengyan enjoy some site-seeing in Guangzhou, China</center><br />
<br />
<h2>'''About UTACCEL'''</h2><br />
<br />
University of Toronto Association for Canada-China Exchange of Leadership (UTACCEL) is an officially recognized student group at the University of Toronto. The organization’s goal is to develop programs that facilitate cultural and knowledge exchange between students in Canada and China. One of their main programs is an annual academic conference in China. Through an intensive interview process, UTACCEL recruits Seminar Leaders from the University of Toronto student body. Seminar Leaders design seminars courses and deliver them to Chinese students during a 10-day conference. Seminar Leaders and delegates also interact with each other in various other ways such as workshops, team competitions and social activities. This year's conference was held at Huamei International High School in Guangzhou, China from July 24th to Aug 2nd.<br />
<br />
<h2>'''Adventures in Biotechnology - Course Description'''</h2><br />
The course introduces key concepts in biology which will be used as a basis to explore current topics in bioinformatics, systems biology and synthetic biology. High throughput science is generating data at an ever increasing rate and computers and software tools are now allowing us to better interpret this data. Our understanding of biological systems has matured. We no longer think of cells as largely random collections of ‘ugly bags of mostly water’. Rather, life is now understood to consist of complex, integrated systems of circuits and components. Our ability to understand complex systems coincides with extraordinary technologies which now allow us to not only read the genetic code but actually write it. This opens up enormous possibilities in biotechnology. The question is, what will we do with this power?<br />
<br />
<ul><br />
<li>[[Media:Toronto_UTACCEL_Evolution.ppt]]<br />
<li>[[Media:Toronto_UTACCEL_Cell_biology.ppt]]<br />
<li>[[Media:Toronto_UTACCEL_Genetics.ppt]]<br />
<li>[[Media:Toronto_UTACCEL_Bioinformatics.ppt]]<br />
<li>[[Media:Toronto_UTACCEL_Systems_Biology.ppt]]<br />
<li>[[Media:Toronto_UTACCEL_Synthetic_Biology.ppt]]<br />
</ul><br />
<br />
<h2>'''A typical day at the conference'''</h2><br />
Wake up at 6 a.m. to the sound of blaring classical music. Hand shower (for some only cold water available) and head to breakfast - typically buns, warm soy milk and hard boiled eggs (widely agreed by the seminar leaders to be the best meal of the day). Weather pushing 40 degrees C with nearly 100% humidity, bring an umbrella! Now climb six flights of stairs to your assigned classroom and turn on the air conditioner (does it work today? 50/50). Teach until lunchtime. Steamed rice and assorted sides. Make plans with other seminar leaders to venture off campus to avoid cafeteria food for dinner. Afternoon "free-time" a.k.a. time to refine lectures, handouts for next class and hold office hours. Supervise evening program consisting of skills workshops and challenges. Roll into bed by midnight. Do it all again tomorrow.<br />
<br />
<h2>'''Why China?'''</h2><br />
The global demand for energy is steadily increasing worldwide. However, the inhabitants of developing nations like China whose soaring economy and large population are beginning to demand access to resources akin to those enjoyed by their western counterparts will push the demand for energy to new heights. Meeting this demand will mean continued dependence on fossil reserves despite the desire to move to alternative and renewable sources of power. We felt that first hand experience of China would complement our text book understanding of the human issues surrounding this ongoing energy debate.<br />
<br />
<h2>'''What did they learn'''</h2><br />
Students were introduced to important foundational concepts in evolution, cell biology, genetics, molecular biology, bioinformatics, systems biology and finally synthetic biology. They learned to define synthetic biology and were encouraged to think about issues raised by the power of the technology within an informed context. In addition, students were exposed to a western style of teaching akin to what they would face if they travel abroad for a university degree which many of the students attending the conference were considering.<br />
<br />
[[image:Toronto_China_2.jpg|600px|center]]<br />
<center>Delegates and their instructors at the UTACCEL conference</center><br />
<br />
<h2>'''What did we learn'''</h2><br />
There is no substitute for first hand experience. China is, in a word, crowded. But, conveying this in words is different from feeling it, walking the streets and seeing her people at work and at play. At once a mixture of old and new, archaic and sophisticated yet with deep history and pride, China is a nation faced with issues we will all face as world population increases. Those of us who also had an opportunity to attend the World EXPO in Shang-hai got a glimpse of various visions for a better world powered by new knowledge, technological advances and creative will. Clearly, energy demand will not pause while we carefully work out the details. Inevitably, mistakes and compromises will be made and this will leave us with a legacy of picking up environmental pieces despite our best intentions.<br />
<br />
[[image:Toronto_China_3.jpg|600px|center]]<br />
<center>Suspended assortment of junk at the World EXPO in Shanghai becomes the image of a modern city</center></div>Gcromarhttp://2010.igem.org/Team:Toronto/TeamTeam:Toronto/Team2010-10-27T07:57:31Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
|}<br />
<br />
<br />
<h1>'''Who we are'''</h1><br />
<br clear="all" /><br />
<br />
=Instructors=<br />
<table><br />
<tr><br />
<th width=50%></th><br />
<th width=50%></th><br />
</tr><br />
<tr><br />
<td style="padding: 10px; vertical-align: top"><br />
==Graham Cromar==<br />
[[Image:Toronto-profile_graham.jpg|x200px|left|thumb|Graham Cromar]]<br />
I am a 5th year Ph.D. student in the Department of Molecular Structure and Function at the Hospital for Sick Children in Toronto. At various times in the past I have studied cell and molecular biology (B.Sc. Toronto), molecular biology and genetics (M.Sc. Guelph), computer programming and systems analysis (dip. Inst. Computer Studies, Toronto), bioinformatics (cert. Canadian Genetic Disease Network) and, systems and matrix biology (present). I have worked as a lab technician, teaching assistant, computer programmer/systems analyst and IS manager. But, what I would most like to do is what I wrote in my high school yearbook about 20 years ago... Design organisms for export to far away galaxies. Thanks to iGEM I'm 1/4 of the way there...<br />
</td><br />
<td style="padding: 10px; vertical-align: top"><br />
==Kris Hon==<br />
[[Image:Toronto-profile_kris.jpg|x200px|left|thumb|Kris Hon]]<br />
I am currently a 2nd year masters student in the Department of Biochemistry at the Unviersity of Toronto, having graduated with a BSc. Honours in Molecular Genetics and Microbiology at the University of Toronto. I am currently studying the effects of micro-compartmentalization in metabolic channeling. The creative freedom and the chance of making a lasting difference drew me to synthetic biology. The fact the iGEM allows for both certainly does help! <br />
</td><br />
</tr><br />
<br />
<tr><br />
<br />
<td style="padding: 10px; vertical-align: top"><br />
<br />
==Amin Zia==<br />
[[Image:Toronto-profile_amin.jpg|x200px|left|thumb|Amin Zia]]<br />
I am a post-doctoral fellow at the Dept. of Cell and Systems Biology with a background in Engineering.<br />
I'm currently working on a couple of cool ideas to tailor engineering methods for Bioinformatics and Systems Biology.<br />
My interest in synthetic biology comes naturally with my background...<br />
</td><br />
</tr><br />
</table><br />
<br />
<br clear="all" /><br />
<br />
=Students=<br />
<br />
<table><br />
<tr><br />
<th width=50%></th><br />
<th width=50%></th><br />
</tr><br />
<br />
<tr><br />
<td style="padding: 10px; vertical-align: top"><br />
==Anish Kapadia==<br />
[[Image:Toronto-profile_anish.jpg|x200px|left|thumb|Anish Kapadia]]<br />
It is fascinating to apply our current knowledge of genetic systems to solving practical problems. I am particularly interested in the potential applications of bio-engineered systems in medicine. I am a currently studying Medicine at University of Toronto.<br />
<br />
</td><br />
<td style="padding: 10px; vertical-align: top"><br />
<br />
==Kenny Zhan==<br />
[[Image:Toronto-profile_kenny.jpg|x200px|left|thumb|Kenny Zhan]]<br />
Hi everyone my name is Kenny Zhan and I am a fourth year undergraduate student. I am currently pursuing a specialization in biochemistry at the University of Toronto. This year I have been part of the roster team that works in the lab to bring the design into reality. I find synthetic biology fascinating as I have an interest in developing new techniques and procedures to support the rapidly expanding field of biological sciences. In my free time I enjoy activities such as hiking, biking, ultimate and geocaching. Hope to see you all down at the jamboree in Boston come November. <br />
</td><br />
</tr><br />
<br />
<tr><br />
<td style="padding: 10px; vertical-align: top"><br />
<br />
==Farhan Raja==<br />
[[Image:Toronto-profile_farhan.jpg|x200px|left|thumb|Farhan Raja]]<br />
I am a graduate student in Biochemistry and my research involves applying computational techniques (eg. FBA) towards the reconstruction and analysis of pathogenic metabolism. I helped with the modeling portion of this year's iGEM team.<br />
</td><br />
<td style="padding: 10px; vertical-align: top"><br />
<br />
==Elena Pasko==<br />
[[Image:Toronto-profile_elena.jpg|x200px|left|thumb|Elena Pasko]]<br />
I am attending my fourth year at the University of Toronto where I am double majoring in Human Biology and Nutritional Sciences. Synthetic biology is a new and expanding field and iGEM has given me an opportunity to be involved in it. <br />
This year I was part of the wet lab team where I have gained tremendous laboratory experience. In my spare time I love to read, swim, and practice yoga.<br />
</td><br />
</tr><br />
<br />
<tr><br />
<br />
<td style="padding: 10px; vertical-align: top"><br />
<br />
==Mengyan Li==<br />
[[Image:Toronto-profile_mengyan.jpg|x200px|left|thumb|Mengyan Li]]<br />
Profile for Mengyan Li.<br />
</td><br />
<td style="padding: 10px; vertical-align: top"><br />
==Yen Leung==<br />
[[Image:Toronto-profile_yen.jpg|x200px|left|thumb|Yen Leung]]<br />
Hi there! My name is Yen and I am in my final undergraduate year at the University of<br />
Toronto. I am specializing in Neuroscience and majoring in Nutritional Sciences. My<br />
research interests include the role of nutrition in neuroscience and aging. Synthetic<br />
biology has also been of interest to me since I joined iGEM last year. My job on the team<br />
this year was wet lab work and training new team members.<br />
</td><br />
</tr><br />
</table><br />
<br />
<br clear="all" /><br />
<br />
=Advisors=<br />
<br />
<table><br />
<tr><br />
<th width=50%></th><br />
<th width=50%></th><br />
</tr><br />
<br />
<tr><br />
<td style="padding: 10px; vertical-align: top"><br />
==Daniel Wong==<br />
[[Image:Toronto-profile_dan.jpg|x200px|left|thumb|Daniel Wong]]<br />
Daniel Wong received his BASc in Engineering Science biomedical option at the University of Toronto. He is currently with the Institute of Biomaterials and Biomedical Engineering, working toward his PhD. He works in the Cochlear Implant Laboratory at the Hospital for Sick Children, developing neuroimaging algorithms for the analysis of multichannel EEG data. As design team lead for this year's iGEM team, he guided the design team in developing the protocols for creating the team's biobrick parts. His academic interests include neuroimaging and systems modelling. To relax, he enjoys a good round of golf or social salsa dancing.<br />
</td><br />
<td style="padding: 10px; vertical-align: top"><br />
==Stacy Hung==<br />
[[Image:Toronto-profile_stacy.jpg|x200px|left|thumb|Stacy Hung]]<br />
With a B.Sc. Honours Biology and Bioinformatics from the University of Waterloo, Stacy is currently a 4th year Ph.D. student at the University of Toronto. Her main focus is to identify novel enzyme drug targets against malaria by studying the metabolic networks of these parasites. She is inspired by anything that is new, exciting, and can have a long-term impact for making the world a better place -- besides her research, iGEM is certainly one of these things!<br />
</td><br />
</tr><br />
<br />
<tr><br />
<td style="padding: 10px; vertical-align: top"><br />
<br />
==John Parkinson==<br />
[[Image:Toronto-profile_john.jpg|x200px|left|thumb|John Parkinson]]<br />
Graduated from the University of Bath with a bachelor of science in Applied Biology in 1990. After receiving a PhD in Biochemistry from the University of Manchester in 1995, I completed a NATO fellowship at the University of Manitoba. From 1997 to 2000, I was awarded a fellowship at the Edinburgh Centre for Protein Technology, University of Edinburgh and from 2000 to 2003 completed another fellowship at the University of Edinburgh on Nematode genomics. I am currently a scientist at The Hospital for Sick Children Research Institute. The research in our laboratory is aimed at understanding how molecular information can give rise to complex biological behaviour. Using computational methods, we study the organization and dynamics of cellular components within the context of integrated biological systems. Comparative genomics methods are also being applied to provide insights into how these systems may have evolved from the remote origins of life.<br />
</td><br />
</tr><br />
</table><br />
<br />
=Contributions=<br />
Graham Cromar, Kris Hon, Anish Kapadia, Elena Pasko and Mengyan Li conceived the project. Parts were designed by Anish Kapadia, Elena Pasko and Mengyan Li with supervision by Kris Hon. Computational prediction of metabolic channeling candidates was performed by Kris Hon. Dynamic simulations on multimeric enzyme assembly were carried out by Danial Wong. Flux balance analysis and other simulations were carried out by Farhan Raja and Amin Zia. Wet lab experiments were carried out by Anish Kapadia, Elena Pasko, Mengyan Li under the guidance of Kris Hon. Graham Cromar performed additional experiments with the Catechol tolerance assay. Kris Hon created the encapsulin expression construct and verified its expression. Kenny Zhan and Yen Leung provided additional assistance with laboratory experiments and parts construction. Stacy Hung provided assistance with figures and graphics. Elena Pasko laid out the wiki. Wiki content was provided by all members of the team. The team greatly acknowledges John Parkinson who provided research space and materials.<br />
<br />
<br />
<br clear="all" /></div>Gcromarhttp://2010.igem.org/Team:Toronto/TeamTeam:Toronto/Team2010-10-27T07:55:08Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
|}<br />
<br />
<br />
<h1>'''Who we are'''</h1><br />
<br clear="all" /><br />
<br />
=Instructors=<br />
<table><br />
<tr><br />
<th width=50%></th><br />
<th width=50%></th><br />
</tr><br />
<tr><br />
<td style="padding: 10px; vertical-align: top"><br />
==Graham Cromar==<br />
[[Image:Toronto-profile_graham.jpg|x200px|left|thumb|Graham Cromar]]<br />
I am a 5th year Ph.D. student in the Department of Molecular Structure and Function at the Hospital for Sick Children in Toronto. At various times in the past I have studied cell and molecular biology (B.Sc. Toronto), molecular biology and genetics (M.Sc. Guelph), computer programming and systems analysis (dip. Inst. Computer Studies, Toronto), bioinformatics (cert. Canadian Genetic Disease Network) and, systems and matrix biology (present). I have worked as a lab technician, teaching assistant, computer programmer/systems analyst and IS manager. But, what I would most like to do is what I wrote in my high school yearbook about 20 years ago... Design organisms for export to far away galaxies. Thanks to iGEM I'm 1/4 of the way there...<br />
</td><br />
<td style="padding: 10px; vertical-align: top"><br />
==Kris Hon==<br />
[[Image:Toronto-profile_kris.jpg|x200px|left|thumb|Kris Hon]]<br />
I am currently a 2nd year masters student in the Department of Biochemistry at the Unviersity of Toronto, having graduated with a BSc. Honours in Molecular Genetics and Microbiology at the University of Toronto. I am currently studying the effects of micro-compartmentalization in metabolic channeling. The creative freedom and the chance of making a lasting difference drew me to synthetic biology. The fact the iGEM allows for both certainly does help! <br />
</td><br />
</tr><br />
<br />
<tr><br />
<br />
<td style="padding: 10px; vertical-align: top"><br />
<br />
==Amin Zia==<br />
[[Image:Toronto-profile_amin.jpg|x200px|left|thumb|Amin Zia]]<br />
I am a post-doctoral fellow at the Dept. of Cell and Systems Biology with a background in Engineering.<br />
I'm currently working on a couple of cool ideas to tailor engineering methods for Bioinformatics and Systems Biology.<br />
My interest in synthetic biology comes naturally with my background...<br />
</td><br />
</tr><br />
</table><br />
<br />
<br clear="all" /><br />
<br />
=Students=<br />
<br />
<table><br />
<tr><br />
<th width=50%></th><br />
<th width=50%></th><br />
</tr><br />
<br />
<tr><br />
<td style="padding: 10px; vertical-align: top"><br />
==Anish Kapadia==<br />
[[Image:Toronto-profile_anish.jpg|x200px|left|thumb|Anish Kapadia]]<br />
It is fascinating to apply our current knowledge of genetic systems to solving practical problems. I am particularly interested in the potential applications of bio-engineered systems in medicine. I am a currently studying Medicine at University of Toronto.<br />
<br />
</td><br />
<td style="padding: 10px; vertical-align: top"><br />
<br />
==Kenny Zhan==<br />
[[Image:Toronto-profile_kenny.jpg|x200px|left|thumb|Kenny Zhan]]<br />
Hi everyone my name is Kenny Zhan and I am a fourth year undergraduate student. I am currently pursuing a specialization in biochemistry at the University of Toronto. This year I have been part of the roster team that works in the lab to bring the design into reality. I find synthetic biology fascinating as I have an interest in developing new techniques and procedures to support the rapidly expanding field of biological sciences. In my free time I enjoy activities such as hiking, biking, ultimate and geocaching. Hope to see you all down at the jamboree in Boston come November. <br />
</td><br />
</tr><br />
<br />
<tr><br />
<td style="padding: 10px; vertical-align: top"><br />
<br />
==Farhan Raja==<br />
[[Image:Toronto-profile_farhan.jpg|x200px|left|thumb|Farhan Raja]]<br />
I am a graduate student in Biochemistry and my research involves applying computational techniques (eg. FBA) towards the reconstruction and analysis of pathogenic metabolism. I helped with the modeling portion of this year's iGEM team.<br />
</td><br />
<td style="padding: 10px; vertical-align: top"><br />
<br />
==Elena Pasko==<br />
[[Image:Toronto-profile_elena.jpg|x200px|left|thumb|Elena Pasko]]<br />
I am attending my fourth year at the University of Toronto where I am double majoring in Human Biology and Nutritional Sciences. Synthetic biology is a new and expanding field and iGEM has given me an opportunity to be involved in it. <br />
This year I was part of the wet lab team where I have gained tremendous laboratory experience. In my spare time I love to read, swim, and practice yoga.<br />
</td><br />
</tr><br />
<br />
<tr><br />
<br />
<td style="padding: 10px; vertical-align: top"><br />
<br />
==Mengyan Li==<br />
[[Image:Toronto-profile_mengyan.jpg|x200px|left|thumb|Mengyan Li]]<br />
Profile for Mengyan Li.<br />
</td><br />
<td style="padding: 10px; vertical-align: top"><br />
==Yen Leung==<br />
[[Image:Toronto-profile_yen.jpg|x200px|left|thumb|Yen Leung]]<br />
Hi there! My name is Yen and I am in my final undergraduate year at the University of<br />
Toronto. I am specializing in Neuroscience and majoring in Nutritional Sciences. My<br />
research interests include the role of nutrition in neuroscience and aging. Synthetic<br />
biology has also been of interest to me since I joined iGEM last year. My job on the team<br />
this year was wet lab work and training new team members.<br />
</td><br />
</tr><br />
</table><br />
<br />
<br clear="all" /><br />
<br />
=Advisors=<br />
<br />
<table><br />
<tr><br />
<th width=50%></th><br />
<th width=50%></th><br />
</tr><br />
<br />
<tr><br />
<td style="padding: 10px; vertical-align: top"><br />
==Daniel Wong==<br />
[[Image:Toronto-profile_dan.jpg|x200px|left|thumb|Daniel Wong]]<br />
Daniel Wong received his BASc in Engineering Science biomedical option at the University of Toronto. He is currently with the Institute of Biomaterials and Biomedical Engineering, working toward his PhD. He works in the Cochlear Implant Laboratory at the Hospital for Sick Children, developing neuroimaging algorithms for the analysis of multichannel EEG data. As design team lead for this year's iGEM team, he guided the design team in developing the protocols for creating the team's biobrick parts. His academic interests include neuroimaging and systems modelling. To relax, he enjoys a good round of golf or social salsa dancing.<br />
</td><br />
<td style="padding: 10px; vertical-align: top"><br />
==Stacy Hung==<br />
[[Image:Toronto-profile_stacy.jpg|x200px|left|thumb|Stacy Hung]]<br />
With a B.Sc. Honours Biology and Bioinformatics from the University of Waterloo, Stacy is currently a 4th year Ph.D. student at the University of Toronto. Her main focus is to identify novel enzyme drug targets against malaria by studying the metabolic networks of these parasites. She is inspired by anything that is new, exciting, and can have a long-term impact for making the world a better place -- besides her research, iGEM is certainly one of these things!<br />
</td><br />
</tr><br />
<br />
<tr><br />
<td style="padding: 10px; vertical-align: top"><br />
<br />
==John Parkinson==<br />
[[Image:Toronto-profile_john.jpg|x200px|left|thumb|John Parkinson]]<br />
Graduated from the University of Bath with a bachelor of science in Applied Biology in 1990. After receiving a PhD in Biochemistry from the University of Manchester in 1995, I completed a NATO fellowship at the University of Manitoba. From 1997 to 2000, I was awarded a fellowship at the Edinburgh Centre for Protein Technology, University of Edinburgh and from 2000 to 2003 completed another fellowship at the University of Edinburgh on Nematode genomics. I am currently a scientist at The Hospital for Sick Children Research Institute. The research in our laboratory is aimed at understanding how molecular information can give rise to complex biological behaviour. Using computational methods, we study the organization and dynamics of cellular components within the context of integrated biological systems. Comparative genomics methods are also being applied to provide insights into how these systems may have evolved from the remote origins of life.<br />
</td><br />
</tr><br />
</table><br />
<br />
<H2>Contributions</H2><br />
Graham Cromar, Kris Hon, Anish Kapadia, Elena Pasko and Mengyan Li conceived the project. Parts were designed by Anish Kapadia, Elena Pasko and Mengyan Li with supervision by Kris Hon. Computational prediction of metabolic channeling candidates was performed by Kris Hon. Dynamic simulations on multimeric enzyme assembly were carried out by Danial Wong. Flux balance analysis and other simulations were carried out by Farhan Raja and Amin Zia. Wet lab experiments were carried out by Anish Kapadia, Elena Pasko, Mengyan Li under the guidance of Kris Hon. Graham Cromar performed additional experiments with the Catechol tolerance assay. Kris Hon created the encapsulin expression construct and verified its expression. Kenny Zhan and Yen Leung provided additional assistance with laboratory experiments and parts construction. Stacy Hung provided assistance with figures and graphics. Elena Pasko laid out the wiki. Wiki content was provided by all members of the team. The team greatly acknowledges John Parkinson who provided research space and materials.<br />
<br />
<br />
<br clear="all" /></div>Gcromarhttp://2010.igem.org/Team:Toronto/Human_PracticesTeam:Toronto/Human Practices2010-10-27T06:57:31Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
|}<br />
<br />
<br />
<br />
<h1>'''Human Practices'''</h1><br />
<br />
This year, the Toronto iGEM team partnered with UTACCEL to bring synthetic biology to high school students in China! We designed a six-hour course entitled "Adventures in Biotechnology" which was delivered to two dozen high school students over the course of a 10-day academic conference. Our goals were threefold:<br />
<br />
<ol><br />
<li>Spread the word about synthetic biology.<br />
<li>Experience a different perspective on the demand for energy.<br />
<li>Have fun!<br />
</ol><br />
<br />
[[image:Toronto_China_1.jpg|600px|center]]<br />
<center>Friend Justin and iGEM team members Graham, Kenny and Mengyan enjoy some site-seeing in Guangzhou, China</center><br />
<br />
<h2>'''About UTACCEL'''</h2><br />
<br />
University of Toronto Association for Canada-China Exchange of Leadership (UTACCEL) is an officially recognized student group at the University of Toronto. The organization’s goal is to develop programs that facilitate cultural and knowledge exchange between students in Canada and China. One of their main programs is an annual academic conference in China. Through an intensive interview process, UTACCEL recruits Seminar Leaders from the University of Toronto student body. Seminar Leaders design seminars courses and deliver them to Chinese students during a 10-day conference. Seminar Leaders and delegates also interact with each other in various other ways such as workshops, team competitions and social activities. This year's conference was held at Huamei International High School in Guangzhou, China from July 24th to Aug 2nd.<br />
<br />
<h2>'''Adventures in Biotechnology - Course Description'''</h2><br />
The course introduces key concepts in biology which will be used as a basis to explore current topics in bioinformatics, systems biology and synthetic biology. High throughput science is generating data at an ever increasing rate and computers and software tools are now allowing us to better interpret this data. Our understanding of biological systems has matured. We no longer think of cells as largely random collections of ‘ugly bags of mostly water’. Rather, life is now understood to consist of complex, integrated systems of circuits and components. Our ability to understand complex systems coincides with extraordinary technologies which now allow us to not only read the genetic code but actually write it. This opens up enormous possibilities in biotechnology. The question is, what will we do with this power?<br />
<br />
<ul><br />
<li>[[Media:Toronto_UTACCEL_Evolution.ppt]]<br />
<li>[[Media:Toronto_UTACCEL_Cell_biology.ppt]]<br />
<li>[[Media:Toronto_UTACCEL_Genetics.ppt]]<br />
<li>[[Media:Toronto_UTACCEL_Bioinformatics.ppt]]<br />
<br />
<li>[[Media:Toronto_UTACCEL_Synthetic_Biology.ppt]]<br />
<li>[[Media:Toronto_UTACCEL_Systems_Biology.ppt]]<br />
</ul><br />
<br />
<h2>'''A typical day at the conference'''</h2><br />
Wake up at 6 a.m. to the sound of blaring classical music. Hand shower (for some only cold water available) and head to breakfast - typically buns, warm soy milk and hard boiled eggs (widely agreed by the seminar leaders to be the best meal of the day). Weather pushing 40 degrees C with nearly 100% humidity, bring an umbrella! Now climb six flights of stairs to your assigned classroom and turn on the air conditioner (does it work today? 50/50). Teach until lunchtime. Steamed rice and assorted sides. Make plans with other seminar leaders to venture off campus to avoid cafeteria food for dinner. Afternoon "free-time" a.k.a. time to refine lectures, handouts for next class and hold office hours. Supervise evening program consisting of skills workshops and challenges. Roll into bed by midnight. Do it all again tomorrow.<br />
<br />
<h2>'''Why China?'''</h2><br />
The global demand for energy is steadily increasing worldwide. However, the inhabitants of developing nations like China whose soaring economy and large population are beginning to demand access to resources akin to those enjoyed by their western counterparts will push the demand for energy to new heights. Meeting this demand will mean continued dependence on fossil reserves despite the desire to move to alternative and renewable sources of power. We felt that first hand experience of China would complement our text book understanding of the human issues surrounding this ongoing energy debate.<br />
<br />
<h2>'''What did they learn'''</h2><br />
Students were introduced to important foundational concepts in evolution, cell biology, genetics, molecular biology, bioinformatics, systems biology and finally synthetic biology. They learned to define synthetic biology and were encouraged to think about issues raised by the power of the technology within an informed context. In addition, students were exposed to a western style of teaching akin to what they would face if they travel abroad for a university degree which many of the students attending the conference were considering.<br />
<br />
[[image:Toronto_China_2.jpg|600px|center]]<br />
<center>Delegates and their instructors at the UTACCEL conference</center><br />
<br />
<h2>'''What did we learn'''</h2><br />
There is no substitute for first hand experience. China is, in a word, crowded. But, conveying this in words is different from feeling it, walking the streets and seeing her people at work and at play. At once a mixture of old and new, archaic and sophisticated yet with deep history and pride, China is a nation faced with issues we will all face as world population increases. Those of us who also had an opportunity to attend the World EXPO in Shang-hai got a glimpse of various visions for a better world powered by new knowledge, technological advances and creative will. Clearly, energy demand will not pause while we carefully work out the details. Inevitably, mistakes and compromises will be made and this will leave us with a legacy of picking up environmental pieces despite our best intentions.<br />
<br />
[[image:Toronto_China_3.jpg|600px|center]]<br />
<center>Suspended assortment of junk at the World EXPO in Shanghai becomes the image of a modern city</center></div>Gcromarhttp://2010.igem.org/Team:Toronto/Human_PracticesTeam:Toronto/Human Practices2010-10-27T06:36:44Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
|}<br />
<br />
<br />
<br />
<h1>'''Human Practices'''</h1><br />
<br />
This year, the Toronto iGEM team partnered with UTACCEL to bring synthetic biology to high school students in China! We designed a six-hour course entitled "Adventures in Biotechnology" which was delivered to two dozen high school students over the course of a 10-day academic conference. Our goals were threefold:<br />
<br />
<ol><br />
<li>Spread the word about synthetic biology.<br />
<li>Experience a different perspective on the demand for energy.<br />
<li>Have fun!<br />
</ol><br />
<br />
[[image:Toronto_China_1.jpg|600px|center]]<br />
<center>Friend Justin and iGEM team members Graham, Kenny and Mengyan enjoy some site-seeing in Guangzhou, China</center><br />
<br />
<h2>'''About UTACCEL'''</h2><br />
<br />
University of Toronto Association for Canada-China Exchange of Leadership (UTACCEL) is an officially recognized student group at the University of Toronto. The organization’s goal is to develop programs that facilitate cultural and knowledge exchange between students in Canada and China. One of their main programs is an annual academic conference in China. Through an intensive interview process, UTACCEL recruits Seminar Leaders from the University of Toronto student body. Seminar Leaders design seminars courses and deliver them to Chinese students during a 10-day conference. Seminar Leaders and delegates also interact with each other in various other ways such as workshops, team competitions and social activities. This year's conference was held at Huamei International High School in Guangzhou, China from July 24th to Aug 2nd.<br />
<br />
<h2>'''Adventures in Biotechnology - Course Description'''</h2><br />
The course introduces key concepts in biology which will be used as a basis to explore current topics in bioinformatics, systems biology and synthetic biology. High throughput science is generating data at an ever increasing rate and computers and software tools are now allowing us to better interpret this data. Our understanding of biological systems has matured. We no longer think of cells as largely random collections of ‘ugly bags of mostly water’. Rather, life is now understood to consist of complex, integrated systems of circuits and components. Our ability to understand complex systems coincides with extraordinary technologies which now allow us to not only read the genetic code but actually write it. This opens up enormous possibilities in biotechnology. The question is, what will we do with this power?<br />
<br />
<ul><br />
<li>[[Media:Toronto_UTACCEL_Evolution.ppt]]<br />
<li>[[Media:Toronto_UTACCEL_Cell_biology.ppt]]<br />
<li>[[Media:Toronto_UTACCEL_Genetics.ppt]]<br />
<li>[[Media:Toronto_UTACCEL_Bioinformatics.ppt]]<br />
<li>[[Media:Toronto_UTACCEL_Systems_Biology.ppt]]<br />
<li>[[Media:Toronto_UTACCEL_Synthetic_Biology.ppt]]<br />
</ul><br />
<br />
<h2>'''A typical day at the conference'''</h2><br />
Wake up at 6 a.m. to the sound of blaring classical music. Hand shower (for some only cold water available) and head to breakfast - typically buns, warm soy milk and hard boiled eggs (widely agreed by the seminar leaders to be the best meal of the day). Weather pushing 40 degrees C with nearly 100% humidity, bring an umbrella! Now climb six flights of stairs to your assigned classroom and turn on the air conditioner (does it work today? 50/50). Teach until lunchtime. Steamed rice and assorted sides. Make plans with other seminar leaders to venture off campus to avoid cafeteria food for dinner. Afternoon "free-time" a.k.a. time to refine lectures, handouts for next class and hold office hours. Supervise evening program consisting of skills workshops and challenges. Roll into bed by midnight. Do it all again tomorrow.<br />
<br />
<h2>'''Why China?'''</h2><br />
The global demand for energy is steadily increasing worldwide. However, the inhabitants of developing nations like China whose soaring economy and large population are beginning to demand access to resources akin to those enjoyed by their western counterparts will push the demand for energy to new heights. Meeting this demand will mean continued dependence on fossil reserves despite the desire to move to alternative and renewable sources of power. We felt that first hand experience of China would complement our text book understanding of the human issues surrounding this ongoing energy debate.<br />
<br />
<h2>'''What did they learn'''</h2><br />
Students were introduced to important foundational concepts in evolution, cell biology, genetics, molecular biology, bioinformatics, systems biology and finally synthetic biology. They learned to define synthetic biology and were encouraged to think about issues raised by the power of the technology within an informed context. In addition, students were exposed to a western style of teaching akin to what they would face if they travel abroad for a university degree which many of the students attending the conference were considering.<br />
<br />
[[image:Toronto_China_2.jpg|600px|center]]<br />
<center>Delegates and their instructors at the UTACCEL conference</center><br />
<br />
<h2>'''What did we learn'''</h2><br />
There is no substitute for first hand experience. China is, in a word, crowded. But, conveying this in words is different from feeling it, walking the streets and seeing her people at work and at play. At once a mixture of old and new, archaic and sophisticated yet with deep history and pride, China is a nation faced with issues we will all face as world population increases. Those of us who also had an opportunity to attend the World EXPO in Shang-hai got a glimpse of various visions for a better world powered by new knowledge, technological advances and creative will. Clearly, energy demand will not pause while we carefully work out the details. Inevitably, mistakes and compromises will be made and this will leave us with a legacy of picking up environmental pieces despite our best intentions.<br />
<br />
[[image:Toronto_China_3.jpg|600px|center]]<br />
<center>Suspended assortment of junk at the World EXPO in Shanghai becomes the image of a modern city</center></div>Gcromarhttp://2010.igem.org/Team:Toronto/Human_PracticesTeam:Toronto/Human Practices2010-10-27T06:35:03Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
|}<br />
<br />
<br />
<br />
<h1>'''Human Practices'''</h1><br />
<br />
This year, the Toronto iGEM team partnered with UTACCEL to bring synthetic biology to high school students in China! We designed a six-hour course entitled "Adventures in Biotechnology" which was delivered to two dozen high school students over the course of a 10-day academic conference. Our goals were threefold:<br />
<br />
<ol><br />
<li>Spread the word about synthetic biology.<br />
<li>Experience a different perspective on the demand for energy.<br />
<li>Have fun!<br />
</ol><br />
<br />
[[image:Toronto_China_1.jpg|600px|center]]<br />
<center>Friend Justin and iGEM team members Graham, Kenny and Mengyan enjoy some site-seeing in Guangzhou, China</center><br />
<br />
<h2>'''About UTACCEL'''</h2><br />
<br />
University of Toronto Association for Canada-China Exchange of Leadership (UTACCEL) is an officially recognized student group at the University of Toronto. The organization’s goal is to develop programs that facilitate cultural and knowledge exchange between students in Canada and China. One of their main programs is an annual academic conference in China. Through an intensive interview process, UTACCEL recruits Seminar Leaders from the University of Toronto student body. Seminar Leaders design seminars courses and deliver them to Chinese students during a 10-day conference. Seminar Leaders and delegates also interact with each other in various other ways such as workshops, team competitions and social activities. This year's conference was held at Huamei International High School in Guangzhou, China from July 24th to Aug 2nd.<br />
<br />
<h2>'''Adventures in Biotechnology - Course Description'''</h2><br />
The course introduces key concepts in biology which will be used as a basis to explore current topics in bioinformatics, systems biology and synthetic biology. High throughput science is generating data at an ever increasing rate and computers and software tools are now allowing us to better interpret this data. Our understanding of biological systems has matured. We no longer think of cells as largely random collections of ‘ugly bags of mostly water’. Rather, life is now understood to consist of complex, integrated systems of circuits and components. Our ability to understand complex systems coincides with extraordinary technologies which now allow us to not only read the genetic code but actually write it. This opens up enormous possibilities in biotechnology. The question is, what will we do with this power?<br />
<br />
[[Media:Toronto_UTACCEL_Evolution.ppt]]<br />
[[Media:Toronto_UTACCEL_Cell_biology.ppt]]<br />
[[Media:Toronto_UTACCEL_Genetics.ppt]]<br />
[[Media:Toronto_UTACCEL_Bioinformatics.ppt]]<br />
[[Media:Toronto_UTACCEL_Systems_Biology.ppt]]<br />
[[Media:Toronto_UTACCEL_Synthetic_Biology.ppt]]<br />
<br />
<h2>'''A typical day at the conference'''</h2><br />
Wake up at 6 a.m. to the sound of blaring classical music. Hand shower (for some only cold water available) and head to breakfast - typically buns, warm soy milk and hard boiled eggs (widely agreed by the seminar leaders to be the best meal of the day). Weather pushing 40 degrees C with nearly 100% humidity, bring an umbrella! Now climb six flights of stairs to your assigned classroom and turn on the air conditioner (does it work today? 50/50). Teach until lunchtime. Steamed rice and assorted sides. Make plans with other seminar leaders to venture off campus to avoid cafeteria food for dinner. Afternoon "free-time" a.k.a. time to refine lectures, handouts for next class and hold office hours. Supervise evening program consisting of skills workshops and challenges. Roll into bed by midnight. Do it all again tomorrow.<br />
<br />
<h2>'''Why China?'''</h2><br />
The global demand for energy is steadily increasing worldwide. However, the inhabitants of developing nations like China whose soaring economy and large population are beginning to demand access to resources akin to those enjoyed by their western counterparts will push the demand for energy to new heights. Meeting this demand will mean continued dependence on fossil reserves despite the desire to move to alternative and renewable sources of power. We felt that first hand experience of China would complement our text book understanding of the human issues surrounding this ongoing energy debate.<br />
<br />
<h2>'''What did they learn'''</h2><br />
Students were introduced to important foundational concepts in evolution, cell biology, genetics, molecular biology, bioinformatics, systems biology and finally synthetic biology. They learned to define synthetic biology and were encouraged to think about issues raised by the power of the technology within an informed context. In addition, students were exposed to a western style of teaching akin to what they would face if they travel abroad for a university degree which many of the students attending the conference were considering.<br />
<br />
[[image:Toronto_China_2.jpg|600px|center]]<br />
<center>Delegates and their instructors at the UTACCEL conference</center><br />
<br />
<h2>'''What did we learn'''</h2><br />
There is no substitute for first hand experience. China is, in a word, crowded. But, conveying this in words is different from feeling it, walking the streets and seeing her people at work and at play. At once a mixture of old and new, archaic and sophisticated yet with deep history and pride, China is a nation faced with issues we will all face as world population increases. Those of us who also had an opportunity to attend the World EXPO in Shang-hai got a glimpse of various visions for a better world powered by new knowledge, technological advances and creative will. Clearly, energy demand will not pause while we carefully work out the details. Inevitably, mistakes and compromises will be made and this will leave us with a legacy of picking up environmental pieces despite our best intentions.<br />
<br />
[[image:Toronto_China_3.jpg|600px|center]]<br />
<center>Suspended assortment of junk at the World EXPO in Shanghai becomes the image of a modern city</center></div>Gcromarhttp://2010.igem.org/File:Toronto_Dilutions.jpgFile:Toronto Dilutions.jpg2010-10-27T06:30:36Z<p>Gcromar: </p>
<hr />
<div></div>Gcromarhttp://2010.igem.org/Team:Toronto/ProtocolsTeam:Toronto/Protocols2010-10-27T06:29:52Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Protocols'''</h1><br />
<br />
<h2>'''Tolerance Assay'''</h2><br />
<br />
A 0.5 M (10x) stock solution of catechol was made in a 5 ml volume of autoclaved, distilled water. This solution was added to 25 ml of sterile MM2 media to make up a volume of 30 ml. This was added to 20 ml of MM2 innoculated with E.coli DH5a (approximate density 1x10e8 cells per ml) for a final volume of 50 ml (catechol concentration 50mM). Control samples were created as above minus catechol. Treated and untreated samples were placed in a 37C shaker incubator and aliquots were removed every two hours. 0.5 ml aliquots were serially diluted in 4.5 ml of MM2 media and four dilutions representing 1 x 10<sup>-3</sup>, 1 x 10<sup>-4</sup>, 1 x 10<sup>-5</sup> and 1x10<sup>-6</sup> were plated by spreading 200 ul of the diluted sample on LB agar plates. After drying, plates were incubated in an inverted position in a 37C incubator overnight. Plates with between 30 and 300 colonies were counted to determine the number of colony forming units using the method described by Imperial College (iGEM 2009 OpenWetWare).<br />
<br />
[[image:Toronto_Dilutions.jpg|center]]<br />
<br />
<h2>'''Making Chemically Competent Cells'''</h2><br />
<br />
<p>Day 1</p><br />
<p>1. Start a 5 mL LB overnight culture in the 37C incubator from either a colony streaked plate or with one of the last frozen competent stocks of the strains you want to make competent.</p><br />
<p>Day 2</p><br />
<p>1. Inoculate the 5 mL overnight culture into a new flask with 200 mL of fresh LB.</p><br />
<p>2. Grow until O.D. 600 = 0.4.</p><br />
<p>a. To check O.D., take 1 mL of the bacterial culture and put it in a plastic cuvette.</p><br />
<p>b. Take another plastic cuvette and add in 1 mL of LB.</p><br />
<p>c. Change the wavelength of the spectrophotometer to 600 nm.</p><br />
<p>d. Blank using the LB cuvette.</p><br />
<p>e. Measure and record the absorbance of the cuvette containing the culture.</p><br />
<p>f. If the O.D. is below 0.4, place the flask back in the 37°C incubator and repeat ‘e’ at a later time point. Dispose of the cuvette with the culture but keep the cuvette with the LB as a blank.</p><br />
<p>3. Transfer 50 mL of the culture into a pre-chilled Falcon tube. Repeat for each of the remaining 3 Falcon tubes.</p><br />
<p>4. Place the Falcon tubes in ice for 15 minutes to cool the cultures.</p><br />
<p>5. Pellet the cells using a Beckman centrifuge at 4000 rpm for 10 minutes at 4°C.</p><br />
<p>6. Pour out the media in the sink and stand the Falcon tubes in an inverted position on paper towels for 1 min to drain away traces of liquid.</p><br />
<p>7. Re-suspend the pellets in each tube with 10 mL of ice cold 0.1M CaCl2 by lightly pipetteing with a P1000.</p><br />
<p>8. Store on ice for 10 min.</p><br />
<p>9. Pellet the cells using a Beckman centrifuge at 4000 rpm for 10 minutes at 4°C.</p><br />
<p>10. Pour out the media in the sink and stand the Falcon tubes in an inverted position on paper towels for 1 min to drain away traces of liquid.</p><br />
<p>11. Re-suspend the pellets in each tube with 2 mL of ice cold 0.1M CaCl2 by lightly pipetteing with a P1000.</p><br />
<p>12. Pool the aliquots and keep on ice for 1 hour.</p><br />
<p>13. Make a dry ice/ethanol bath.</p><br />
<p>a. Dry ice is on the 14th floor in the corresponding room where the incubators are at on our floor.</p><br />
<p>b. Ethanol is under the fumehood in a cabinet labelled “Flammables” beside the imaging room.</p><br />
<p>14. Add in 4.8 mL of ice cold 40% glycerol and very gently invert the tube to mix well.</p><br />
<p>15. Divide the mixture into the pre-chilled microfuge tubes in 400 µL aliquots.</p><br />
<p>16. Flash freeze the aliquots in the dry ice bath.</p><br />
<p>17. Store in the -80°C freezer.</p><br />
<br />
<h2>'''Transforming Chemically Competent Cells'''</h2><br />
<p>1. Thaw a stock of competent cells that you want to transform into on ice for 10 minutes. DO NOT REMOVE IT FROM THE ICE DURING THIS TIME.</p><br />
<p>2. If you are doing more than 1 transformation, take 100 µL of cells from the freezer stock and add it to prechilled microfuge tubes. If not, take 100 µL of cells to use as transformation controls.</p><br />
<p>3. Add 1 µL of DNA to the cells you want transformed. Stir gently with the pipette as any serious agitation will decrease transformation by a lot.</p><br />
<p>4. Leave mixtures on ice for 30 minutes.</p><br />
<p>5. Heat shock cells for 30s at 42°C. Different times and temperatures will decrease transformation efficiency.</p><br />
<p>6. Place mixtures on ice for 5 min.</p><br />
<p>7. Add LB to each tube until the total volume is 1 mL. (i.e. If there is 100 µL of cells, add 900 µL of fresh LB)</p><br />
<p>8. Place the tubes in the 37°C shaker for 1 hour.</p><br />
<p>9. Pre-warm and label (date, initials, what DNA is transformed with what cells, amount plated) selection plates in the 37°C incubator with the sign “BACTERIA ONLY”.</p><br />
<p>10. Spread plate 2 amounts of transformants (10 µL and 50 µL) onto the corresponding selection plates.</p><br />
<p>11. Place overnight in the 37°C incubator with the sign “BACTERIA ONLY”.</p><br />
<br />
<h2>'''Digesting DNA'''</h2><br />
<p>1. Thaw the NEBuffer(s) that you will need to digest your DNA of choice.</p><br />
<p>a. The NEBuffer will have to be chosen carefully if digesting with 2 or more enzymes. </p><br />
<p>b. Always try to maximize the percent activity (indicated in the NEB info slips for each restriction enzyme). </p> <br />
<p>c. Be on the lookout for star activity in different buffers.</p><br />
<p>d. If you can’t find a common NEBuffer, you can also digest sequentially.</p><br />
<p>2. Add your components in the following order (for a 25 µL reaction)</p><br />
<p>a. ddH2O</p><br />
<p>b. 10X NEBuffer (2.5 µL)</p><br />
<p>c. DNA</p><br />
<p>d. Restriction Enzyme (5 units)</p><br />
<p>i. If you scale up the reaction volumes, you won’t need to add more enzyme because 5 units is already way more than enough (unless you digesting 5+ µg)</p><br />
<p>3. Turn on the PCR machine and place the tubes inside one of the 48 well blocks. Select the program Kris > DIGEST, and the block your sample is in (A or B).</p><br />
<p>a. If you need to run the samples on a gel, add 5 µL of 6x Loading dye and either freeze the sample in -20°C.</p><br />
<p>4. If the enzymes can be heat inactivated (check the NEB slips), select the program in Kris > INACTIV.</p><br />
<p>a. If not, use the PCR purification kit to remove the restriction enzyme from the mixture.</p><br />
<p>b. Inactivation of enzymes is important if you want to ligate your DNA (otherwise, the enzyme will just digest right after the ligase works on it).</p><br />
<br />
<h2>'''Ligating DNA'''</h2><br />
<p>1. Thaw the 10X T4 DNA Ligase Buffer.</p><br />
<p>2. Add the components in the following order (10 µL reaction):</p><br />
<p>a. 10X T4 DNA Ligase Buffer (1 µL)</p><br />
<p>b. Vector DNA (10 ng)</p><br />
<p>c. Insert DNA (6 insert:1 vector molar ratio)</p><br />
<p>d. ddH2O (to make the volume 9.5 µL total, if necessary)</p><br />
<p>e. T4 DNA Ligase Buffer (0.5 µL)</p><br />
<p>3. Leave at room temperature for 30 minutes.</p><br />
<p>4. Freeze the mixture in the -20°C freezer for future use or use 1 µL in a transformation immediately.</p><br />
<br />
<h2>'''Gel Electrophoresis'''</h2><br />
<p>1. Based on what percentage gel you want to run, add half the percentage in grams to 50 mL of 1X TBE in a 125 mL / 250 mL flask.</p><br />
<p>a. For example, if you want to make a 1% gel, add in 0.5g of agarose into 50 mL of 1X TBE.</p><br />
<p>2. Place the flask in the microwave and heat it for 45 seconds. If it starts bubbling, immediately open the microwave door.</p><br />
<p>3. Take out the flask and swirl the mixture for 5-10 seconds. Use gloves or a folded dry paper towel to hold the flask to avoid getting burned.</p><br />
<p>4. Place the flask in the microwave and heat for 15-30 seconds. If it starts bubbling, immediately open the microwave door. Let the flask and mixture cool until it is warm when you touch it.</p><br />
<p>5. Add 2.5 µL of ethidium bromide into the cooling mixture and mix well.</p><br />
<p>a. Be VERY careful not to get any EtBr on your skin/clothes.</p><br />
<p>6. Take the small tray and tape both ends. Be sure to wrap the tape tightly around edges and corners so that no fluid will leak out.</p><br />
<p>7. Slowly pour the gel into the tray and see if it leaks. If it does, try to add more tape in the area to stop the leak.</p><br />
<p>8. Once the gel is poured, place the comb into preset ridges on the top of the tray.</p><br />
<p>9. Once the gel has solidified, carefully remove the comb and the tape.</p><br />
<p>10. Place the gel tray (with the gel still on it) into the electrophoresis cell. Place the wells facing the negative electrode (black).</p><br />
<p>11. Fill the electrophoresis cell with enough 1X TBE to cover the gel.</p><br />
<p>12. Add in 10 µL of to the side where the positive electrode (red) is located.</p><br />
<p>13. Load your samples and ladder(s); record what samples are in what wells.</p><br />
<p>14. Close the cover of the electrophoresis cell and plug it into the power supply box.</p><br />
<p>15. Set the voltage to 110V (you can push it to 120V, but don’t go any higher as the gel will melt) and turn off the power supply when the dye reaches at least ¾ down the gel (usually 45 min – 1 hr).</p><br />
<br />
<h2>'''Polymerase Chain Reaction'''</h2><br />
<p>1. Let the dNTPs, primers, DNA template(s) and 10X reaction buffer thaw at room temperature</p><br />
<p>2. Keep the Taq polymerase on ice at all times.</p><br />
<p>3. Add the reagents in the following order for a 25 µL reaction:</p><br />
<p>a. 10X reaction buffer (2.5 µL)</p><br />
<p>b. ddH2O (filling volume up to 25 µL, following these values it would be 17.15 µL)</p><br />
<p>c. Forward primer (0.2 µL)</p><br />
<p>d. Reverse primer (0.2 uL)</p><br />
<p>e. 2 mM dNTPs (3.75 µL)</p><br />
<p>f. DNA template (1 µL)</p><br />
<p>g. Taq polymerase (0.2 µL)</p><br />
<p>4. Repeat step 3 for all DNA samples you want amplified. Make the appropriate controls.</p><br />
<p>a. For the negative control, replace the DNA template with 1 µL of ddH2O.</p><br />
<p>5. Place the tubes in the PCR machine (in the same block) and select the PCR program you want to run. If you don’t have a pre-made program, make the following program in your own folder:</p><br />
<p>a. Set the lid temperature to 105°C</p><br />
<p>b. Step 1: 95°C for 15 min</p><br />
<p>c. Step 2: 94°C for 30 seconds</p><br />
<p>d. Step 3: (Primer Tm – 3) for 30 seconds</p><br />
<p>e. Step 4: 72°C for (1 min per kb of product)</p><br />
<p>f. Step 5: Repeat steps 2-4 39 times.</p><br />
<p>g. Step 6: 72°C for 20 min</p><br />
<p>h. Step 7: 4°C forever</p><br />
<p>6. Run it in a gel or use the PCR purification kit as necessary.</p></div>Gcromarhttp://2010.igem.org/Team:Toronto/DesignTeam:Toronto/Design2010-10-27T06:09:28Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Project Design'''</h1><br />
<p>In order to identify enzymes in the catechol degradation pathway (ortho cleavage) that benefit from the introduction of metabolic channelling, we performed ''in silico'' analyses with software developed in our lab (Sanford et al.). Preliminary results from our simulations suggests that the 3rd (muconolactone D-isomerase) and 4th (3-oxodipate-enol lactone hydrolase) enzymes in the pathway would benefit most from channelling (Figure 1). Muconolactone D-isomerase catalyzes an energetically unfavourable reaction, while 3-oxodipate-enol lactone hydrolase catalyzes a very energetically favourable reaction. By introducing channeling between these two enzymes, the energetically unfavourable product of the mucanolactone D-isomerase-catalyzed reaction would be converted to the product of 3-oxodipate-enol lactone hydrolase before mucanolactone D-isomerase converts the energetically unfavourable product back into the energetically favourable substrate. The net effect would be an increased flux through the catechol degradation pathway. </p><br />
<br />
<br />
<br />
[[Image:Catechol degradation 5.png|center]]<br />
<br />
<blockquote> Figure 1: Ortho degradation of catechol (1,2-Benzenediol) proceeds in six steps. Only the last enzyme is present in E. coli. Enzyme candidates with the largest predicted channeling effect as predicted by Cell++ are boxed in red.</blockquote><br />
<br />
<br/><br />
<p>The five enzymes in the catechol degradation pathway not naturally present in ''E. coli'' will be introduced into ''E. coli'' as BioBrick parts. The parts will then be used to construct five recombinant strains of ''E. coli'' (Figure 2). These include a full reconstruction of the pathway (control strain) and several partial reconstructions (test strains). The latter will contain pathway 'holes' corresponding to all combinations of sequential pairs of enzymes, with complementary fusion pairs. The test strain with the fusion of enzymes 3 and 4 will be of prime interest, while the other test strains will be used validate the predictions made by Cell++. </p><br />
<br />
Many of the enzymes in the system are multimeric. For this reason we propose expressing the fusion proteins alongside their native (un-fused) counterparts to promote linkage of the enzymes while allowing for assembly of unlinked monomers onto the fusions which we refer to as 'seeds'. The relative expression of free monomers and fusion seeds is predicted to be important since too many seeds relative to free monomers will lead to tangles whereas too many free monomers relative to seeds will ablate any channeling effects. The assembly dynamics and desired relative expression ratios of seeds and free monomers are being explored using ''in silico'' modeling as described in the modeling section. Below is a diagramatic representation of the proposed expression system (Figure 3). The exact choice of promoters will depend upon predictions from our modeling results.<br />
<br />
[[Image:design 6.png|center]]<br />
<blockquote>Figure 2: Diagramatic representation of proposed gene fusion experiments. Pathway reconstructions are shown in black for the control and test strains with pathway holes for the partial reconstructions shown in red. Yellow stars mark proposed gene fusions to be tested to validate computational predictions. The blue circle marks the best candidate for enzyme channeling based on ''in silico'' experiments </blockquote><br />
<br/><br />
<p>In order to demonstrate the breakdown of catechol by these recombinant strains, catechol will be added into the growth media of these strains. A colorimetric assay, previously developed to quantify the amount of catechol in solution, will be utilized to assay the rate of catechol degradation by the different strains (Tao et al.). In addition, a survival assay will be used to determine the threshold for catechol exposure for non recombinant ''E. coli'' and to evaluate the effect that the recombinantly-expressed enzymes in the catechol pathway will have on ''E. coli'' survival. </p><br />
<br />
<br><br />
[[Image:Construct Diagram.jpg|center|800 px]]<br />
<blockquote>Figure 3: An overview of the recombinant fusion construct and experimental system. Muconolactone D-isomerase (enzyme 3; green) and 3-oxodipate-enol lactone hydrolase (enzyme 4; sky blue) subunits will be linked via an alpha-helical linker. The linker is expected to serves a dual purpose: 1) The fusion product seeds the formation of multimeric proteins required for enzymatic function, and 2) The linker spatially localizes the two enzymes to aid channeling. The fusion construct (terR) along with a redundant pair of non-fused enzymes 3 and 4 (inset; lac) are placed under different inducible promoters. This allows for the assessment of potential channeling effects by selectively inducing the fusion protein relative to the induction of redundant copies of the same un-fused enzymes. </blockquote></div>Gcromarhttp://2010.igem.org/File:Toronto_UTACCEL_Synthetic_Biology.pptFile:Toronto UTACCEL Synthetic Biology.ppt2010-10-27T05:26:36Z<p>Gcromar: </p>
<hr />
<div></div>Gcromarhttp://2010.igem.org/File:Toronto_UTACCEL_Bioinformatics.pptFile:Toronto UTACCEL Bioinformatics.ppt2010-10-27T05:25:02Z<p>Gcromar: </p>
<hr />
<div></div>Gcromarhttp://2010.igem.org/File:Toronto_UTACCEL_Genetics.pptFile:Toronto UTACCEL Genetics.ppt2010-10-27T05:24:36Z<p>Gcromar: </p>
<hr />
<div></div>Gcromarhttp://2010.igem.org/File:Toronto_UTACCEL_Cell_biology.pptFile:Toronto UTACCEL Cell biology.ppt2010-10-27T05:24:03Z<p>Gcromar: </p>
<hr />
<div></div>Gcromarhttp://2010.igem.org/Team:Toronto/Human_PracticesTeam:Toronto/Human Practices2010-10-27T05:23:23Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<br />
<h1>'''Human Practices'''</h1><br />
<br />
This year, the Toronto iGEM team partnered with UTACCEL to bring synthetic biology to high school students in China! We designed a six-hour course entitled "Adventures in Biotechnology" which was delivered to two dozen high school students over the course of a 10-day academic conference. Our goals were threefold:<br />
<br />
1. Spread the word about synthetic biology.<br />
2. Experience a different perspective on the demand for energy.<br />
3. Have fun!<br />
<br />
[[image:Toronto_China_1.jpg|600px|center]]<br />
<center>Friend Justin and iGEM team members Graham, Kenny and Mengyan enjoy some site-seeing in Guangzhou, China</center><br />
<br />
<h2>'''About UTACCEL'''</h2><br />
<br />
University of Toronto Association for Canada-China Exchange of Leadership (UTACCEL) is an officially recognized student group at the University of Toronto. The organization’s goal is to develop programs that facilitate cultural and knowledge exchange between students in Canada and China. One of their main programs is an annual academic conference in China. Through an intensive interview process, UTACCEL recruits Seminar Leaders from the University of Toronto student body. Seminar Leaders design seminars courses and deliver them to Chinese students during a 10-day conference. Seminar Leaders and delegates also interact with each other in various other ways such as workshops, team competitions and social activities. This year's conference was held at Huamei International High School in Guangzhou, China from July 24th to Aug 2nd.<br />
<br />
<h2>'''Adventures in Biotechnology - Course Description'''</h2><br />
The course introduces key concepts in biology which will be used as a basis to explore current topics in bioinformatics, systems biology and synthetic biology. High throughput science is generating data at an ever increasing rate and computers and software tools are now allowing us to better interpret this data. Our understanding of biological systems has matured. We no longer think of cells as largely random collections of ‘ugly bags of mostly water’. Rather, life is now understood to consist of complex, integrated systems of circuits and components. Our ability to understand complex systems coincides with extraordinary technologies which now allow us to not only read the genetic code but actually write it. This opens up enormous possibilities in biotechnology. The question is, what will we do with this power?<br />
<br />
[[Media:Toronto_UTACCEL_Evolution.ppt]]<br />
[[Media:Toronto_UTACCEL_Cell_biology.ppt]]<br />
[[Media:Toronto_UTACCEL_Genetics.ppt]]<br />
[[Media:Toronto_UTACCEL_Bioinformatics.ppt]]<br />
[[Media:Toronto_UTACCEL_Systems_Biology.ppt]]<br />
[[Media:Toronto_UTACCEL_Synthetic_Biology.ppt]]<br />
<br />
<h2>'''A typical day at the conference'''</h2><br />
Wake up at 6 a.m. to the sound of blaring classical music. Hand shower (for some only cold water available) and head to breakfast - typically buns, warm soy milk and hard boiled eggs (widely agreed by the seminar leaders to be the best meal of the day). Weather pushing 40 degrees C with nearly 100% humidity, bring an umbrella! Now climb six flights of stairs to your assigned classroom and turn on the air conditioner (does it work today? 50/50). Teach until lunchtime. Steamed rice and assorted sides. Make plans with other seminar leaders to venture off campus to avoid cafeteria food for dinner. Afternoon "free-time" a.k.a. time to refine lectures, handouts for next class and hold office hours. Supervise evening program consisting of skills workshops and challenges. Roll into bed by midnight. Do it all again tomorrow.<br />
<br />
<h2>'''Why China?'''</h2><br />
The global demand for energy is steadily increasing worldwide. However, the inhabitants of developing nations like China whose soaring economy and large population are beginning to demand access to resources akin to those enjoyed by their western counterparts will push the demand for energy to new heights. Meeting this demand will mean continued dependence on fossil reserves despite the desire to move to alternative and renewable sources of power. We felt that first hand experience of China would complement our text book understanding of the human issues surrounding this ongoing energy debate.<br />
<br />
<h2>'''What did they learn'''</h2><br />
Students were introduced to important foundational concepts in evolution, cell biology, genetics, molecular biology, bioinformatics, systems biology and finally synthetic biology. They learned to define synthetic biology and were encouraged to think about issues raised by the power of the technology within an informed context. In addition, students were exposed to a western style of teaching akin to what they would face if they travel abroad for a university degree which many of the students attending the conference were considering.<br />
<br />
[[image:Toronto_China_2.jpg|600px|center]]<br />
<center>Delegates and their instructors at the UTACCEL conference</center><br />
<br />
<h2>'''What did we learn'''</h2><br />
There is no substitute for first hand experience. China is, in a word, crowded. But, conveying this in words is different from feeling it, walking the streets and seeing her people at work and at play. At once a mixture of old and new, archaic and sophisticated yet with deep history and pride, China is a nation faced with issues we will all face as world population increases. Those of us who also had an opportunity to attend the World EXPO in Shang-hai got a glimpse of various visions for a better world powered by new knowledge, technological advances and creative will. Clearly, energy demand will not pause while we carefully work out the details. Inevitably, mistakes and compromises will be made and this will leave us with a legacy of picking up environmental pieces despite our best intentions.<br />
<br />
[[image:Toronto_China_3.jpg|600px|center]]<br />
<center>Suspended assortment of junk at the World EXPO in Shanghai becomes the image of a modern city</center></div>Gcromarhttp://2010.igem.org/File:Toronto_UTACCEL_Evolution.pptFile:Toronto UTACCEL Evolution.ppt2010-10-27T05:21:43Z<p>Gcromar: </p>
<hr />
<div></div>Gcromarhttp://2010.igem.org/Team:Toronto/Human_PracticesTeam:Toronto/Human Practices2010-10-27T05:06:42Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<br />
<h1>'''Human Practices'''</h1><br />
<br />
This year, the Toronto iGEM team partnered with UTACCEL to bring synthetic biology to high school students in China! We designed a six-hour course entitled "Adventures in Biotechnology" which was delivered to two dozen high school students over the course of a 10-day academic conference. Our goals were threefold:<br />
<br />
1. Spread the word about synthetic biology.<br />
2. Experience a different perspective on the demand for energy.<br />
3. Have fun!<br />
<br />
[[image:Toronto_China_1.jpg|600px|center]]<br />
<center>Friend Justin and iGEM team members Graham, Kenny and Mengyan enjoy some site-seeing in Guangzhou, China</center><br />
<br />
<h2>'''About UTACCEL'''</h2><br />
<br />
University of Toronto Association for Canada-China Exchange of Leadership (UTACCEL) is an officially recognized student group at the University of Toronto. The organization’s goal is to develop programs that facilitate cultural and knowledge exchange between students in Canada and China. One of their main programs is an annual academic conference in China. Through an intensive interview process, UTACCEL recruits Seminar Leaders from the University of Toronto student body. Seminar Leaders design seminars courses and deliver them to Chinese students during a 10-day conference. Seminar Leaders and delegates also interact with each other in various other ways such as workshops, team competitions and social activities. This year's conference was held at Huamei International High School in Guangzhou, China from July 24th to Aug 2nd.<br />
<br />
<h2>'''Adventures in Biotechnology - Course Description'''</h2><br />
The course introduces key concepts in biology which will be used as a basis to explore current topics in bioinformatics, systems biology and synthetic biology. High throughput science is generating data at an ever increasing rate and computers and software tools are now allowing us to better interpret this data. Our understanding of biological systems has matured. We no longer think of cells as largely random collections of ‘ugly bags of mostly water’. Rather, life is now understood to consist of complex, integrated systems of circuits and components. Our ability to understand complex systems coincides with extraordinary technologies which now allow us to not only read the genetic code but actually write it. This opens up enormous possibilities in biotechnology. The question is, what will we do with this power?<br />
<br />
<h2>'''A typical day at the conference'''</h2><br />
Wake up at 6 a.m. to the sound of blaring classical music. Hand shower (for some only cold water available) and head to breakfast - typically buns, warm soy milk and hard boiled eggs (widely agreed by the seminar leaders to be the best meal of the day). Weather pushing 40 degrees C with nearly 100% humidity, bring an umbrella! Now climb six flights of stairs to your assigned classroom and turn on the air conditioner (does it work today? 50/50). Teach until lunchtime. Steamed rice and assorted sides. Make plans with other seminar leaders to venture off campus to avoid cafeteria food for dinner. Afternoon "free-time" a.k.a. time to refine lectures, handouts for next class and hold office hours. Supervise evening program consisting of skills workshops and challenges. Roll into bed by midnight. Do it all again tomorrow.<br />
<br />
<h2>'''Why China?'''</h2><br />
The global demand for energy is steadily increasing worldwide. However, the inhabitants of developing nations like China whose soaring economy and large population are beginning to demand access to resources akin to those enjoyed by their western counterparts will push the demand for energy to new heights. Meeting this demand will mean continued dependence on fossil reserves despite the desire to move to alternative and renewable sources of power. We felt that first hand experience of China would complement our text book understanding of the human issues surrounding this ongoing energy debate.<br />
<br />
<h2>'''What did they learn'''</h2><br />
Students were introduced to important foundational concepts in evolution, cell biology, genetics, molecular biology, bioinformatics, systems biology and finally synthetic biology. They learned to define synthetic biology and were encouraged to think about issues raised by the power of the technology within an informed context. In addition, students were exposed to a western style of teaching akin to what they would face if they travel abroad for a university degree which many of the students attending the conference were considering.<br />
<br />
[[image:Toronto_China_2.jpg|600px|center]]<br />
<center>Delegates and their instructors at the UTACCEL conference</center><br />
<br />
<h2>'''What did we learn'''</h2><br />
There is no substitute for first hand experience. China is, in a word, crowded. But, conveying this in words is different from feeling it, walking the streets and seeing her people at work and at play. At once a mixture of old and new, archaic and sophisticated yet with deep history and pride, China is a nation faced with issues we will all face as world population increases. Those of us who also had an opportunity to attend the World EXPO in Shang-hai got a glimpse of various visions for a better world powered by new knowledge, technological advances and creative will. Clearly, energy demand will not pause while we carefully work out the details. Inevitably, mistakes and compromises will be made and this will leave us with a legacy of picking up environmental pieces despite our best intentions.<br />
<br />
[[image:Toronto_China_3.jpg|600px|center]]<br />
<center>Suspended assortment of junk at the World EXPO in Shanghai becomes the image of a modern city</center></div>Gcromarhttp://2010.igem.org/Team:Toronto/Human_PracticesTeam:Toronto/Human Practices2010-10-27T04:57:35Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<br />
<h1>'''Human Practices'''</h1><br />
<br />
This year, the Toronto iGEM team partnered with UTACCEL to bring synthetic biology to high school students in China! We designed a six-hour course entitled "Adventures in Biotechnology" which was delivered to two dozen high school students over the course of a 10-day academic conference. Our goals were threefold:<br />
<br />
1. Spread the word about synthetic biology.<br />
2. Experience a different perspective on the demand for energy.<br />
3. Have fun!<br />
<br />
[[image:Toronto_China_1.jpg|600px|center]]<br />
<br />
<h2>'''About UTACCEL'''</h2><br />
<br />
University of Toronto Association for Canada-China Exchange of Leadership (UTACCEL) is an officially recognized student group at the University of Toronto. The organization’s goal is to develop programs that facilitate cultural and knowledge exchange between students in Canada and China. One of their main programs is an annual academic conference in China. Through an intensive interview process, UTACCEL recruits Seminar Leaders from the University of Toronto student body. Seminar Leaders design seminars courses and deliver them to Chinese students during a 10-day conference. Seminar Leaders and delegates also interact with each other in various other ways such as workshops, team competitions and social activities. This year's conference was held at Huamei International High School in Guangzhou, China from July 24th to Aug 2nd.<br />
<br />
<h2>'''Adventures in Biotechnology - Course Description'''</h2><br />
The course introduces key concepts in biology which will be used as a basis to explore current topics in bioinformatics, systems biology and synthetic biology. High throughput science is generating data at an ever increasing rate and computers and software tools are now allowing us to better interpret this data. Our understanding of biological systems has matured. We no longer think of cells as largely random collections of ‘ugly bags of mostly water’. Rather, life is now understood to consist of complex, integrated systems of circuits and components. Our ability to understand complex systems coincides with extraordinary technologies which now allow us to not only read the genetic code but actually write it. This opens up enormous possibilities in biotechnology. The question is, what will we do with this power?<br />
<br />
<h2>'''A typical day at the conference'''</h2><br />
Wake up at 6 a.m. to the sound of blaring classical music. Hand shower (for some only cold water available) and head to breakfast - typically buns, warm soy milk and hard boiled eggs (widely agreed by the seminar leaders to be the best meal of the day). Weather pushing 40 degrees C with nearly 100% humidity, bring an umbrella! Now climb six flights of stairs to your assigned classroom and turn on the air conditioner (does it work today? 50/50). Teach until lunchtime. Steamed rice and assorted sides. Make plans with other seminar leaders to venture off campus to avoid cafeteria food for dinner. Afternoon "free-time" a.k.a. time to refine lectures, handouts for next class and hold office hours. Supervise evening program consisting of skills workshops and challenges. Roll into bed by midnight. Do it all again tomorrow.<br />
<br />
<h2>'''Why China?'''</h2><br />
The global demand for energy is steadily increasing worldwide. However, the inhabitants of developing nations like China whose soaring economy and large population are beginning to demand access to resources akin to those enjoyed by their western counterparts will push the demand for energy to new heights. Meeting this demand will mean continued dependence on fossil reserves despite the desire to move to alternative and renewable sources of power. We felt that first hand experience of China would complement our text book understanding of the human issues surrounding this ongoing energy debate.<br />
<br />
<h2>'''What did they learn'''</h2><br />
Students were introduced to important foundational concepts in evolution, cell biology, genetics, molecular biology, bioinformatics, systems biology and finally synthetic biology. They learned to define synthetic biology and were encouraged to think about issues raised by the power of the technology within an informed context. In addition, students were exposed to a western style of teaching akin to what they would face if they travel abroad for a university degree which many of the students attending the conference were considering.<br />
<br />
[[image:Toronto_China_2.jpg|600px|center]]<br />
<br />
<h2>'''What did we learn'''</h2><br />
There is no substitute for first hand experience. China is, in a word, crowded. But, conveying this in words is different from feeling it, walking the streets and seeing her people at work and at play. At once a mixture of old and new, archaic and sophisticated yet with deep history and pride, China is a nation faced with issues we will all face as world population increases. Those of us who also had an opportunity to attend the World EXPO in Shang-hai got a glimpse of various visions for a better world powered by new knowledge, technological advances and creative will. Clearly, energy demand will not pause while we carefully work out the details. Inevitably, mistakes and compromises will be made and this will leave us with a legacy of picking up environmental pieces despite our best intentions.<br />
<br />
[[image:Toronto_China_3.jpg|600px|center]]</div>Gcromarhttp://2010.igem.org/File:Toronto_China_3.jpgFile:Toronto China 3.jpg2010-10-27T04:56:05Z<p>Gcromar: World EXPO. In the eye of the beholder...what do you see?</p>
<hr />
<div>World EXPO. In the eye of the beholder...what do you see?</div>Gcromarhttp://2010.igem.org/File:Toronto_China_2.jpgFile:Toronto China 2.jpg2010-10-27T04:53:49Z<p>Gcromar: conference attendees in session</p>
<hr />
<div>conference attendees in session</div>Gcromarhttp://2010.igem.org/Team:Toronto/Human_PracticesTeam:Toronto/Human Practices2010-10-27T04:52:21Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<br />
<h1>'''Human Practices'''</h1><br />
<br />
This year, the Toronto iGEM team partnered with UTACCEL to bring synthetic biology to high school students in China! We designed a six-hour course entitled "Adventures in Biotechnology" which was delivered to two dozen high school students over the course of a 10-day academic conference. Our goals were threefold:<br />
<br />
1. Spread the word about synthetic biology.<br />
2. Experience a different perspective on the demand for energy.<br />
3. Have fun!<br />
<br />
[[image:Toronto_China_1.jpg|500px]]<br />
<br />
<h2>'''About UTACCEL'''</h2><br />
<br />
University of Toronto Association for Canada-China Exchange of Leadership (UTACCEL) is an officially recognized student group at the University of Toronto. The organization’s goal is to develop programs that facilitate cultural and knowledge exchange between students in Canada and China. One of their main programs is an annual academic conference in China. Through an intensive interview process, UTACCEL recruits Seminar Leaders from the University of Toronto student body. Seminar Leaders design seminars courses and deliver them to Chinese students during a 10-day conference. Seminar Leaders and delegates also interact with each other in various other ways such as workshops, team competitions and social activities. This year's conference was held at Huamei International High School in Guangzhou, China from July 24th to Aug 2nd.<br />
<br />
<h2>'''Adventures in Biotechnology - Course Description'''</h2><br />
The course introduces key concepts in biology which will be used as a basis to explore current topics in bioinformatics, systems biology and synthetic biology. High throughput science is generating data at an ever increasing rate and computers and software tools are now allowing us to better interpret this data. Our understanding of biological systems has matured. We no longer think of cells as largely random collections of ‘ugly bags of mostly water’. Rather, life is now understood to consist of complex, integrated systems of circuits and components. Our ability to understand complex systems coincides with extraordinary technologies which now allow us to not only read the genetic code but actually write it. This opens up enormous possibilities in biotechnology. The question is, what will we do with this power?<br />
<br />
<h2>'''A typical day at the conference'''</h2><br />
Wake up at 6 a.m. to the sound of blaring classical music. Hand shower (for some only cold water available) and head to breakfast - typically buns, warm soy milk and hard boiled eggs (widely agreed by the seminar leaders to be the best meal of the day). Weather pushing 40 degrees C with nearly 100% humidity, bring an umbrella! Now climb six flights of stairs to your assigned classroom and turn on the air conditioner (does it work today? 50/50). Teach until lunchtime. Steamed rice and assorted sides. Make plans with other seminar leaders to venture off campus to avoid cafeteria food for dinner. Afternoon "free-time" a.k.a. time to refine lectures, handouts for next class and hold office hours. Supervise evening program consisting of skills workshops and challenges. Roll into bed by midnight. Do it all again tomorrow.<br />
<br />
<h2>'''Why China?'''</h2><br />
The global demand for energy is steadily increasing worldwide. However, the inhabitants of developing nations like China whose soaring economy and large population are beginning to demand access to resources akin to those enjoyed by their western counterparts will push the demand for energy to new heights. Meeting this demand will mean continued dependence on fossil reserves despite the desire to move to alternative and renewable sources of power. We felt that first hand experience of China would complement our text book understanding of the human issues surrounding this ongoing energy debate.<br />
<br />
<h2>'''What did they learn'''</h2><br />
Students were introduced to important foundational concepts in evolution, cell biology, genetics, molecular biology, bioinformatics, systems biology and finally synthetic biology. They learned to define synthetic biology and were encouraged to think about issues raised by the power of the technology within an informed context. In addition, students were exposed to a western style of teaching akin to what they would face if they travel abroad for a university degree which many of the students attending the conference were considering.<br />
<br />
[[image:Toronto_China_2.jpg|500px|center]]<br />
<br />
<h2>'''What did we learn'''</h2><br />
There is no substitute for first hand experience. China is, in a word, crowded. But, conveying this in words is different from feeling it, walking the streets and seeing her people at work and at play. At once a mixture of old and new, archaic and sophisticated yet with deep history and pride, China is a nation faced with issues we will all face as world population increases. Those of us who also had an opportunity to attend the World EXPO in Shang-hai got a glimpse of various visions for a better world powered by new knowledge, technological advances and creative will. Clearly, energy demand will not pause while we carefully work out the details. Inevitably, mistakes and compromises will be made and this will leave us with a legacy of picking up environmental pieces despite our best intentions.<br />
<br />
[[image:Toronto_China_3.jpg|500px]]</div>Gcromarhttp://2010.igem.org/File:Toronto_China_1.jpgFile:Toronto China 1.jpg2010-10-27T04:45:21Z<p>Gcromar: iGEM team members Kenny, Mengyan, Graham with friend Justin</p>
<hr />
<div>iGEM team members Kenny, Mengyan, Graham with friend Justin</div>Gcromarhttp://2010.igem.org/Team:Toronto/Human_PracticesTeam:Toronto/Human Practices2010-10-27T04:25:45Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<br />
<h1>'''Human Practices'''</h1><br />
<br />
This year, the Toronto iGEM team partnered with UTACCEL to bring synthetic biology to high school students in China! We designed a six-hour course entitled "Adventures in Biotechnology" which was delivered to two dozen high school students over the course of a 10-day academic conference. Our goals were threefold:<br />
<br />
1. Spread the word about synthetic biology.<br />
2. Experience a different perspective on the demand for energy.<br />
3. Have fun!<br />
<br />
<h2>'''About UTACCEL'''</h2><br />
<br />
University of Toronto Association for Canada-China Exchange of Leadership (UTACCEL) is an officially recognized student group at the University of Toronto. The organization’s goal is to develop programs that facilitate cultural and knowledge exchange between students in Canada and China. One of their main programs is an annual academic conference in China. Through an intensive interview process, UTACCEL recruits Seminar Leaders from the University of Toronto student body. Seminar Leaders design seminars courses and deliver them to Chinese students during a 10-day conference. Seminar Leaders and delegates also interact with each other in various other ways such as workshops, team competitions and social activities. This year's conference was held at Huamei International High School in Guangzhou, China from July 24th to Aug 2nd.<br />
<br />
<h2>'''Adventures in Biotechnology - Course Description'''</h2><br />
The course introduces key concepts in biology which will be used as a basis to explore current topics in bioinformatics, systems biology and synthetic biology. High throughput science is generating data at an ever increasing rate and computers and software tools are now allowing us to better interpret this data. Our understanding of biological systems has matured. We no longer think of cells as largely random collections of ‘ugly bags of mostly water’. Rather, life is now understood to consist of complex, integrated systems of circuits and components. Our ability to understand complex systems coincides with extraordinary technologies which now allow us to not only read the genetic code but actually write it. This opens up enormous possibilities in biotechnology. The question is, what will we do with this power?<br />
<br />
<h2>'''A typical day at the conference'''</h2><br />
Wake up at 6 a.m. to the sound of blaring classical music. Hand shower (for some only cold water available) and head to breakfast - typically buns, warm soy milk and hard boiled eggs (widely agreed by the seminar leaders to be the best meal of the day). Weather pushing 40 degrees C with nearly 100% humidity, bring an umbrella! Now climb six flights of stairs to your assigned classroom and turn on the air conditioner (does it work today? 50/50). Teach until lunchtime. Steamed rice and assorted sides. Make plans with other seminar leaders to venture off campus to avoid cafeteria food for dinner. Afternoon "free-time" a.k.a. time to refine lectures, handouts for next class and hold office hours. Supervise evening program consisting of skills workshops and challenges. Roll into bed by midnight. Do it all again tomorrow.<br />
<br />
<h2>'''Why China?'''</h2><br />
The global demand for energy is steadily increasing worldwide. However, the inhabitants of developing nations like China whose soaring economy and large population are beginning to demand access to resources akin to those enjoyed by their western counterparts will push the demand for energy to new heights. Meeting this demand will mean continued dependence on fossil reserves despite the desire to move to alternative and renewable sources of power. We felt that first hand experience of China would complement our text book understanding of the human issues surrounding this ongoing energy debate.<br />
<br />
<h2>'''What did they learn'''</h2><br />
Students were introduced to important foundational concepts in evolution, cell biology, genetics, molecular biology, bioinformatics, systems biology and finally synthetic biology. They learned to define synthetic biology and were encouraged to think about issues raised by the power of the technology within an informed context. In addition, students were exposed to a western style of teaching akin to what they would face if they travel abroad for a university degree which many of the students attending the conference were considering.<br />
<br />
<h2>'''What did we learn'''</h2><br />
There is no substitute for first hand experience. China is, in a word, crowded. But, conveying this in words is different from feeling it, walking the streets and seeing her people at work and at play. At once a mixture of old and new, archaic and sophisticated yet with deep history and pride, China is a nation faced with issues we will all face as world population increases. Those of us who also had an opportunity to attend the World EXPO in Shang-hai got a glimpse of various visions for a better world powered by new knowledge, technological advances and creative will. Clearly, energy demand will not pause while we carefully work out the details. Inevitably, mistakes and compromises will be made and this will leave us with a legacy of picking up environmental pieces despite our best intentions.</div>Gcromarhttp://2010.igem.org/Team:Toronto/ResultsTeam:Toronto/Results2010-10-27T04:11:33Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
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!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
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!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Results'''</h1><br />
<br />
<h2>'''''E. coli'' DH5a is sensitive to Catechol exposure'''</h2><br />
<br />
To measure the effect of our pathway manipulations on the viability of ''E.coli'' exposed to catechol we plan to perform a tolerance test. In preliminary experiments we established the baseline sensitivity of ''E.coli'' (DH5a) exposed to 50mM catechol in liquid media over a six hour time course. Aliquots of this mixture were plated on LB media at various dilutions and colonies were counted and compared with untreated controls (see "Protocols"). There was no measurable difference between control and treated cells when bacteria were exposed to catechol in a growth media (data not shown). However, in minimal media (MM2) growth of control cells was moderate while exposed cells exhibited a marked decrease in survival (Figure 1). Although the effects of Catechol exposure in ''E.coli'' and ''P. putida'' have previously been investigated [Park et al. 2001] completion of these baseline experiments will enable us to determine the effect of pathway manipulations on the proposed engineered ''E.coli'' strains containing various configurations of the pathway.<br />
<br />
<p><br />
'''A'''<br />
[[Image:Toronto-LB-Plate_1.jpg|400px]] [[Image:Toronto-LB-Plate_2.jpg|400px]]<br />
</p><br />
<p><br />
'''B'''[[Image:Toronto-results-catechol_tolerance.png|center]]<br />
</p><br />
<blockquote>'''Figure 1:''' A. Growth of ''E.coli'' DH5a on LB plates (1x10<sup>-5</sup> dilution) following exposure to minimal media and minimal media with 50mM catechol in solution. Plates shown are for the last time points plotted. B. Survival curves of ''E. coli'' DH5a exposed to catechol over a six hour time course.</blockquote><br />
<br />
<H2>'''Synthetic construction of a functioning catechol ortho degradation pathway in E.coli is predicted to increase cell growth rate'''</H2><br />
To predict the effects of adding the catechol ortho-cleavage pathway on ''E.coli'' metabolism we used a previously developed genome-scale reconstruction of E. coli (iJR904) and applied flux balance analysis (FBA) to compare likely growth outcomes for normal and optimized pathways. We found that optimization of this pathway in the presence of glucose and sufficient oxygen leads to a prediction of increased growth rate. This result bodes well for the success of engineered bacteria introduced into mixed culture within the tailings ponds as they might be expected to have a survival advantage over their native counterparts. However, it should be pointed out that a corresponding genome-scale reconstruction of the target organism ''P. putida'' is not available at this time. These methods and results are discussed at length in the Modeling section.<br />
<br />
<h2>'''Colocalization of muconolactone D-isomerase (EC:5.3.3.4) and 3-oxoadipate enol-lactone hydrolase (EC:3.1.1.24) are predicted to lead to increased catechol degradation through metabolic channeling'''</h2><br />
In order to determine which enzymes would most benefit from metabolic channeling in the catechol degradation pathway, we used a cellular simulation tool, developed in our lab, called Cell++. It allows us to place enzymes of choice in a compartment within a cellular environment and calculates the effect of localization on metabolite concentrations in a user-defined biochemical pathway (Sanford et al. 2006). We gathered kinetic data of the five enzymes in the degradation pathway and investigated the effects of localizing pairs of sequential enzymes (i.e. catechol 1, 2-dioxygenase and muconate cycloisomeras) on metabolite concentrations. Four co-localization simulations were performed in Cell++ along with a control simulation where no enzymes were co-localized. The initial metabolite in the simulation environment, catechol, was added into the simulation environment and the simulation environment was run for 10,000 iterations to observe the effect on catechol degradation in each simulation. The results are displayed as a table indicating the concentration of all metabolites at 1000 equidistant time points during the simulation. <br />
<p>To make use of this information, we extracted the maximum metabolite concentration and the maximum rate of metabolite formation for all metabolites during each simulation. These values are normalized from a scale of 0 to 1, where 0 indicates the lowest value of the concentration/rate of formation (C/RoF) in the simulations and 1 is the highest. These values were further evaluated against the control simulation to the find the percentage change of the C/RoF values of each metabolite in the four co-localization simulations. In order to classify a particular enzyme co-localization as beneficial, we need to see an increase in the maximum concentration and rate of formation of the final metabolite in the pathway (meta6). These increases would represent an increased flux through the pathway. Of the four co-localization simulations, simulation 3 (which co-localizes the enzymes muconolactone D-isomerase and 3-oxodipate-enol lactone hydrolase) was the only simulation that matched the criteria for a beneficial co-localization. From these simulations, we decided to co-localize these two enzymes for our wet lab experiments to see if Cell++ accurately depicted the effects on catechol degradation.</p><br />
<br />
''' '''[[Image:Cellppheatmap.jpg|center]]<br />
<br />
<blockquote>'''Figure 2.''' Simulating the effects of enzyme co-localization on catechol degradation efficiency.<br />
Red indicates increase in the value of the kinetic parameter relative to the simulation where no enzymes are co-localized (0.0), while green indicates a decrease in the kinetic parameter. 1.0, 2.0, 3.0 and 4.0 represent pair-wise fusions of the 1st and 2nd, 2nd and 3rd, 3rd and 4th, and 4th and 5th enzymes respectively. “Max[]” and “Vmax” represent the maximum concentration and the maximum rate of formation of a particular metabolite in a particular simulation. The fusion of the 3rd and 4th enzymes (3.0) resulted in an increase of the production of the final pathway product, while the other fusion pairs demonstrate no increase or even reductions in the production of the final metabolite in the simulated pathway.</blockquote></div>Gcromarhttp://2010.igem.org/Toronto/23_October_2010Toronto/23 October 20102010-10-26T16:08:28Z<p>Gcromar: New page: Retrieve plates from incubator and count colonies on control and treated (50mM catechol exposure) as described in methods. Calculate CFU's and plot.</p>
<hr />
<div>Retrieve plates from incubator and count colonies on control and treated (50mM catechol exposure) as described in methods. Calculate CFU's and plot.</div>Gcromarhttp://2010.igem.org/Toronto/22_October_2010Toronto/22 October 20102010-10-26T16:06:22Z<p>Gcromar: New page: Innoculate 0.5 ml overnight culture (x2) into 500 ml flasks containing 200ml LB broth, grow to O.D.<sub>600</sub>=1.0 representing 10<sup>9</sup> cells per ml. Pour LB plates and allow to...</p>
<hr />
<div>Innoculate 0.5 ml overnight culture (x2) into 500 ml flasks containing 200ml LB broth, grow to O.D.<sub>600</sub>=1.0 representing 10<sup>9</sup> cells per ml.<br />
<br />
Pour LB plates and allow to cool. Invert and label plates with exposure, time and dilution factors.<br />
<br />
Innoculate 0.5 ml of culture (x2) into two 100 ml flasks containing 20 ml MM2 media. Add 30 ml MM2 media, one containing Catechol at 50mM concentration and incubate with shaking at 37C for six hours. Remove aliquots and plate dilutions every two hours and place plates in 37c incubator overnight.</div>Gcromarhttp://2010.igem.org/Toronto/21_October_2010Toronto/21 October 20102010-10-26T15:58:53Z<p>Gcromar: New page: Preparation for Catechol tolerance assay: Innoculate overnight cultures of ''E.coli'' DH5a Prepare LB+agar and MM2 (liquid) media and autoclave.</p>
<hr />
<div>Preparation for Catechol tolerance assay:<br />
Innoculate overnight cultures of ''E.coli'' DH5a<br />
Prepare LB+agar and MM2 (liquid) media and autoclave.</div>Gcromarhttp://2010.igem.org/Team:Toronto/DesignTeam:Toronto/Design2010-10-26T15:52:56Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
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!align="center"|[[Team:Toronto|Home]]<br />
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!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
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!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Project Design'''</h1><br />
<p>In order to identify enzymes in the catechol degradation pathway (ortho cleavage) that benefit from the introduction of metabolic channelling, we performed ''in silico'' analyses with software developed in our lab (Sanford et al.). Preliminary results from our simulations suggests that the 3rd (muconolactone D-isomerase) and 4th (3-oxodipate-enol lactone hydrolase) enzymes in the pathway would benefit most from channelling (Figure 2). Muconolactone D-isomerase catalyzes an energetically unfavourable reaction, while 3-oxodipate-enol lactone hydrolase catalyzes a very energetically favourable reaction. By introducing channeling between these two enzymes, the energetically unfavourable product of the mucanolactone D-isomerase-catalyzed reaction would be converted to the product of 3-oxodipate-enol lactone hydrolase before mucanolactone D-isomerase converts the energetically unfavourable product back into the energetically favourable substrate. The net effect would be an increased flux through the catechol degradation pathway. </p><br />
<br />
<br />
<br />
[[Image:Catechol degradation 5.png|center]]<br />
<br />
<blockquote> Figure 2: Ortho degradation of catechol (1,2-Benzenediol) proceeds in six steps. Only the last enzyme is present in E. coli. Enzyme candidates with the largest predicted channeling effect as predicted by Cell++ are boxed in red.</blockquote><br />
<br />
<br/><br />
<p>The five enzymes in the catechol degradation pathway not naturally present in ''E. coli'' will be introduced into ''E. coli'' as BioBrick parts. The parts will then be used to construct five recombinant strains of ''E. coli'' (Figure 3). These include a full reconstruction of the pathway (control strain) and several partial reconstructions (test strains). The latter will contain pathway 'holes' corresponding to all combinations of sequential pairs of enzymes, with complementary fusion pairs. The test strain with the fusion of enzymes 3 and 4 will be of prime interest, while the other test strains will be used validate the predictions made by Cell++. </p><br />
<br />
<br />
<br />
<br />
[[Image:design 6.png|center]]<br />
<blockquote>Figure 3: Diagramatic representation of proposed gene fusion experiments. Pathway reconstructions are shown in black for the control and test strains with pathway holes for the partial reconstructions shown in red. Yellow stars mark proposed gene fusions to be tested to validate computational predictions. The blue circle marks the best candidate for enzyme channeling based on ''in silico'' experiments </blockquote><br />
<br/><br />
<p>In order to demonstrate the breakdown of catechol by these recombinant strains, catechol will be added into the growth media of these strains. A colorimetric assay, previously developed to quantify the amount of catechol in solution, will be utilized to assay the rate of catechol degradation by the different strains (Tao et al.). In addition, a survival assay will be used to determine the threshold for catechol exposure for non recombinant ''E. coli'' and to evaluate the effect that the recombinantly-expressed enzymes in the catechol pathway will have on ''E. coli'' survival. </p></div>Gcromarhttp://2010.igem.org/Team:Toronto/DesignTeam:Toronto/Design2010-10-26T15:51:47Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
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!align="center"|[[Team:Toronto|Home]]<br />
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!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
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!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Project Design'''</h1><br />
<p>In order to identify enzymes in the catechol degradation pathway (ortho cleavage) that benefit from the introduction of metabolic channelling, we performed ''in silico'' analyses with software developed in our lab (Sanford et al.). Preliminary results from our simulations suggests that the 3rd (muconolactone D-isomerase) and 4th (3-oxodipate-enol lactone hydrolase) enzymes in the pathway would benefit most from channelling (Figure 2). Muconolactone D-isomerase catalyzes an energetically unfavourable reaction, while 3-oxodipate-enol lactone hydrolase catalyzes a very energetically favourable reaction. By introducing channeling between these two enzymes, the energetically unfavourable product of the mucanolactone D-isomerase-catalyzed reaction would be converted to the product of 3-oxodipate-enol lactone hydrolase before mucanolactone D-isomerase converts the energetically unfavourable product back into the energetically favourable substrate. The net effect would be an increased flux through the catechol degradation pathway. </p><br />
<br />
<br />
<br />
[[Image:Catechol degradation 5.png|center]]<br />
<br />
<blockquote> Figure 2: Ortho degradation of catechol (1,2-Benzenediol) proceeds in six steps. Only the last enzyme is present in E. coli. Enzyme candidates with the largest predicted channeling effect as predicted by Cell++ are boxed in red.</blockquote><br />
<br />
<br/><br />
<p>The five enzymes in the catechol degradation pathway not naturally present in ''E. coli'' will be introduced into ''E. coli'' as BioBrick parts. The parts will then be used to construct five recombinant strains of ''E. coli'' (Figure 3). These include a full reconstruction of the pathway (control strain) and several partial reconstructions (test strains). The latter will contain pathway 'holes' corresponding to all combinations of sequential pairs of enzymes, with complementary fusion pairs. The test strain with the fusion of enzymes 3 and 4 will be of prime interest, while the other test strains will be used validate the predictions made by Cell++. </p><br />
<br />
<br />
<br />
<br />
[[Image:design 6.png|center]]<br />
<blockquote>Figure 3: Diagramatic representation of proposed gene fusion experiments. Pathway reconstructions are shown in black for the control and test strains with pathway holes for the partial reconstructions shown in red. Yellow stars mark proposed gene fusions to be tested to validate computational predictions. The blue circle marks the best candidate for enzyme channeling based on in silico experiments </blockquote><br />
<br/><br />
<p>In order to demonstrate the breakdown of catechol by these recombinant strains, catechol will be added into the growth media of these strains. A colorimetric assay, previously developed to quantify the amount of catechol in solution, will be utilized to assay the rate of catechol degradation by the different strains (Tao et al.). In addition, a survival assay will be used to determine the threshold for catechol exposure for non recombinant E. coli and to evaluate the effect that the recombinantly-expressed enzymes in the catechol pathway will have on E. coli survival. </p></div>Gcromarhttp://2010.igem.org/Team:Toronto/DesignTeam:Toronto/Design2010-10-26T15:47:49Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
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!align="center"|[[Team:Toronto|Home]]<br />
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!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
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!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Project Design'''</h1><br />
<p>In order to identify enzymes in the catechol degradation pathway (ortho cleavage) that benefit from the introduction of metabolic channelling, we performed in silico analyses with software developed in our lab (Sanford et al.). Preliminary results from our simulations suggests that the 3rd (muconolactone D-isomerase) and 4th (3-oxodipate-enol lactone hydrolase) enzymes in the pathway would benefit most from channelling (Figure 2). Muconolactone D-isomerase catalyzes an energetically unfavourable reaction, while 3-oxodipate-enol lactone hydrolase catalyzes a very energetically favourable reaction. By introducing channeling between these two enzymes, the energetically unfavourable product of the mucanolactone D-isomerase-catalyzed reaction would be converted to the product of 3-oxodipate-enol lactone hydrolase before mucanolactone D-isomerase converts the energetically unfavourable product back into the energetically favourable substrate. The net effect would be an increased flux through the catechol degradation pathway. </p><br />
<br />
<br />
<br />
[[Image:Catechol degradation 5.png|center]]<br />
<br />
<blockquote> Figure 2: Ortho degradation of catechol (1,2-Benzenediol) proceeds in six steps. Only the last enzyme is present in E. coli. Enzyme candidates with the largest predicted channeling effect as predicted by Cell++ are boxed in red.</blockquote><br />
<br />
<br/><br />
<p>The five enzymes in the catechol degradation pathway not naturally present in E. coli will be introduced into E. coli as BioBrick parts. The parts will then be used to construct five recombinant strains of E. coli (Figure 3). These include a full reconstruction of the pathway (control strain) and several partial reconstructions (test strains). The latter will contain pathway 'holes' corresponding to all combinations of sequential pairs of enzymes, with complementary fusion pairs. The test strain with the fusion of enzymes 3 and 4 will be of prime interest, while the other test strains will be used validate the predictions made by Cell++. </p><br />
<br />
<br />
<br />
<br />
[[Image:design 6.png|center]]<br />
<blockquote>Figure 3: Diagramatic representation of proposed gene fusion experiments. Pathway reconstructions are shown in black for the control and test strains with pathway holes for the partial reconstructions shown in red. Yellow stars mark proposed gene fusions to be tested to validate computational predictions. The blue circle marks the best candidate for enzyme channeling based on in silico experiments </blockquote><br />
<br/><br />
<p>In order to demonstrate the breakdown of catechol by these recombinant strains, catechol will be added into the growth media of these strains. A colorimetric assay, previously developed to quantify the amount of catechol in solution, will be utilized to assay the rate of catechol degradation by the different strains (Tao et al.). In addition, a survival assay will be used to determine the threshold for catechol exposure for non recombinant E. coli and to evaluate the effect that the recombinantly-expressed enzymes in the catechol pathway will have on E. coli survival. </p></div>Gcromarhttp://2010.igem.org/Team:Toronto/ProjectTeam:Toronto/Project2010-10-26T15:45:18Z<p>Gcromar: </p>
<hr />
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!align="center"|[[Team:Toronto|Home]]<br />
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!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
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!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
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|}<br />
<br />
<br />
<h1>'''Project Description'''</h1><hr/><br />
<br />
<h2> '''Background'''</h2><br />
<br />
<h3>'''Oil Sands'''</h3><br />
<br />
<p> Oil sands are naturally occurring mixtures of sand or clay, water and an extremely dense and viscous form of petroleum called bitumen. [[Image:CompositionofOilSand40.jpg|right]]Bitumen is a collection of heavy hydrocarbons, which results from preferential microbial degradation of lighter hydrocarbons in oil reserves over geological time scales. These heavy oil reserves are some of the largest fossil fuel stores in the world with more stored energy than traditional oil reserves.</p><br />
<p>Oil sands represent approximately 67% of the world’s total petroleum resource, with the two largest deposits being in Canada and Venezuela. Together they contain approximately 3.6 trillion barrels of oil, compared to 1.75 trillion barrels of conventional oil worldwide. The oil sands in the Athabasca Basin in northeastern Alberta, Canada represent the world’s largest resource estimated to contain over 1.7 trillion barrels of bitumen.</p><br />
<br />
<br />
<br />
<h3>'''Tailings Ponds'''</h3><br />
<p>Oil sands are developed either through open-pit mining or deep underground (in situ) production. In open-pit mining, hot water is used to separate bitumen from the mixture of sand and clay.</p><br />
<p>Each cubic meter of mined oil sands requires up to 3 m3 of water and produces about 4 m3 of slurry wastes containing sand, clay, water, unrecovered bitumen and dissolved inorganic and organic compounds. This mixture is then sent to a discontinued mine pit, referred to as a tailings pond. It is a large man-made collection of waste, which has a negative impact on the environment.</p> <br />
<p>Over 70% of the water demand is met by recycling the recovered water. Even so, these wastewaters are accumulating at a rate of about 0.1-0.2 m3 per ton of oil sands processed. Recycling of the water back to the extraction process further concentrates contaminants as more waste material is added to the already contaminated water. It has been estimated that over 1 billion m3 of tailings pond water will be generated by the year 2025.</p><br />
<p>They are composed of a wide variety of chemical toxins. Amongst these are polycyclic aromatic hydrocarbons (PAHs), with Naphthalenes, Anthracenes and Fluorenes present at the highest concentrations. These compounds are toxic to the surrounding aquatic ecosystems, and have been shown to have an adverse effect on human health. PAHs are carcinogenic and are also known to have a negative impact on immune function, kidney function, liver function, reproduction and development.</p><br />
<br />
<br />
<br />
<h3>'''Bioremediation'''</h3><br />
<br />
<p>Bioremediation is the use of microorganisms to return the natural environment altered by contaminants to its original condition. It has been proposed as a possible method for the reclamation of land and water impacted by oil sands development. Although natural examples of these processes exist, they tend to be characterized by slow reaction rates and/or undesirable side reactions.</p><br />
<p>Over expression of rate limiting enzymes has enjoyed moderate success in overcoming some pathway limitations. However, because enzymes operate within complex, highly organized systems, there is increasing recognition within the metabolic engineering community that spatial organization offers an additional route for optimizing pathway efficiency.<br />
</p><br />
<br />
<br />
<br />
<h3>'''Catechol'''</h3><br />
<br />
<p>Pseudomonas putida, a microbe naturally present in tailings ponds, has the metabolic machinery needed to metabolize many toxins within the tailings ponds. This includes the capability to degrade certain PAHs in tailings ponds such as naphthalenes, anthracenes and fluorenes. These three major PAHs are degraded to form a common compound called catechol. However, this process is slow and inefficient. Since catechol is the common breakdown product in the breakdown of these PAHs, enhancing the breakdown of catechol may lead to an increase in the rate of PAH degradation. This project will look to evaluate how we can increase the degradation rate of catechol in order to achieve faster PAH breakdown. </p><br />
<br />
<p>The metabolic network present in P. putida for degrading catechol is highlighted by the black box in Figure 1. (http://www.genome.jp/kegg-bin/show_pathway?ko00362+C00090). </p><br />
<br />
<br />
<br />
[[Image:putida!!.png|center]]<br />
<br />
<br />
<blockquote>Figure 1. The degradation of catechol (ortho cleavage pathway) leads to common cellular metabolites, acetyl-CoA and Succinyl-CoA , through the action of five enzyme catalyzed reactions in ''Pseudomonas putida''.</blockquote><br />
<br />
<br />
<h3>'''Metabolic Channeling'''</h3><br />
<p>Metabolic channeling is the process whereby metabolic intermediates are channelled between the active sites of consecutive enzymes in a biochemical pathway. This serves to increase reaction efficiency by limiting the diffusion of metabolites into the surrounded cellular intracellular environment. Channelling provides a biochemical system with benefits such as:</p><br />
<ul><br />
<li> Enhancing unfavourable reactions</li><br />
<li> Sequestering toxic intermediates </li><br />
<li> Protecting unstable intermediates</li><br />
<li> Preventing inhibition of other enzymes by the channelling intermediate</li><br />
</ul><br />
<p>Channeling has been utilized in bioengineering to enhance the efficiency of desired biochemical systems. Some methods which have been utilized to mimic examples of metabolic channelling in nature are through:</p><br />
<ul><br />
<li> Immobilized Enzymes</li><br />
<li> Fusion Proteins </li><br />
<li> Scaffolds </li><br />
</ul><br />
<br />
<br/><br/><br />
<br />
[[Image:immobilized enzymes 2.png|border=|Immobilized Enzymes]] [[Image:Fusion Protein 3.png|border|Fusion Protein]] [[Image:Scaffolding 4.png|border|Scaffolding]]<br />
<pre>Immobilized Enzymes Fusion Protein Scaffolding</pre><br />
<br />
<br />
<h3>'''The Project'''</h3><br />
<br/><br />
<p><br />
<br />
Our project aims to take advantage of the catechol degradation pathway present in P. Putida by introducing metabolic channeling to increase the efficiency with which catechol is degraded. Besides being toxic in it's own right, we anticipate that accelerating the degradation of catechol, a common product of polyaromatic hydrocarbon (PAH) degradation, will result in increased PAH degradation. This hypothesis, supported by computer modeling will be tested in an ''E.coli'' model using the synthetic construction of several variations of the pathway involving the channeling of different pairs of enzymes by protein fusion.</p><br />
<br />
<br />
<h4>'''Future Goals'''</h4><br />
<br />
<p>Once we complete the characterization of the “Encapsulator” micro-compartment from the 2009 iGEM project, we will evaluate whether compartmentalization can further enhance the effects of metabolic channeling as compared to gene fusions. Ultimately, channeling will be introduced to target pathways in endogenous tailings ponds species (e.g. P. Putida) for application in the bioremediation of the tailings ponds.</p></div>Gcromarhttp://2010.igem.org/Team:Toronto/ProjectTeam:Toronto/Project2010-10-26T15:40:22Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Project Description'''</h1><hr/><br />
<br />
<h2> '''Background'''</h2><br />
<br />
<h3>'''Oil Sands'''</h3><br />
<br />
<p> Oil sands are naturally occurring mixtures of sand or clay, water and an extremely dense and viscous form of petroleum called bitumen. [[Image:CompositionofOilSand40.jpg|right]]Bitumen is a collection of heavy hydrocarbons, which results from preferential microbial degradation of lighter hydrocarbons in oil reserves over geological time scales. These heavy oil reserves are some of the largest fossil fuel stores in the world with more stored energy than traditional oil reserves.</p><br />
<p>Oil sands represent approximately 67% of the world’s total petroleum resource, with the two largest deposits being in Canada and Venezuela. Together they contain approximately 3.6 trillion barrels of oil, compared to 1.75 trillion barrels of conventional oil worldwide. The oil sands in the Athabasca Basin in northeastern Alberta, Canada represent the world’s largest resource estimated to contain over 1.7 trillion barrels of bitumen.</p><br />
<br />
<br />
<br />
<h3>'''Tailings Ponds'''</h3><br />
<p>Oil sands are developed either through open-pit mining or deep underground (in situ) production. In open-pit mining, hot water is used to separate bitumen from the mixture of sand and clay.</p><br />
<p>Each cubic meter of mined oil sands requires up to 3 m3 of water and produces about 4 m3 of slurry wastes containing sand, clay, water, unrecovered bitumen and dissolved inorganic and organic compounds. This mixture is then sent to a discontinued mine pit, referred to as a tailings pond. It is a large man-made collection of waste, which has a negative impact on the environment.</p> <br />
<p>Over 70% of the water demand is met by recycling the recovered water. Even so, these wastewaters are accumulating at a rate of about 0.1-0.2 m3 per ton of oil sands processed. Recycling of the water back to the extraction process further concentrates contaminants as more waste material is added to the already contaminated water. It has been estimated that over 1 billion m3 of tailings pond water will be generated by the year 2025.</p><br />
<p>They are composed of a wide variety of chemical toxins. Amongst these are polycyclic aromatic hydrocarbons (PAHs), with Naphthalenes, Anthracenes and Fluorenes present at the highest concentrations. These compounds are toxic to the surrounding aquatic ecosystems, and have been shown to have an adverse effect on human health. PAHs are carcinogenic and are also known to have a negative impact on immune function, kidney function, liver function, reproduction and development.</p><br />
<br />
<br />
<br />
<h3>'''Bioremediation'''</h3><br />
<br />
<p>Bioremediation is the use of microorganisms to return the natural environment altered by contaminants to its original condition. It has been proposed as a possible method for the reclamation of land and water impacted by oil sands development. Although natural examples of these processes exist, they tend to be characterized by slow reaction rates and/or undesirable side reactions.</p><br />
<p>Over expression of rate limiting enzymes has enjoyed moderate success in overcoming some pathway limitations. However, because enzymes operate within complex, highly organized systems, there is increasing recognition within the metabolic engineering community that spatial organization offers an additional route for optimizing pathway efficiency.<br />
</p><br />
<br />
<br />
<br />
<h3>'''Catechol'''</h3><br />
<br />
<p>Pseudomonas putida, a microbe naturally present in tailings ponds, has the metabolic machinery needed to metabolize many toxins within the tailings ponds. This includes the capability to degrade certain PAHs in tailings ponds such as naphthalenes, anthracenes and fluorenes. These three major PAHs are degraded to form a common compound called catechol. However, this process is slow and inefficient. Since catechol is the common breakdown product in the breakdown of these PAHs, enhancing the breakdown of catechol may lead to an increase in the rate of PAH degradation. This project will look to evaluate how we can increase the degradation rate of catechol in order to achieve faster PAH breakdown. </p><br />
<br />
<p>The metabolic network present in P. putida for degrading catechol is highlighted by the black box in Figure 1. (http://www.genome.jp/kegg-bin/show_pathway?ko00362+C00090). </p><br />
<br />
<br />
<br />
[[Image:putida!!.png|center]]<br />
<br />
<br />
<blockquote>Figure 1. The degradation of catechol to common cellular metabolites, acetyl-CoA and Succinyl-CoA , involves five enzyme catalyzed reactions in Pseudomonas putida.</blockquote><br />
<br />
<br />
<h3>'''Metabolic Channeling'''</h3><br />
<p>Metabolic channeling is the process whereby metabolic intermediates are channelled between the active sites of consecutive enzymes in a biochemical pathway. This serves to increase reaction efficiency by limiting the diffusion of metabolites into the surrounded cellular intracellular environment. Channelling provides a biochemical system with benefits such as:</p><br />
<ul><br />
<li> Enhancing unfavourable reactions</li><br />
<li> Sequestering toxic intermediates </li><br />
<li> Protecting unstable intermediates</li><br />
<li> Preventing inhibition of other enzymes by the channelling intermediate</li><br />
</ul><br />
<p>Channeling has been utilized in bioengineering to enhance the efficiency of desired biochemical systems. Some methods which have been utilized to mimic examples of metabolic channelling in nature are through:</p><br />
<ul><br />
<li> Immobilized Enzymes</li><br />
<li> Fusion Proteins </li><br />
<li> Scaffolds </li><br />
</ul><br />
<br />
<br/><br/><br />
<br />
[[Image:immobilized enzymes 2.png|border=|Immobilized Enzymes]] [[Image:Fusion Protein 3.png|border|Fusion Protein]] [[Image:Scaffolding 4.png|border|Scaffolding]]<br />
<pre>Immobilized Enzymes Fusion Protein Scaffolding</pre><br />
<br />
<br />
<h3>'''The Project'''</h3><br />
<br/><br />
<p><br />
<br />
Our project aims to take advantage of the catechol degradation pathway present in P. Putida by introducing metabolic channeling to increase the efficiency with which catechol is degraded. Besides being toxic in it's own right, we anticipate that accelerating the degradation of catechol, a common product of polyaromatic hydrocarbon (PAH) degradation, will result in increased PAH degradation. This hypothesis, supported by computer modeling will be tested in an ''E.coli'' model using the synthetic construction of several variations of the pathway involving the channeling of different pairs of enzymes by protein fusion.</p><br />
<br />
<br />
<h4>'''Future Goals'''</h4><br />
<br />
<p>Once we complete the characterization of the “Encapsulator” micro-compartment from the 2009 iGEM project, we will evaluate whether compartmentalization can further enhance the effects of metabolic channeling as compared to gene fusions. Ultimately, channeling will be introduced to target pathways in endogenous tailings ponds species (e.g. P. Putida) for application in the bioremediation of the tailings ponds.</p></div>Gcromarhttp://2010.igem.org/Team:Toronto/PartsTeam:Toronto/Parts2010-10-26T03:05:39Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Parts Submitted to the Registry'''</h1><br />
<br />
<br />
===Parts===<br />
<br />
<groupparts>iGEM010 Toronto</groupparts></div>Gcromarhttp://2010.igem.org/Team:Toronto/ProtocolsTeam:Toronto/Protocols2010-10-26T03:02:15Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
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!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Protocols'''</h1><br />
<br />
<h2>'''Tolerance Assay'''</h2><br />
<br />
A 0.5 M (10x) stock solution of catechol was made in a 5 ml volume of autoclaved, distilled water. This solution was added to 25 ml of sterile MM2 media to make up a volume of 30 ml. This was added to 20 ml of MM2 innoculated with E.coli DH5a (approximate density 1x10e8 cells per ml) for a final volume of 50 ml (catechol concentration 50mM). Control samples were created as above minus catechol. Treated and untreated samples were placed in a 37C shaker incubator and aliquots were removed every two hours. 0.5 ml aliquots were serially diluted in 4.5 ml of MM2 media and four dilutions representing 1 x 10<sup>-3</sup>, 1 x 10<sup>-4</sup>, 1 x 10<sup>-5</sup> and 1x10<sup>-6</sup> were plated by spreading 200 ul of the diluted sample on LB agar plates. After drying, plates were incubated in an inverted position in a 37C incubator overnight. Plates with between 30 and 300 colonies were counted to determine the number of colony forming units using the method described by Imperial College (iGEM 2009 OpenWetWare).</div>Gcromarhttp://2010.igem.org/Team:Toronto/ResultsTeam:Toronto/Results2010-10-26T02:59:04Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
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!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Results'''</h1><br />
<br />
<h2>'''''E. coli'' DH5a is sensitive to Catechol exposure'''</h2><br />
<br />
To measure the effect of our pathway manipulations on the viability of ''E.coli'' exposed to catechol we plan to perform a tolerance test. In preliminary experiments we established the baseline sensitivity of ''E.coli'' (DH5a) exposed to 50mM catechol in liquid media over a six hour time course. Aliquots of this mixture were plated on LB media at various dilutions and colonies were counted and compared with untreated controls (see "Protocols"). There was no measurable difference between control and treated cells when bacteria were exposed to catechol in a growth media (data not shown). However, in minimal media (MM2) growth of control cells was moderate while exposed cells exhibited a marked decrease in survival (Figure 1). Although the effects of Catechol exposure in ''E.coli'' and ''P. putida'' have previously been investigated [Park et al. 2001] completion of these baseline experiments will enable us to determine the effect of pathway manipulations on the proposed engineered ''E.coli'' strains containing various configurations of the pathway.<br />
<br />
<p><br />
A<br />
[[Image:Toronto-LB-Plate_1.jpg|400px]] [[Image:Toronto-LB-Plate_2.jpg|400px]]<br />
</p><br />
<p><br />
B[[Image:Toronto-results-catechol_tolerance.png]]<br />
</p><br />
'''Figure 1: A. Growth of ''E.coli'' DH5a on LB plates (1x10<sup>-5</sup> dilution) following exposure to minimal media and minimal media with 50mM catechol in solution. Plates shown are for the last time points plotted. B. Survival curves of ''E. coli'' DH5a exposed to catechol over a six hour time course.'''<br />
<br />
<H2>'''Synthetic construction of a functioning catechol ortho degradation pathway in E.coli is predicted to increase cell growth rate'''</H2><br />
Justification and summary of flux balance results which are described in more detail in the modeling section but highlighted here. The important point to make is that this is good news because, if true, it means that acceleration of the native pathway in the target organism P. putida would likely result in bacteria capable of outcompeting their normal counterparts, reducing the need to re-apply.<br />
<br />
<h2>'''Colocalization of muconolactone D-isomerase (EC:5.3.3.4) and 3-oxoadipate enol-lactone hydrolase (EC:3.1.1.24) are predicted to lead to increased catechol degradation through metabolic channeling'''</h2><br />
Kris, you need a blurb here on this early Cell++ result.<br />
<br />
<H2>'''Recombinantly expressed microcompartments assemble ''in vivo'''''</H2><br />
Kris your EM goes here with the appropriate description.</div>Gcromarhttp://2010.igem.org/Team:Toronto/ResultsTeam:Toronto/Results2010-10-26T02:54:03Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
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!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Results'''</h1><br />
<br />
<h2>'''''E. coli'' DH5a is sensitive to Catechol exposure'''</h2><br />
<br />
To measure the effect of our pathway manipulations on the viability of ''E.coli'' exposed to catechol we plan to perform a tolerance test. In preliminary experiments we established the baseline sensitivity of ''E.coli'' (DH5a) exposed to 50mM catechol in liquid media over a six hour time course. Aliquots of this mixture were plated on LB media at various dilutions and colonies were counted and compared with untreated controls (see "Protocols"). There was no measurable difference between control and treated cells when bacteria were exposed to catechol in a growth media (data not shown). However, in minimal media (MM2) growth of control cells was moderate while exposed cells exhibited a marked decrease in survival (Figure 1). Although the effects of Catechol exposure in ''E.coli'' and ''P. putida'' have previously been investigated [Park et al. 2001] completion of these baseline experiments will enable us to determine the effect of pathway manipulations on the proposed engineered ''E.coli'' strains containing various configurations of the pathway.<br />
<br />
<p><br />
A<br />
[[Image:Toronto-LB-Plate_1.jpg|400px]] [[Image:Toronto-LB-Plate_2.jpg|400px]]<br />
</p><br />
<p><br />
B[[Image:Toronto-results-catechol_tolerance.png]]<br />
</p><br />
'''Figure 1: A. Growth of ''E.coli'' DH5a on LB plates (1x10<sup>-5</sup> dilution) following exposure to minimal media and minimal media with 50mM catechol in solution. Plates shown are for the last time points plotted. B. Survival curves of ''E. coli'' DH5a exposed to catechol over a six hour time course.'''<br />
<br />
<h2>'''Colocalization of enzymes blat and blat are predicted to lead to increased catechol degradation through metabolic channeling'''</h2><br />
Kris, you need a blurb here on this early Cell++ result.<br />
<br />
<H2>'''Synthetic construction of a functioning catechol ortho degradation pathway in E.coli is predicted to increase cell growth rate'''</H2><br />
Justification and summary of flux balance results which are described in more detail in the modeling section but highlighted here. The important point to make is that this is good news because, if true, it means that acceleration of the native pathway in the target organism P. putida would likely result in bacteria capable of outcompeting their normal counterparts, reducing the need to re-apply.<br />
<br />
<H2>'''Recombinantly expressed microcompartments assemble ''in vivo'''''</H2><br />
Kris your EM goes here with the appropriate description.</div>Gcromarhttp://2010.igem.org/Team:Toronto/ResultsTeam:Toronto/Results2010-10-26T02:50:52Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
{|style="color:black;background-color:#FFFFFF;" cellpadding="2" cellspacing="0" border="1" width="100%" align="center"<br />
!align="center"|[[Team:Toronto|Home]]<br />
!align="center"|[[Team:Toronto/Project|Project]]<br />
!align="center"|[[Team:Toronto/Design|Design]]<br />
!align="center"|[[Team:Toronto/Protocols|Protocols]]<br />
!align="center"|[[Team:Toronto/Notebook|Notebook]]<br />
!align="center"|[[Team:Toronto/Results|Results]]<br />
!align="center"|[[Team:Toronto/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Toronto/Modeling|Modeling]]<br />
!align="center"|[[Team:Toronto/Software|Software Used]]<br />
!align="center"|[[Team:Toronto/Human Practices|Human Practices]]<br />
!align="center"|[[Team:Toronto/Safety|Safety]]<br />
!align="center"|[[Team:Toronto/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Toronto Official Team Profile]<br />
!align="center"|[[Team:Toronto/FAQ|FAQ]]<br />
!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Results'''</h1><br />
<br />
<h2>'''''E. coli'' DH5a is sensitive to Catechol exposure'''</h2><br />
<br />
To measure the effect of our pathway manipulations on the viability of ''E.coli'' exposed to catechol we plan to perform a tolerance test. In preliminary experiments we established the baseline sensitivity of ''E.coli'' (DH5a) exposed to 50mM catechol in liquid media over a six hour time course. Aliquots of this mixture were plated on LB media at various dilutions and colonies were counted and compared with untreated controls (see "Protocols"). There was no measurable difference between control and treated cells when bacteria were exposed to catechol in a growth media (data not shown). However, in minimal media (MM2) growth of control cells was moderate while exposed cells exhibited a marked decrease in survival (Figure 1). Although the effects of Catechol exposure in ''E.coli'' and ''P. putida'' have previously been investigated [Park et al. 2001] completion of these baseline experiments will enable us to determine the effect of pathway manipulations on the proposed engineered ''E.coli'' strains containing various configurations of the pathway.<br />
<br />
<p><br />
A<br />
[[Image:Toronto-LB-Plate_1.jpg|400px]] [[Image:Toronto-LB-Plate_2.jpg|400px]]<br />
</p><br />
<p><br />
B[[Image:Toronto-results-catechol_tolerance.png]]<br />
</p><br />
'''Figure 1: A. Growth of ''E.coli'' DH5a on LB plates (1x10<sup>-5</sup> dilution) following exposure to minimal media and minimal media with 50mM catechol in solution. Plates shown are for the last time points plotted. B. Survival curves of ''E. coli'' DH5a exposed to catechol over a six hour time course.'''<br />
<br />
<h2>'''Colocalization of enzymes blat and blat are predicted to lead to increased catechol degradation through metabolic channeling'''</h2><br />
Kris, you need a blurb here on this early Cell++ result.<br />
<br />
<H2>'''Synthetic construction of functioning blat pathway in E.coli is predicted to increase cell growth rate'''</H2><br />
Justification and summary of flux balance results which are described in more detail in the modeling section but highlighted here. The important point to make is that this is good news because, if true, it means that acceleration of the native pathway in the target organism P. putida would likely result in bacteria capable of outcompeting their normal counterparts, reducing the need to re-apply.<br />
<br />
<H2>'''Recombinantly expressed microcompartments assemble ''in vivo'''''</H2><br />
Kris your EM goes here with the appropriate description.</div>Gcromarhttp://2010.igem.org/Team:Toronto/ResultsTeam:Toronto/Results2010-10-26T02:34:09Z<p>Gcromar: </p>
<hr />
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|}<br />
<br />
<br />
<h1>'''Results'''</h1><br />
<h2>'''E. coli DH5a is sensitive to Catechol exposure'''</h2><br />
<br />
To measure the effect of our pathway manipulations on the viability of ''E.coli'' exposed to catechol we plan to perform a tolerance test. In preliminary experiments we established the baseline sensitivity of ''E.coli'' (DH5a) exposed to 50mM catechol in liquid media over a six hour time course. Aliquots of this mixture were plated on LB media at various dilutions and colonies were counted and compared with untreated controls (see "Protocols"). There was no measurable difference between control and treated cells when bacteria were exposed to catechol in a growth media (data not shown). However, in minimal media (MM2) growth of control cells was moderate while exposed cells exhibited a marked decrease in survival (Figure 1). Although the effects of Catechol exposure in ''E.coli'' and ''P. putida'' have previously been investigated [Park et al. 2001] completion of these baseline experiments will enable us to determine the effect of pathway manipulations on the proposed engineered ''E.coli'' strains containing various configurations of the pathway.<br />
<br />
<p><br />
A<br />
[[Image:Toronto-LB-Plate_1.jpg|400px]] [[Image:Toronto-LB-Plate_2.jpg|400px]]<br />
</p><br />
<p><br />
B[[Image:Toronto-results-catechol_tolerance.png]]<br />
</p><br />
'''Figure 1: A. Growth of ''E.coli'' DH5a on LB plates (1x10<sup>-5</sup> dilution) following exposure to minimal media and minimal media with 50mM catechol in solution. Plates shown are for the last time points plotted. B. Survival curves of ''E. coli'' DH5a exposed to catechol over a six hour time course.'''<br />
<br />
'''Recombinantly expressed microcompartments assemble ''in vivo'''''<br />
Kris your EM goes here with the appropriate description</div>Gcromarhttp://2010.igem.org/Team:Toronto/ResultsTeam:Toronto/Results2010-10-26T02:29:31Z<p>Gcromar: </p>
<hr />
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|}<br />
<br />
<br />
<h1>'''Results'''</h1><br />
<h2>'''E. coli DH5a is sensitive to Catechol exposure'''</h2><br />
<br />
To measure the effect of our pathway manipulations on the viability of ''E.coli'' exposed to catechol we plan to perform a tolerance test. In preliminary experiments we established the baseline sensitivity of ''E.coli'' (DH5a) exposed to 50mM catechol in liquid media over a six hour time course. Aliquots of this mixture were plated on LB media at various dilutions and colonies were counted and compared with untreated controls (see "Protocols"). There was no measurable difference between control and treated cells when bacteria were exposed to catechol in a growth media (data not shown). However, in minimal media (MM2) growth of control cells was moderate while exposed cells exhibited a marked decrease in survival (Figure 1). Although the effects of Catechol exposure in ''E.coli'' and ''P. putida'' have previously been investigated [Park et al. 2001] completion of these baseline experiments will enable us to determine the effect of pathway manipulations on the proposed engineered ''E.coli'' strains containing various configurations of the pathway.<br />
<br />
<p><br />
A<br />
[[Image:Toronto-LB-Plate_1.jpg|400px]] [[Image:Toronto-LB-Plate_2.jpg|400px]]<br />
</p><br />
<p><br />
B[[Image:Toronto-results-catechol_tolerance.png]]<br />
</p><br />
'''Figure 1: A. Growth of ''E.coli'' DH5a on LB plates (1x10<sup>-5</sup> dilution) following exposure to minimal media and minimal media with 50mM catechol in solution. Plates shown are for the last time points plotted. B. Survival curves of ''E. coli'' DH5a exposed to catechol over a six hour time course.'''</div>Gcromarhttp://2010.igem.org/Team:Toronto/ResultsTeam:Toronto/Results2010-10-26T02:24:37Z<p>Gcromar: </p>
<hr />
<div>[[Image:Toronto_logo.png]]<br />
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!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Results'''</h1><br />
<h2>'''E. coli DH5a is sensitive to Catechol exposure'''</h2><br />
<br />
To measure the effect of our pathway manipulations on the viability of E.coli exposed to catechol we plan to perform a tolerance test. In preliminary experiments we established the baseline sensitivity of E.coli (DH5a) exposed to 50mM catechol in liquid media over a six hour time course. Aliquots of this mixture were plated on LB media at various dilutions and colonies were counted and compared with untreated controls (see "Protocols"). There was no measurable difference between control and treated cells when bacteria were exposed to catechol in a growth media (data not shown). However, in minimal media (MM2) growth of control cells was moderate while exposed cells exhibited a marked decrease in survival (Figure 1). <br />
<p><br />
A<br />
[[Image:Toronto-LB-Plate_1.jpg|400px]] [[Image:Toronto-LB-Plate_2.jpg|400px]]<br />
</p><br />
<p><br />
B[[Image:Toronto-results-catechol_tolerance.png]]<br />
</p><br />
'''Figure 1: A. Growth of E.coli DH5a on LB plates (1x10e-5 dilution) following exposure to minimal media and minimal media with 50mM catechol in solution. Plates shown are for the last time points plotted. B. Survival curves of E. coli DH5a exposed to catechol over a six hour time course.'''<br />
<br />
Although the effects of Catechol exposure in E.coli and P. putida have previously been investigated [Park et al. 2001] completion of these baseline experiments will enable us to determine the effect of pathway manipulations on the proposed engineered E.coli strains containing various configurations of the pathway.</div>Gcromarhttp://2010.igem.org/File:Toronto-LB-Plate_1.jpgFile:Toronto-LB-Plate 1.jpg2010-10-26T02:01:37Z<p>Gcromar: </p>
<hr />
<div></div>Gcromarhttp://2010.igem.org/File:Toronto-LB-Plate_2.jpgFile:Toronto-LB-Plate 2.jpg2010-10-26T02:00:27Z<p>Gcromar: </p>
<hr />
<div></div>Gcromarhttp://2010.igem.org/Team:Toronto/ResultsTeam:Toronto/Results2010-10-26T01:59:11Z<p>Gcromar: </p>
<hr />
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!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Results'''</h1><br />
<h2>'''E. coli DH5a is sensitive to Catechol exposure'''</h2><br />
<br />
To measure the effect of our pathway manipulations on the viability of E.coli exposed to catechol we plan to perform a tolerance test. In preliminary experiments we established the baseline sensitivity of E.coli (DH5a) exposed to 50mM catechol in liquid media over a six hour time course. Aliquots of this mixture were plated on LB media at various dilutions and colonies were counted and compared with untreated controls (Figure 1; see "Protocols").<br />
<p><br />
[[Image:Toronto-LB-Plate_1.jpg]] [[Image:Toronto-LB-Plate_2.jpg]]<br />
</p><br />
There was no measurable difference between control and treated cells when bacteria were exposed to catechol in a growth media (LB). However, in minimal media (MM2) growth of control cells was moderate while exposed cells exhibited a marked decrease in survival (Figure 2). <br />
<br />
[[Image:Toronto-results-catechol_tolerance.png]]<br />
<br />
<br />
Although the effects of Catechol exposure in E.coli and P. putida have previously been investigated, completion of these baseline experiments will enable us to determine the effect of pathway manipulations on the proposed engineered E.coli strains.</div>Gcromarhttp://2010.igem.org/Team:Toronto/ProjectTeam:Toronto/Project2010-10-26T01:57:19Z<p>Gcromar: </p>
<hr />
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|}<br />
<br />
<br />
<h1>'''Project Description'''</h1><hr/><br />
<br />
<h2> '''Background'''</h2><br />
<br />
<h3>'''Oil Sands'''</h3><br />
<br />
<p> Oil sands are naturally occurring mixtures of sand or clay, water and an extremely dense and viscous form of petroleum called bitumen. [[Image:CompositionofOilSand40.jpg|right]]Bitumen is a collection of heavy hydrocarbons, which results from preferential microbial degradation of lighter hydrocarbons in oil reserves over geological time scales. These heavy oil reserves are some of the largest fossil fuel stores in the world with more stored energy than traditional oil reserves.</p><br />
<p>Oil sands represent approximately 67% of the world’s total petroleum resource, with the two largest deposits being in Canada and Venezuela. Together they contain approximately 3.6 trillion barrels of oil, compared to 1.75 trillion barrels of conventional oil worldwide. The oil sands in the Athabasca Basin in northeastern Alberta, Canada represent the world’s largest resource estimated to contain over 1.7 trillion barrels of bitumen.</p><br />
<br />
<br />
<br />
<h3>'''Tailings Ponds'''</h3><br />
<p>Oil sands are developed either through open-pit mining or deep underground (in situ) production. In open-pit mining, hot water is used to separate bitumen from the mixture of sand and clay.</p><br />
<p>Each cubic meter of mined oil sands requires up to 3 m3 of water and produces about 4 m3 of slurry wastes containing sand, clay, water, unrecovered bitumen and dissolved inorganic and organic compounds. This mixture is then sent to a discontinued mine pit, referred to as a tailings pond. It is a large man-made collection of waste, which has a negative impact on the environment.</p> <br />
<p>Over 70% of the water demand is met by recycling the recovered water. Even so, these wastewaters are accumulating at a rate of about 0.1-0.2 m3 per ton of oil sands processed. Recycling of the water back to the extraction process further concentrates contaminants as more waste material is added to the already contaminated water. It has been estimated that over 1 billion m3 of tailings pond water will be generated by the year 2025.</p><br />
<p>They are composed of a wide variety of chemical toxins. Amongst these are polycyclic aromatic hydrocarbons (PAHs), with Naphthalenes, Anthracenes and Fluorenes present at the highest concentrations. These compounds are toxic to the surrounding aquatic ecosystems, and have been shown to have an adverse effect on human health. PAHs are carcinogenic and are also known to have a negative impact on immune function, kidney function, liver function, reproduction and development.</p><br />
<br />
<br />
<br />
<h3>'''Bioremediation'''</h3><br />
<br />
<p>Bioremediation is the use of microorganisms to return the natural environment altered by contaminants to its original condition. It has been proposed as a possible method for the reclamation of land and water impacted by oil sands development. Although natural examples of these processes exist, they tend to be characterized by slow reaction rates and/or undesirable side reactions.</p><br />
<p>Over expression of rate limiting enzymes has enjoyed moderate success in overcoming some pathway limitations. However, because enzymes operate within complex, highly organized systems, there is increasing recognition within the metabolic engineering community that spatial organization offers an additional route for optimizing pathway efficiency.<br />
</p><br />
<br />
<br />
<br />
<h3>'''Catechol'''</h3><br />
<br />
<p>Pseudomonas putida, a microbe naturally present in tailings ponds, has the metabolic machinery needed to metabolize many toxins within the tailings ponds. This includes the capability to degrade certain PAHs in tailings ponds such as naphthalenes, anthracenes and fluorenes. These three major PAHs are degraded to form a common compound called catechol. However, this process is slow and inefficient. Since catechol is the common breakdown product in the breakdown of these PAHs, enhancing the breakdown of catechol may lead to an increase in the rate of PAH degradation. This project will look to evaluate how we can increase the degradation rate of catechol in order to achieve faster PAH breakdown. </p><br />
<br />
<p>The metabolic network present in P. putida for degrading catechol is highlighted by the black box in Figure 1. (http://www.genome.jp/kegg-bin/show_pathway?ko00362+C00090). </p><br />
<br />
<br />
<br />
[[Image:putida!!.png|center]]<br />
<br />
<br />
<blockquote>Figure 1. The degradation of catechol to common cellular metabolites, acetyl-CoA and Succinyl-CoA , involves five enzyme catalyzed reactions in Pseudomonas putida.</blockquote><br />
<br />
<br />
<h3>'''Metabolic Channeling'''</h3><br />
<p>Metabolic channeling is the process whereby metabolic intermediates are channelled between the active sites of consecutive enzymes in a biochemical pathway. This serves to increase reaction efficiency by limiting the diffusion of metabolites into the surrounded cellular intracellular environment. Channelling provides a biochemical system with benefits such as:</p><br />
<ul><br />
<li> Enhancing unfavourable reactions</li><br />
<li> Sequestering toxic intermediates </li><br />
<li> Protecting unstable intermediates</li><br />
<li> Preventing inhibition of other enzymes by the channelling intermediate</li><br />
</ul><br />
<p>Channeling has been utilized in bioengineering to enhance the efficiency of desired biochemical systems. Some methods which have been utilized to mimic examples of metabolic channelling in nature are through:</p><br />
<ul><br />
<li> Immobilized Enzymes</li><br />
<li> Fusion Proteins </li><br />
<li> Scaffolds </li><br />
</ul><br />
<br />
<br/><br/><br />
<br />
[[Image:immobilized enzymes 2.png|border=|Immobilized Enzymes]] [[Image:Fusion Protein 3.png|border|Fusion Protein]] [[Image:Scaffolding 4.png|border|Scaffolding]]<br />
<pre>Immobilized Enzymes Fusion Protein Scaffolding</pre><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<h3>'''The Project'''</h3><br />
<br/><br />
<p><br />
<br />
This current project aims to take advantage of the catechol degradation pathway present in P. Putida by introducing channelling to increase the efficiency with which catechol is degraded. We anticipate that degrading catechol, an intermediate formed in polyaromatic hydrocarbon (PAH) degradation, will result in an imbalance in cellular catechol concentrations. In turn, we believe there will be increased PAH degradation in order to restore the original cellular catechol concentrations.</p><br />
<br />
<p>In order to identify enzymes in the catechol degradation pathway that benefit from the introduction of metabolic channelling, we performed in silico analyses with software developed in our lab (Sanford et al.). Preliminary results from our simulations suggests that the 3rd (muconolactone D-isomerase) and 4th (3-oxodipate-enol lactone hydrolase) enzymes in the pathway would benefit most from channelling (Figure 2). Muconolactone D-isomerase catalyzes an energetically unfavourable reaction, while 3-oxodipate-enol lactone hydrolase catalyzes a very energetically favourable reaction. By introducing channeling between these two enzymes, the energetically unfavourable product of the mucanolactone D-isomerase-catalyzed reaction would be converted to the product of 3-oxodipate-enol lactone hydrolase before mucanolactone D-isomerase converts the energetically unfavourable product back into the energetically favourable substrate. The net effect would be an increased flux through the catechol degradation pathway. </p><br />
<br />
<br />
<br />
[[Image:Catechol degradation 5.png|center]]<br />
<br />
<blockquote> Figure 2: Ortho degradation of catechol (1,2-Benzenediol) proceeds in six steps. Only the last enzyme is present in E. coli. Enzyme candidates with the largest predicted channeling effect as predicted by Cell++ are boxed in red.</blockquote><br />
<br />
<br/><br />
<p>The five enzymes in the catechol degradation pathway not naturally present in E. coli will be introduced into E. coli as BioBrick parts. The parts will then be used to construct five recombinant strains of E. coli (Figure 3). These include a full reconstruction of the pathway (control strain) and several partial reconstructions (test strains). The latter will contain pathway 'holes' corresponding to all combinations of sequential pairs of enzymes, with complementary fusion pairs. The test strain with the fusion of enzymes 3 and 4 will be of prime interest, while the other test strains will be used validate the predictions made by Cell++. </p><br />
<br />
<br />
<br />
<br />
[[Image:design 6.png|center]]<br />
<blockquote>Figure 3: Diagramatic representation of proposed gene fusion experiments. Pathway reconstructions are shown in black for the control and test strains with pathway holes for the partial reconstructions shown in red. Yellow stars mark proposed gene fusions to be tested to validate computational predictions. The blue circle marks the best candidate for enzyme channeling based on in silico experiments </blockquote><br />
<br/><br />
<p>In order to demonstrate the breakdown of catechol by these recombinant strains, catechol will be added into the growth media of these strains. A colorimetric assay, previously developed to quantify the amount of catechol in solution, will be utilized to assay the rate of catechol degradation by the different strains (Tao et al.). In addition, a survival assay will be used to determine the threshold for catechol exposure for non recombinant E. coli and to evaluate the effect that the recombinantly-expressed enzymes in the catechol pathway will have on E. coli survival. </p><br />
<br />
<br />
<br />
<h4>'''Future Goals'''</h4><br />
<br />
<p>Once we complete the characterization of the “Encapsulator” micro-compartment from the 2009 iGEM project, we will evaluate whether compartmentalization can further enhance the effects of metabolic channeling as compared to gene fusions. Ultimately, channeling will be introduced to target pathways in endogenous tailings ponds species (e.g. P. Putida) for application in the bioremediation of the tailings ponds.</p></div>Gcromarhttp://2010.igem.org/Team:Toronto/ResultsTeam:Toronto/Results2010-10-25T22:05:45Z<p>Gcromar: </p>
<hr />
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!align="center"|[[Team:Toronto/Acknowledgments|Acknowledgments]]<br />
|}<br />
<br />
<br />
<h1>'''Results'''</h1><br />
<h2><'''E. coli DH5a is sensitive to Catechol exposure'''</h2><br />
<br />
To measure the effect of our pathway manipulations on the viability of E.coli exposed to catechol we plan to perform a tolerance test. In preliminary experiments we established the baseline sensitivity of E.coli (DH5a) exposed to 50mM catechol in liquid media over a six hour time course. Aliquots of this mixture were plated on LB media at various dilutions and colonies were counted and compared with untreated controls (Figure 1; see "Protocols").<br />
<p><br />
[[Image:Toronto-LB-Plate_1.jpg]] [[Image:Toronto-LB-Plate_2.jpg]]<br />
</p><br />
There was no measurable difference between control and treated cells when bacteria were exposed to catechol in a growth media (LB). However, in minimal media (MM2) growth of control cells was moderate while exposed cells exhibited a marked decrease in survival (Figure 2). <br />
<br />
[[Image:Toronto-results-catechol_tolerance.png]]<br />
<br />
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Although the effects of Catechol exposure in E.coli and P. putida have previously been investigated, completion of these baseline experiments will enable us to determine the effect of pathway manipulations on the proposed engineered E.coli strains.</div>Gcromarhttp://2010.igem.org/Team:Toronto/ResultsTeam:Toronto/Results2010-10-25T22:04:34Z<p>Gcromar: </p>
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<h1>'''Results'''</h1><br />
<h2><'''E. coli DH5a is sensitive to Catechol exposure'''/h2><br />
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To measure the effect of our pathway manipulations on the viability of E.coli exposed to catechol we plan to perform a tolerance test. In preliminary experiments we established the baseline sensitivity of E.coli (DH5a) exposed to 50mM catechol in liquid media over a six hour time course. Aliquots of this mixture were plated on LB media at various dilutions and colonies were counted and compared with untreated controls (Figure 1; see "Protocols").<br />
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[[Image:Toronto-LB-Plate_1.jpg]] [[Image:Toronto-LB-Plate_2.jpg]]<br />
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There was no measurable difference between control and treated cells when bacteria were exposed to catechol in a growth media (LB). However, in minimal media (MM2) growth of control cells was moderate while exposed cells exhibited a marked decrease in survival (Figure 2). <br />
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[[Image:Toronto-results-catechol_tolerance.png]]<br />
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Although the effects of Catechol exposure in E.coli and P. putida have previously been investigated, completion of these baseline experiments will enable us to determine the effect of pathway manipulations on the proposed engineered E.coli strains.</div>Gcromar