http://2009.igem.org/wiki/index.php?title=Special:Contributions/Prmagomes&feed=atom&limit=50&target=Prmagomes&year=&month=2009.igem.org - User contributions [en]2024-03-28T20:16:34ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:UCSF/PartsTeam:UCSF/Parts2009-10-22T02:47:03Z<p>Prmagomes: </p>
<hr />
<div>The Parts that were submitted to the registry can be found here - http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=UCSF<br />
<br />
<br />
{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/PartsTeam:UCSF/Parts2009-10-22T02:46:31Z<p>Prmagomes: Replacing page with 'The Parts that were submitted to the registry can be found here - http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=UCSF'</p>
<hr />
<div>The Parts that were submitted to the registry can be found here - http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=UCSF</div>Prmagomeshttp://2009.igem.org/Team:UCSF/Human_PracticesTeam:UCSF/Human Practices2009-10-22T01:16:04Z<p>Prmagomes: </p>
<hr />
<div>=='''Abstract'''==<br />
<br />
Biotechnology is exponentially growing in popularity over the years due to the great potential of new scientific inventions like alternative biofuels and various lifesaving vaccines. Biotechnology companies are emerging all over the California coast especially the Bay Area. Surprisingly, within this field of science, another source is bustling for attention: High Schools. At Abraham Lincoln High School, students learn about biotech and its real-world applications in an exclusive two-year advanced biotechnology class. The credentials needed to teach a biotechnology course in high school has raised questions as the work is somewhat similar to that of DYI Bio. The issue is that both DYI Bio and high school biotechnology courses can be potentially dangerous as well as hazardous to the environment if an outbreak were to occur. The safer alternative would be to work in a lab where the qualifications must be met and guidelines must be followed. But back in the classroom, what are the teacher’s qualifications? What preparations must be done to ensure safety of the students? There is no definite answer for these questions because biotechnology is a brand new subject in schools and has yet to be regulated. But that doesn’t mean there is no attention brought to it; teachers and members of the BABEC often meet to answer these questions and create a safe and ethical high school curriculum for the most beneficial learning experience.<br />
<br />
=='''Section 1 – Biotech classes in High School'''==<br />
<br />
The world of biotechnology has never been as big as it is now. This fairly new field has already been responsible for the birth of biofuels and many vital vaccines used today. In fact, many believe that biotechnology has the potential to solve many of our everyday challenges. With the promises it is expected to bring, biotech has already started to appear in high schools across the world. At San Francisco’s Abraham Lincoln High School, the hype for this new course always brings about long waiting lists. Students feel that pursuing biotechnology or biotech will result in an influx of different job opportunities. More importantly, having biotech in high schools allow students to gain the baseline knowledge required to solve everyday problems, and learning the material early on allows students to become future leaders in the biotechnology industry.<br />
<br />
Abraham Lincoln High School offers five first-year biotechnology classes and one second-year biotechnology class. Each class is able to hold around thirty students. In first-year biotech, students are taught the fundamentals of using scientific instruments like micropipettes. The hands-on course teaches the very same techniques that are used in today’s industry and academic labs. After a few weeks of detailed lectures, students are directed to their lab bench, and learn how to make plasmids, recombinant DNA, and more. These experiments are not as simple as following protocols, but actually encourage the students to think outside the box. By the end of the first year, students will have the ability to do transformations, work with enzymes and run gel electrophoresis.<br />
<br />
Second year biotech expands on the knowledge taught in first-year. Because of limited resources, thirty students are chosen from the first-year pool to continue on with the studies. This second course is much more lab intensive. Whereas the majority of first year biotechnology was spent on theory and basic lab skills, second year biotechnology teaches students the applications of first year biotechnology. Intense lectures accompanied by demos are followed by the labs themselves. The ability to perform each experiment is thoroughly tested by the teacher after every lesson. At the end of the year, students will be able to perform techniques such as the Bradford assay, western blotting, and protein purifications. Looking back, the skills that we acquired through second-year biotech played a tremendous role at our time with the iGEM team. <br />
Skills like protein extraction and ELISA are not ones that can be taught by a under qualified teacher. San Francisco has three high schools that offer biotechnology as a class, and Abraham Lincoln High School is fortunate to be one of them. San Mateo High School, located in the Bay Area, is the first to have a three-year program; they even have their own greenhouse! Few years prior, biotech courses were practically non-existent. With the popularity of biotech quickly entering the high school doors, more inexperienced teachers are thinking about teaching biotech. The interest of more schools having courses like biotech also brings about concerns. Although the benefits are obvious, the effect of having inexperienced teachers educating a class of students could prove to be risky.<br />
<br />
=='''Section 2 – Biotech classes in HS Versus DIY BIO'''==<br />
<br />
A teacher interested in teaching biotechnology must also have sufficient knowledge in the field of molecular biology. Most of the powerful tools explained in biotech courses expand from the foundations of molecular biology. This complex subject takes years to master even at the graduate level. Currently, the qualifications for new teachers wanting to teach a biotech course are not specifically designed for the course. Instead, the standards for teaching an advance class like biotech are much like any other science class. This brings up the concern that upcoming teachers are not only undereducated in the content they are teaching, but will also avoid the importance of bioethics. While these lessons might sound repetitive, potential dangers can occur if the educator does not stress the importance of it. <br />
<br />
The knowledge given at current high school courses is similar to the work of DIY (Do-It-Yourself) Bio. Although biotech courses are not meant to be dangerous, the basics taught is enough to create a potential hazard. In today’s world, the dangers of hidden labs in the garages of people’s homes are enough to alarm the FBI and CIA. Online forums are already available for those who want to do biology at home. Some would doubt the possibility of doing dangerous labs because of all the equipment required. But surprisingly, some of these forums also include instructions on how to make your own equipment. With all these resources easily available to the average person, the FBI is fully aware of the possibility of biohackers lurking in everyday homes. The concerns with DIY bio is that “organisms in the hands of amateurs could escape and cause outbreaks of incurable diseases or unpredictable environmental damage(Jim Thomas of ETC group).” Could the dangers of biosecurity also apply to the work being done in high schools? Questions like these are slowly coming up now that the field of biotechnology has expanded. Yes, the work being done at the high school level could be considered DIY bio, but they also have standards set by inspectors. However, these standards are still very brief, and with the possibility of inexperienced and under-qualified teachers quickly emerging, these standards deserve to be looked at once again.<br />
<br />
How is the work being done at research labs any different from the ones done at high schools or in public homes? At the high level of science labs at hospitals, universities, and medical schools, the standards are developed by the National Institute of Health (NIH). These labs usually carry a lot of sophisticated equipment, and strict guidelines are put in place to ensure the safety of the employees. NIH has set guidelines from no eating in the lab to allowing only certified storing cabinets to be used. NIH feels that lab conditions must be kept in a consistent matter. Any violations to the guidelines could result in hefty fines and/or the closure of the lab. Even as temporary employees, each iGEM member had to go through a safety certification procedure before we were allowed to do any lab work. The online program consisted of reading about the disposal of dangerous chemicals, emergency procedures, and ‘the rules’ of working in a lab; each reading was wrapped up with a quiz. Each member had to successfully pass the quiz or else any related lab work was prohibited. At the classroom level, it is important for the educator to have some sort of freedom in teaching the class. George Cachianes (biotech teacher) of Abraham Lincoln High School admit that the lack of strict guidelines is one of the best things about being a teacher. Having written his own curriculum, Cachianes ensures that his lessons and labs are safe in a high school environment. One of the top priorities while making his curriculum is the safety of the students. Not having to worry about standards allows him to focus more on a certain topic, rather than being time pressured to move onto another lesson. As a former employee of the biotech giant Genentech, and member of the Bay Area Biotechnology Education Consortium(BABEC), Cachianes admits that not everyone is qualified to pursue a teaching career in science, “But if you haven’t had college and graduate school, you’re not trained to control that kind of environment. My concern is when a teacher only has a bachelor’s degree, they may know how to do it, but they don’t understand how to.” <br />
<br />
=='''Section 3 – The Future of Teachings of Biotech'''==<br />
<br />
This brings about the important questions of how to regulate teaching biotech in schools. What should the school requirements be? The safety of the facilities, faculty, and students must be observed at all times, and the safety of the general public outside the classroom must be observed as well. A teacher interested in teaching biotechnology must take into consideration safety bioethics. They need to be certified, and the qualifications of becoming a biotech teacher have not yet been defined. However, a science related bachelor’s degree is required. Some fear that the standards of a bachelor’s degree in science will be lowered in order to make teaching a more attractive career. Another option would be taking a very rigorous test that proves the prospective teacher is from industry. Bioethics is a must if teachers plan to teach, and Mr.Cachianes used to have debate projects at the end of each year, so students could go into more detail on the ethical sides of biotech. Now he shows Gattaca, a powerful movie discussing many ethical issues that can potentially arise from biotech. <br />
<br />
Aside from basic lab skills such as gel electrophoresis, students should also be learning and discussing the perils and benefits of Biotech. For example, there has been much debate about Genetically Modified Organisms or GMOs. GMOs come with a long list of both benefits and disadvantages, and much of biotech comes with a list of pros and cons. Students therefore should be educated, and be able to exercise good judgment on what they think is right. In order for that to happen, they need qualified teachers. Teachers from the bay area meet once every other month and are in an organization Bay Area Biotechnology Education Consortium or BABEC. People from industry are present as well, and together, they develop a high school curriculum. These meetings help pull resources by sharing materials and protocols. Furthermore, teachers tome to the workshops offered at BABEC to learn the techniques they will teach their students. The issue of ethics is covered at these meetings as well.<br />
<br />
Some fear that the FBI will create rules and regulations that will limit the ability of high schoolers to do research and experiments in classes. However, Mr.Cachianes feels that the FBI will not try to enforce rules that will apply regular citizens, because it will be too difficult to monitor. However, if it was to come down to that, they should try to enforce it at the purchasing level. For example, make buying things like enzymes more difficult by having a legitimate license. Mr.Cachianes also feels that high schools will remain mostly unaffected, because the experiments performed are made to be safe. It will be possible to have the freedom of teaching a class without having to follow rules in the future, because if the rules are reasonable then there shouldn’t be a problem. Today, there are already some rules in place, like students cannot do any experiments that involve their blood. These rules are reasonable, because things like blood are a biohazard. However, if rules get out of hand, Mr.Cachianes has said that he will not be afraid to quit teaching. Hopefully, it will never come down to that.<br />
<br />
<br />
{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Raquel_GomesRaquel Gomes2009-10-22T01:13:29Z<p>Prmagomes: New page: I am the UCSF iGEM Program Director. I administer and am responsible for the educational component of the Program. This is my second year at iGEM - It was a crazy intense summer this year....</p>
<hr />
<div>I am the UCSF iGEM Program Director. I administer and am responsible for the educational component of the Program. This is my second year at iGEM - It was a crazy intense summer this year. I am looking forward to the Jamboree where I will be able to see the students presenting their project and will get to meet people from all over the world that are passionate about synthetic biology. It is amazing to witness all the creativity that goes into the projects of the teams that participate at iGEM. <br />
<br />
Lets HAVE FUN!!!</div>Prmagomeshttp://2009.igem.org/Team:UCSF/ProjectTeam:UCSF/Project2009-10-22T01:00:46Z<p>Prmagomes: </p>
<hr />
<div>Our team was inspired by the use of robots which perform tasks that would otherwise be difficult to accomplish. For example, the Mars rovers allow us to make observations and conduct physical experiments in harsh, remote locations otherwise inaccessible to humans. In many ways, the human body represents unexplored territory on a different scale. For example, we may understand many aspects of human disease. However, certain markers of disease are extremely difficult to detect (e.g., primary tumors) and treatment of disease can be hampered by the impracticality of performing invasive surgeries.<br />
<br />
<br />
The "holy grail" of nanomedicine would be to develop microscopic robots that could travel anywhere in the body and perform complex, user-defined tasks. Such devices would have several key advantages over traditional, small-molecule therapies:<br />
<br />
* They could home to specific locations in the body (minimize off-target effects)<br />
* They could make decisions based on their external environment<br />
* They could perform more complicated functions<br />
<br />
<br />
While the idea of microscopic, therapeutic robots may seem far-fetched, there are examples of such machines in nature. For example, neutrophils (a type of white blood cell) are capable of: <br />
<br />
* Detecting and homing to a wide range of chemical signals, at times localizing to very specific sites of inflammation<br />
* Triggering a variety of different pathways (extravasation, phagocytosis, apoptosis) in response to external signals<br />
* Navigating through different types of barriers (endothelial tissue, blood-brain barrier, etc)<br />
<br />
<br />
Taking cues from nature, we were interested in harnessing or hijacking the function of complicated, natural cellular robots such as neutrophils to perform therapeutically useful tasks. In other words, we wanted to use neutrophils (or similarly motile cells) as a chassis for engineering. Minimally, we wanted to control how and when these cells move. Over the course of the summer, were were able to:<br />
<br />
* Control the '''NAVIGATION''' system of our cells (alter what the cells pursue, how strong a signal they require)<br />
* Control the '''SPEED''' of our cells (engineer accelerators and brakes)<br />
* Deliver a '''PAYLOAD''' with our cells<br />
<br />
<br />
Our efforts toward these goals are described in the remainder of this site. Throughout our project, we worked with two model organisms: HL-60 (neutrophil-like) cells and ''Dictyostelium discoideum'', a soil-dwelling amoeba that feeds on bacteria. Both cell types are common models for the study of chemotaxis (directed migration toward chemical signals), and each has its distinct advantages with respect to studying specific questions.<br />
<br />
<br />
'''PROJECT SUMMARY'''<br />
<br />
Part 1 - [[Team:UCSF/Navigation|Engineering NAVIGATION]]<br />
<br />
Part 2 - [[Team:UCSF/SPEED|Engineering SPEED]]<br />
<br />
Part 3 - [[Team:UCSF/PAYLOAD|Carrying a PAYLOAD]]<br />
<br />
Part 4 - [[Team:UCSF/Future Applications|Future Application]]<br />
<br />
<br />
{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/ProjectTeam:UCSF/Project2009-10-22T01:00:22Z<p>Prmagomes: </p>
<hr />
<div>Our team was inspired by the use of robots which perform tasks that would otherwise be difficult to accomplish. For example, the Mars rovers allow us to make observations and conduct physical experiments in harsh, remote locations otherwise inaccessible to humans. In many ways, the human body represents unexplored territory on a different scale. For example, we may understand many aspects of human disease. However, certain markers of disease are extremely difficult to detect (e.g., primary tumors) and treatment of disease can be hampered by the impracticality of performing invasive surgeries.<br />
<br />
<br />
The "holy grail" of nanomedicine would be to develop microscopic robots that could travel anywhere in the body and perform complex, user-defined tasks. Such devices would have several key advantages over traditional, small-molecule therapies:<br />
<br />
* They could home to specific locations in the body (minimize off-target effects)<br />
* They could make decisions based on their external environment<br />
* They could perform more complicated functions<br />
<br />
<br />
While the idea of microscopic, therapeutic robots may seem far-fetched, there are examples of such machines in nature. For example, neutrophils (a type of white blood cell) are capable of: <br />
<br />
* Detecting and homing to a wide range of chemical signals, at times localizing to very specific sites of inflammation<br />
* Triggering a variety of different pathways (extravasation, phagocytosis, apoptosis) in response to external signals<br />
* Navigating through different types of barriers (endothelial tissue, blood-brain barrier, etc)<br />
<br />
<br />
Taking cues from nature, we were interested in harnessing or hijacking the function of complicated, natural cellular robots such as neutrophils to perform therapeutically useful tasks. In other words, we wanted to use neutrophils (or similarly motile cells) as a chassis for engineering. Minimally, we wanted to control how and when these cells move. Over the course of the summer, were were able to:<br />
<br />
* Control the '''NAVIGATION''' system of our cells (alter what the cells pursue, how strong a signal they require)<br />
* Control the '''SPEED''' of our cells (engineer accelerators and brakes)<br />
* Deliver a '''PAYLOAD''' with our cells<br />
<br />
<br />
Our efforts toward these goals are described in the remainder of this site. Throughout our project, we worked with two model organisms: HL-60 (neutrophil-like) cells and ''Dictyostelium discoideum'', a soil-dwelling amoeba that feeds on bacteria. Both cell types are common models for the study of chemotaxis (directed migration toward chemical signals), and each has its distinct advantages with respect to studying specific questions.<br />
<br />
<br />
'''PROJECT'''<br />
<br />
Part 1 - [[Team:UCSF/Navigation|Engineering NAVIGATION]]<br />
<br />
Part 2 - [[Team:UCSF/SPEED|Engineering SPEED]]<br />
<br />
Part 3 - [[Team:UCSF/PAYLOAD|Carrying a PAYLOAD]]<br />
<br />
Part 4 - [[Team:UCSF/Future Applications|Future Application]]<br />
<br />
<br />
{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSFTeam:UCSF2009-10-22T00:51:43Z<p>Prmagomes: </p>
<hr />
<div><!--- The Mission, Experiments ---><br />
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<p align="center"><img src="https://static.igem.org/mediawiki/2009/7/7e/Wiki_2009CellBots.jpg" width="276" height="258" align="middle" /></p><br />
<p align="center" class="style2">Engineering the Movement of Cellular Robots</p><br />
<blockquote><br />
<br><br />
<h2 align="left">Abstract</h2><br />
<p align="left">Some eukaryotic cells, such as white blood cells, have the amazing ability to sense specific external chemical signals and move toward those signals. This behavior, known as chemotaxis, is a fundamental biological process crucial to such diverse functions as development, wound healing and immune response. In our project, we used a synthetic biology approach to manipulate signaling pathways that mediate chemotaxis in two model organisms:<br> HL-60 (neutrophil-like) cells and the slime mold, Dictyostelium discoideum. </p> <br />
<br />
<p align="left">In doing so, <strong>we have demonstrated that we can regulate both the navigation and speed of our cells, as well as harness their ability to carry a payload.</strong></p><br />
<br />
<p align="left">Through our manipulations, we hope to better understand how these systems work, and eventually to build or reprogram cells that can perform useful tasks. Imagine, for example, therapeutic nanorobots that could home to a directed site in the body and execute complex, user-defined functions (e.g., kill tumors, deliver drugs, guide stem cell migration and differentiation). Alternatively, imagine bioremediation nanorobots that could find and retrieve toxic substances. Such cellular robots could be revolutionary biotechnological tools.</p><br />
<p align="right"><a href="https://2009.igem.org/Team:UCSF/Project">More...</a></p><br />
<p align="right">&nbsp;</p><br />
<table width="870" border="0" cellpadding="3"><br />
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<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/0/02/Wiki_2009project.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>BUILDING CELL-BOTS</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Project">Introduction</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Navigation">Step 1 - Engineering NAVIGATION</a></h4><br />
<ul><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:UCSF/Navigation">Inserting New Sensors</a></li><br />
<li><a href="https://2009.igem.org/Team:UCSF/NavigationPart2">Tuning Sensor Sensitivity</a></li><br />
</ul><br />
</ul><br />
<h4><a href="https://2009.igem.org/Team:UCSF/SPEED">Step 2 - Engineering SPEED</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/PAYLOAD">Step 3 - Carrying a PAYLOAD</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Future Applications">Our Vision for the Future</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
</tr><br />
</table><br />
<table width="870" border="0" cellpadding="3"><br />
<tr><br />
<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/d/db/Wiki_2009Team.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>OUR TEAM</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Team">Team Members</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Notebook">Notebooks</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Our_summer_experience">Summer Experience</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Human Practices">Human Practices</a></h4><br />
<h4><a href="http://dspace.mit.edu/handle/1721.1/46721">NEW BIOBRICK Standard RFC28 - Aar1 Cloning System</a></h4><br />
<h4><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=UCSF">Parts submitted to the Registry</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Gold Medal Requisites">GOLD MEDAL Requisites</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
</tr><br />
</table><br />
<br><br></br></br><br />
</blockquote><br />
</body><br />
</html><br />
<br />
<br />
'''UCSF iGEM 2009 is sponsored by...'''<br />
<br />
<br />
<br />
<html><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/8/85/Wiki_2009Sponsors.jpg" width="690" height="419" align="middle" /></p></html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/ProjectTeam:UCSF/Project2009-10-22T00:50:44Z<p>Prmagomes: </p>
<hr />
<div>Our team was inspired by the use of robots which perform tasks that would otherwise be difficult to accomplish. For example, the Mars rovers allow us to make observations and conduct physical experiments in harsh, remote locations otherwise inaccessible to humans. In many ways, the human body represents unexplored territory on a different scale. For example, we may understand many aspects of human disease. However, certain markers of disease are extremely difficult to detect (e.g., primary tumors) and treatment of disease can be hampered by the impracticality of performing invasive surgeries.<br />
<br />
<br />
The "holy grail" of nanomedicine would be to develop microscopic robots that could travel anywhere in the body and perform complex, user-defined tasks. Such devices would have several key advantages over traditional, small-molecule therapies:<br />
<br />
* They could home to specific locations in the body (minimize off-target effects)<br />
* They could make decisions based on their external environment<br />
* They could perform more complicated functions<br />
<br />
<br />
While the idea of microscopic, therapeutic robots may seem far-fetched, there are examples of such machines in nature. For example, neutrophils (a type of white blood cell) are capable of: <br />
<br />
* Detecting and homing to a wide range of chemical signals, at times localizing to very specific sites of inflammation<br />
* Triggering a variety of different pathways (extravasation, phagocytosis, apoptosis) in response to external signals<br />
* Navigating through different types of barriers (endothelial tissue, blood-brain barrier, etc)<br />
<br />
<br />
Taking cues from nature, we were interested in harnessing or hijacking the function of complicated, natural cellular robots such as neutrophils to perform therapeutically useful tasks. In other words, we wanted to use neutrophils (or similarly motile cells) as a chassis for engineering. Minimally, we wanted to control how and when these cells move. Over the course of the summer, were were able to:<br />
<br />
* Control the '''NAVIGATION''' system of our cells (alter what the cells pursue, how strong a signal they require)<br />
* Control the '''SPEED''' of our cells (engineer accelerators and brakes)<br />
* Deliver a '''PAYLOAD''' with our cells<br />
<br />
<br />
Our efforts toward these goals are described in the remainder of this site. Throughout our project, we worked with two model organisms: HL-60 (neutrophil-like) cells and ''Dictyostelium discoideum'', a soil-dwelling amoeba that feeds on bacteria. Both cell types are common models for the study of chemotaxis (directed migration toward chemical signals), and each has its distinct advantages with respect to studying specific questions.<br />
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{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
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!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/BackgroundTeam:UCSF/Background2009-10-22T00:47:37Z<p>Prmagomes: </p>
<hr />
<div>Our team was inspired by the use of robots which perform tasks that would otherwise be difficult to accomplish. For example, the Mars rovers allow us to make observations and conduct physical experiments in harsh, remote locations otherwise inaccessible to humans. In many ways, the human body represents unexplored territory on a different scale. For example, we may understand many aspects of human disease. However, certain markers of disease are extremely difficult to detect (e.g., primary tumors) and treatment of disease can be hampered by the impracticality of performing invasive surgeries.<br />
<br />
<br />
The "holy grail" of nanomedicine would be to develop microscopic robots that could travel anywhere in the body and perform complex, user-defined tasks. Such devices would have several key advantages over traditional, small-molecule therapies:<br />
<br />
* They could home to specific locations in the body (minimize off-target effects)<br />
* They could make decisions based on their external environment<br />
* They could perform more complicated functions<br />
<br />
<br />
While the idea of microscopic, therapeutic robots may seem far-fetched, there are examples of such machines in nature. For example, neutrophils (a type of white blood cell) are capable of: <br />
<br />
* Detecting and homing to a wide range of chemical signals, at times localizing to very specific sites of inflammation<br />
* Triggering a variety of different pathways (extravasation, phagocytosis, apoptosis) in response to external signals<br />
* Navigating through different types of barriers (endothelial tissue, blood-brain barrier, etc)<br />
<br />
<br />
Taking cues from nature, we were interested in harnessing or hijacking the function of complicated, natural cellular robots such as neutrophils to perform therapeutically useful tasks. In other words, we wanted to use neutrophils (or similarly motile cells) as a chassis for engineering. Minimally, we wanted to control how and when these cells move. Over the course of the summer, were were able to:<br />
<br />
* Control the '''NAVIGATION''' system of our cells (alter what the cells pursue, how strong a signal they require)<br />
* Control the '''SPEED''' of our cells (engineer accelerators and brakes)<br />
* Deliver a '''PAYLOAD''' with our cells<br />
<br />
<br />
Our efforts toward these goals are described in the remainder of this site. Throughout our project, we worked with two model organisms: HL-60 (neutrophil-like) cells and ''Dictyostelium discoideum'', a soil-dwelling amoeba that feeds on bacteria. Both cell types are common models for the study of chemotaxis (directed migration toward chemical signals), and each has its distinct advantages with respect to studying specific questions.<br />
<br />
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{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/BackgroundTeam:UCSF/Background2009-10-22T00:46:35Z<p>Prmagomes: </p>
<hr />
<div>Our team was inspired by the use of robots which perform tasks that would otherwise be difficult to accomplish. For example, the Mars rovers allow us to make observations and conduct physical experiments in harsh, remote locations otherwise inaccessible to humans. In many ways, the human body represents unexplored territory on a different scale. For example, we may understand many aspects of human disease. However, certain markers of disease are extremely difficult to detect (e.g., primary tumors) and treatment of disease can be hampered by the impracticality of performing invasive surgeries.<br />
<br />
<br />
The "holy grail" of nanomedicine would be to develop microscopic robots that could travel anywhere in the body and perform complex, user-defined tasks. Such devices would have several key advantages over traditional, small-molecule therapies:<br />
<br />
* They could home to specific locations in the body (minimize off-target effects)<br />
* They could make decisions based on their external environment<br />
* They could perform more complicated functions<br />
<br />
<br />
While the idea of microscopic, therapeutic robots may seem far-fetched, there are examples of such machines in nature. For example, neutrophils (a type of white blood cell) are capable of: <br />
<br />
* Detecting and homing to a wide range of chemical signals, at times localizing to very specific sites of inflammation<br />
* Triggering a variety of different pathways (extravasation, phagocytosis, apoptosis) in response to external signals<br />
* Navigating through different types of barriers (endothelial tissue, blood-brain barrier, etc)<br />
<br />
<br />
Taking cues from nature, we were interested in harnessing or hijacking the function of complicated, natural cellular robots such as neutrophils to perform therapeutically useful tasks. In other words, we wanted to use neutrophils (or similarly motile cells) as a chassis for engineering. Minimally, we wanted to control how and when these cells move. Over the course of the summer, were were able to:<br />
<br />
* Control the '''NAVIGATION''' system of our cells (alter what the cells pursue, how strong a signal they require)<br />
* Control the '''SPEED''' of our cells (engineer accelerators and brakes)<br />
* Deliver a '''PAYLOAD''' with our cells<br />
<br />
<br />
Our efforts toward these goals are described in the remainder of this site. Throughout our project, we worked with two model organisms: HL-60 (neutrophil-like) cells and ''Dictyostelium discoideum'', a soil-dwelling amoeba that feeds on bacteria. Both cell types are common models for the study of chemotaxis (directed migration toward chemical signals), and each has its distinct advantages with respect to studying specific questions.<br />
<br />
<br />
<br />
{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Background|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/PAYLOADTeam:UCSF/PAYLOAD2009-10-21T18:34:31Z<p>Prmagomes: </p>
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<h1 align="left">Making Engineered Cells carry a PAYLOAD</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>Once a cellular robot has detected and traveled to a site of disease in the human body, it should be able to perform a useful therapeutic task. One simple but very useful task that we envision our cells performing in the body is the targeted delivery of drugs or signaling molecules to diseased areas. This would be useful in the delivery of all sorts of substances to targeted areas in the body. Some examples include: 1) cases where a drug is toxic and off-target side effects are dose-limiting (e.g. chemotherapeutics used for cancer), 2) cases where precise delivery of a contrast agent to diseased areas would aid in diagnosis (e.g. sites of cancer metastasis), 3) cases where localized delivery of a specific bioactive molecule to a site would be therapeutically beneficial (e.g. delivery of specific growth factors to areas of wound healing and regeneration). </p><br />
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<p>&nbsp;</p><br />
<p>Toward the end of engineering our cellular robots to carry useful cargos, we designed and began testing a generalizable scheme for tethering fluorescent polymer microspheres (&quot;beads&quot;) to the outside of our cells. </p><br />
<h3>Approach</h3><br />
<p>We designed a modular and generalizable scheme for attaching fluorescent polymer microspheres to the outside of our cells. To make the approach modular, we thought of our strategy as having three main interchangeable parts: 1) Bead attachment part, 2) Linker, 3) Cell attachment part. By ensuring that we make the parts interchangeable, we made the approach generalizable. For example, changing out the cell attachment part for a different one would allow us to attach beads to a different type of cell. Likewise, we could use different types of linkers including photocleavable or enzymatically cleavable ones. Changing out the bead attachment part could allow us to attach not only beads but other cargos to cells. </p><br />
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[[Image:cellbeads_scheme.jpg|400px|thumb|center|'''Modular and generalizable scheme for attaching beads to cells''']]<br />
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<p>&nbsp;</p><br />
<p>Some important early considerations for us were therefore: 1) Best to use a scheme that does not require much (or any) genetic manipulation, so that we can use our scheme quickly and easily with any cell or cell type, 2) Use well-established chemistries and reagents, 3) Be able to swap and interchange modular parts.</p><br />
<p>For our pilot experiments this summer, we used the simplest possible parts that we could think of. For cellular attachment, we used the jack bean lectin protein <a href="http://en.wikipedia.org/wiki/Concanavalin_A">Concanavalin A (ConA)</a>, which binds mainly to internal and nonreducing terminal alpha-mannosyl groups. We left the linker out in some cases, though in others we used<a href="http://en.wikipedia.org/wiki/Polyethylene_glycol"> polyethylene glycol (PEG)</a> linkers. For bead attachment, we used either streptavidin-biotin chemistry, passive adsorption of protein on the surface of polystyrene beads, or <a href="http://en.wikipedia.org/wiki/Carbodiimide">carbodiimide chemistry</a> with the crosslinker EDC.</p><br />
<p>To observe the cells, we coated glass-bottomed <a href="http://www.nuncbrand.com/en/page.aspx?ID=235">Lab-Tek II Chambered Coverglass slides</a> with fibronectin, adhered cells to the slide, and then incubated with varying concentrations of modified beads followed by a wash step. We then visualized the cells using time-lapse microscopy. </p><br />
<h3>Results</h3><br />
<p>We have been able to observe our cells exhibiting motility while tethered to &quot;cargos&quot; of fluorescent beads. Below are two representative movies, each taken using fluorescent beads with ConA (no linker). Frame rate is 15 seconds (real time) per frame. </p><br />
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<p>&nbsp;</p><br />
<p>Here are some stillshots from the movies. </p><br />
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[[Image:cellbead1_enh.jpg]]<br />
[[Image:cellbead2_enh.jpg]]<br />
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<h3>Summary and Outlook</h3><br />
<p>We have shown that <strong>we can tether simple fluorescent bead &quot;cargos&quot; to our cells</strong>. We are using a simple, generalizable, and modular scheme to attach these cargos, and we will continue to try different attachment and linker parts to improve on our initial result. Some things we are working on include efficiency and robustness of attachment and internalization of beads by cells.</p><br />
<p>We would also like to explore the ability of cells to carry larger bead cargos and different types of cargos (non-bead). In addition, we would like to study the physical limitations of these cells in carrying cargo. For example, it would be particular interesting to determine whether the cells can carry carry cargos through complex environments such as three-dimensional hydrogel environments, endothelial cell monolayers, and more. </p><br />
<p>&nbsp;</p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/Future_Applications">NEXT - Future Applications</a></strong></p><br />
<p align="left">&nbsp;</p><br />
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==Methods==<br />
<br />
'''Payload:'''<br />
*[[Media:Protocols_cellbeads_ConAandBeads.pdf|Binding concanavalin A to beads]]<br />
*[[Media:Protocols_cellbeads_BeadsAndCellsMicroscopy.pdf|Attaching beads to cells and observation using time lapse microscopy]]<br />
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==Selected Reading==<br />
<br />
Microoxen: Microorganisms to move microscale loads. Weibel DB, Garstecki P, Ryan D, DiLuzio WR, Mayer M, Seto JE, Whitesides GM. PNAS August 23, 2005 vol. 102 no. 34 11963-11967.<br />
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!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/SPEEDTeam:UCSF/SPEED2009-10-21T18:32:42Z<p>Prmagomes: </p>
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<h1 align="left">Engineering SPEED: Creating Synthetic Brakes and Accelerators</h1><br />
<p align="left">&nbsp;</p><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>Just like we have control over speed in a car – we can brake or accelerate – it would be useful to engineer such behavior into our cellular nanorobots. Just think about it: We could <strong>speed cells up</strong> so that they reach their targets faster and <strong>stop them</strong> once they have arrived or do not behave properly.</p><br />
<p>&nbsp;</p><br />
<h3>Background</h3><br />
<p>For these experiments we chose Dictyostelium discoideum cells to test our prototypical brakes and accelerators quickly. We expect that our brakes and accelerators can be used in a plug and play fashion because Dicty’s way of movement is very similar to a neutrophil’s:</p><br />
<p>When a receptor binds chemoattractant, it induces the conversion of PhosphatidylInositol(4,5)bisphosphate (PIP2) to PhosphatidylInositol(3,4,5)trisphosphate (PIP3) (two signaling lipids in the plasma membrane) at the front of our cells. In a <strong>positive feedback loop</strong> PIP3 triggers the formation of more PIP3 at the front while similarly PIP2 leads to more PIP2 production at the sides and rear of the cell. This system sets the axis of polarity of the cell. The PIP3 patch at the front aligns the actin network and accordingly functions as a ‘turbo boost’ pushing the cell forward. </p><br />
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[[Image:BACKGROUND1.jpg|350px|thumb|center|'''Polarized distribution of PIP3 and PIP2:''' A patch of PIP3 is localized at the front of a cell while PIP2 is at the back. Feedback loops are used to establish and maintain this polarized distribution. ]]<br />
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<h3>Approach</h3><br />
<p>Inspired by nature we tried to build accelerators and brakes by introducing our own synthetic protein based feedback loops. We designed feedback elements by fusing localization and catalytic domains involved in PIP3 production and degradation to modulate localization and level of PIP3 and PIP2 in the cell.</p><br />
<p>Here is an example of a positive feedback loop: a PIP3 binding localization domain fused to a PIP3 producing catalytic domain could produce more PIP3 where there is already PIP3- at the front. This might strengthen polarity and accelerate a cell. </p><br />
<p>Similarly a PIP3 binding domain fused to a PIP2 producing catalytic domain could put PIP2 in place of PIP3 and thereby act as a negative feedback loop.</p><br />
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[[Image:feedbackloops.jpg|600px|thumb|center|'''Synthetic protein based feedback loops:''' <br />
On the left: '''a positive feedback loop''' made of a PIP3 binding domain fused to a PIP3 generating enzyme.<br />
On the right: '''a negative feedback loop''' made of a PIP3 binding domain fused to a PIP2 generating enzyme.]]<br />
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<p align="left">&nbsp;</p><br />
<h3>Results</h3><br />
<p>Over the summer we assembled more than 100 fusions of localization and catalytic domains and screened whether they work. How? We measured the effect our constructs have on motility of Dictyostelium cells: stronger polarity should make cells faster while weaker polarity ought to slow them down!</p><br />
<p><a href="https://2009.igem.org/Matrix_of_Fusion_Constructs">Here is an overview of all feedback loops we screened</a> and the effect they had on the speed of cells. We used automated cell tracking on more than 196 hours worth of movies (note: one movie is 10 minutes!) and identified strains that moved faster or slower at a very stringent statistical cutoff (p&lt;0.0001).</p><br />
<p>This way we were able to identify <strong>7 brakes and 1 accelerator!</strong></p><br />
<p>Check out the movie of one of our strong brakes (<strong><a href="http://www.youtube.com/watch?v=_9od33Nx06Y">BBa_K209066: PTEN-RasC dominant active (da)</a></strong>) compared to <strong><a href="http://www.youtube.com/watch?v=Vtdtf8-zSRs">wildtype</a></strong>.</p><br />
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[[Image:HO230.jpg|400px|thumb|right|'''PTEN fused to RasC da (PIP2 binding - PIP3 generating); speed: 3.5 um/min''' Cells were plated in buffer and basic motile behavior was recorded for 10 minutes taking 1 picture every 15 seconds.]] [[Image:wt.jpg|400px|thumb|left|'''wildtype; speed: 5.9 um/min'''<br />
Cells were plated in buffer and basic motile behavior was recorded for 10 minutes taking 1 picture every 15 seconds.]]<br />
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<p>This is indeed an effect of fusing the particular localization to the catalytic domain as neither of them alone has such a strong effect.<a href="https://2009.igem.org/Further_Results"> Access further data and details here.</a><br />
</p><br />
<p>We hypothesize that this construct acts as a <strong>negative feedback loop on PIP2</strong> - (generating PIP3 where PIP2 should be) thereby confusing the cell with multiple fronts: </p><br />
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<h3>Summary and Outlook</h3><br />
<p>We have screened more than 100 synthetic feedback elements for their ability to accelerate or slow down speed of cell motility. We have isolated a hand full of functional elements. Now we need to confirm the mechanism of action of these elements. In the future we would like to make them inducible by a signal from outside – like a stoplight! </p><br />
<p>&nbsp;</p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/PAYLOAD">NEXT - PAYLOAD</a></strong></p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
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<br />
==Methods==<br />
<br />
'''Engineering Speed:'''<br />
<br />
*[[Media:Aar1_shuffle.pdf|Cloning: Aar1 Shuffle]] - besides BBFRFC28, here is an actual step by step scheme we used for some of our combinatorial assembly of parts<br />
*[[Media:DICTY TRANSFORMATION.pdf|Dicty: Transformation Protocol]] - here is how we get our constructs into Dicty<br />
*[[Media:DICTY-analyzing_motility.pdf|Dicty: Motility Assay Protocol]] - here is how we prepare and film Dicty cells<br />
<br />
<br />
==Selected Reading==<br />
<br />
G protein signaling events are activated at the leading edge of chemotactic cells. Parent CA, Blacklock BJ, Froehlich WM, Murphy DB, Devreotes PN. Cell. 1998 Oct 2;95(1):81-91.<br />
<br />
PI3-kinase signaling contributes to orientation in shallow gradients and enhances speed in steep chemoattractant gradients. Bosgraaf L, Keizer-Gunnink I, Van Haastert PJ. J Cell Sci. 2008 Nov 1;121(Pt 21):3589-97. Epub 2008 Oct 7.<br />
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{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationPart2Team:UCSF/NavigationPart22009-10-21T18:30:39Z<p>Prmagomes: </p>
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<h1 align="left">Engineering NAVIGATION: Tuning Engineered Cells Sensitivity towards New Signals</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>In our experiments, we could control exactly how much ligand was presented to our cells. In &quot;real-life,&quot; however, we would want our cellular robots to be able to respond to a variety of signal strengths: from very low to very high. To accomplish this, we would want to be able to control the sensitivity of our receptors, or how the receptor's output changes when the measured quantity of ligand changes.</p><br />
<h3>Approach</h3><br />
<p>We felt that one key determinant of sensitivity would be the number of receptors present at the plasma membrane of the cell. Therefore, we measured the migration response of a receptor (delta Opioid receptor) whose recycling behavior could be engineered by fusing different recycling interaction modules to the C terminus of the GPCR. We tested a number of such receptor-module fusions for migration response and compared them to receptor alone. The primary assay here was again the Boyden chamber (transwell) assay.</p><br />
<p>&nbsp;</p><br />
<h3>Results</h3><br />
<p>We found that virtually any protein domain/module known to alter the recycling of delta Opioid receptor (DOR) affected cellular migration to a low concentration of ligand (1 nM DADLE). Below, we show two examples of such domains. The actin binding domain from alpha-Actinin-1 (ACTN1ABD) appears to potentiate cellular migration when compared to the wild-type receptor. On the other hand, cellular response is inhibited at this concentration when DOR is bound to a domain of EBP50 (ERMbd) that binds to ERM (ezrin/radixin/moesin) family of proteins. </p><br />
<p>&nbsp;</p><br />
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[[Image:Sensitivity.png|250px|thumb|center|Recycling modules affect sensitivity of DOR]]<br />
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<h3>Summary and Outlook</h3><br />
<p>We have shown that we can tune the sensitivity of a receptor both up and down by fusing it to different recycling modules. Next, we would like to measure recycling directly, and determine whether changes in recycling are necessary for this difference in chemotactic response. All experiments in HL-60 cells would benefit the generation of stable cell lines, which would allow us to more precisely quantify these new behaviors. </p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/SPEED">NEXT - SPEED</a></strong></p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html><br />
<br />
==Methods==<br />
<br />
'''Navigation:'''<br />
<br />
*[[Media:amaxa.pdf|HL-60: Transfection Protocol]] - here is how we get our constructs into HL-60 cells<br />
*[[Media:transwell.pdf|HL-60: Boyden Chamber (Transwell) Protocol]] - here is how we assay chemotaxis in HL-60 cells<br />
*[[Media:ezt.pdf|HL-60: Time-lapse Microscopy Protocol]] - here is how we film HL-60 cells<br />
<br />
<br />
==Selected Reading==<br />
<br />
'''Navigation (New Sensors):'''<br />
<br />
Receptors induce chemotaxis by releasing the betagamma subunit of Gi, not by activating Gq or Gs. Neptune ER, Bourne HR. Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14489-94.<br />
<br />
Delineation of muscarinic receptor domains conferring selectivity of coupling to guanine nucleotide-binding proteins and second messengers. Wess J, Bonner TI, Dörje F, Brann MR. Mol Pharmacol. 1990 Oct;38(4):517-23.<br />
<br />
'''Navigation (Sensor Sensitivity):'''<br />
<br />
Actin-binding protein alpha-actinin-1 interacts with the metabotropic glutamate receptor type 5b and modulates the cell surface expression and function of the receptor. Cabello N, Remelli R, Canela L, Soriguera A, Mallol J, Canela EI, Robbins MJ, Lluis C, Franco R, McIlhinney RA, Ciruela F. J Biol Chem. 2007 Apr 20;282(16):12143-53. Epub 2007 Feb 20.<br />
<br />
Engineered protein connectivity to actin mimics PDZ-dependent recycling of G protein-coupled receptors but not its regulation by Hrs. Lauffer BE, Chen S, Melero C, Kortemme T, von Zastrow M, Vargas GA. J Biol Chem. 2009 Jan 23;284(4):2448-58. Epub 2008 Nov 10.<br />
<br />
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{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationPart2Team:UCSF/NavigationPart22009-10-21T18:29:52Z<p>Prmagomes: </p>
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<h1 align="left">Engineering NAVIGATION: Tuning Engineered Cells Sensitivity towards New Signals</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>In our experiments, we could control exactly how much ligand was presented to our cells. In &quot;real-life,&quot; however, we would want our cellular robots to be able to respond to a variety of signal strengths: from very low to very high. To accomplish this, we would want to be able to control the sensitivity of our receptors, or how the receptor's output changes when the measured quantity of ligand changes.</p><br />
<h3>Approach</h3><br />
<p>We felt that one key determinant of sensitivity would be the number of receptors present at the plasma membrane of the cell. Therefore, we measured the migration response of a receptor (delta Opioid receptor) whose recycling behavior could be engineered by fusing different recycling interaction modules to the C terminus of the GPCR. We tested a number of such receptor-module fusions for migration response and compared them to receptor alone. The primary assay here was again the Boyden chamber (transwell) assay.</p><br />
<p>&nbsp;</p><br />
<h3>Results</h3><br />
<p>We found that virtually any protein domain/module known to alter the recycling of delta Opioid receptor (DOR) affected cellular migration to a low concentration of ligand (1 nM DADLE). Below, we show two examples of such domains. The actin binding domain from alpha-Actinin-1 (ACTN1ABD) appears to potentiate cellular migration when compared to the wild-type receptor. On the other hand, cellular response is inhibited at this concentration when DOR is bound to a domain of EBP50 (ERMbd) that binds to ERM (ezrin/radixin/moesin) family of proteins. </p><br />
<p>&nbsp;</p><br />
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[[Image:Sensitivity.png|250px|thumb|center|Recycling modules affect sensitivity of DOR]]<br />
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<h3>Summary and Outlook</h3><br />
<p>We have shown that we can tune the sensitivity of a receptor both up and down by fusing it to different recycling modules. Next, we would like to measure recycling directly, and determine whether changes in recycling are necessary for this difference in chemotactic response. All experiments in HL-60 cells would benefit the generation of stable cell lines, which would allow us to more precisely quantify these new behaviors. </p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/SPEED">NEXT - SPEED</a></strong></p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html><br />
<br />
=Methods=<br />
<br />
'''Navigation:'''<br />
<br />
*[[Media:amaxa.pdf|HL-60: Transfection Protocol]] - here is how we get our constructs into HL-60 cells<br />
*[[Media:transwell.pdf|HL-60: Boyden Chamber (Transwell) Protocol]] - here is how we assay chemotaxis in HL-60 cells<br />
*[[Media:ezt.pdf|HL-60: Time-lapse Microscopy Protocol]] - here is how we film HL-60 cells<br />
<br />
=Selected Reading=<br />
<br />
'''Navigation (New Sensors):'''<br />
<br />
Receptors induce chemotaxis by releasing the betagamma subunit of Gi, not by activating Gq or Gs. Neptune ER, Bourne HR. Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14489-94.<br />
<br />
Delineation of muscarinic receptor domains conferring selectivity of coupling to guanine nucleotide-binding proteins and second messengers. Wess J, Bonner TI, Dörje F, Brann MR. Mol Pharmacol. 1990 Oct;38(4):517-23.<br />
<br />
'''Navigation (Sensor Sensitivity):'''<br />
<br />
Actin-binding protein alpha-actinin-1 interacts with the metabotropic glutamate receptor type 5b and modulates the cell surface expression and function of the receptor. Cabello N, Remelli R, Canela L, Soriguera A, Mallol J, Canela EI, Robbins MJ, Lluis C, Franco R, McIlhinney RA, Ciruela F. J Biol Chem. 2007 Apr 20;282(16):12143-53. Epub 2007 Feb 20.<br />
<br />
Engineered protein connectivity to actin mimics PDZ-dependent recycling of G protein-coupled receptors but not its regulation by Hrs. Lauffer BE, Chen S, Melero C, Kortemme T, von Zastrow M, Vargas GA. J Biol Chem. 2009 Jan 23;284(4):2448-58. Epub 2008 Nov 10.<br />
<br />
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{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/TeamTeam:UCSF/Team2009-10-21T18:23:54Z<p>Prmagomes: /* Team Members */</p>
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<div><!--- The Mission, Experiments ---><br />
<br />
{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}<br />
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{|align="center"<br />
|[[Image:Igemteamucsf.jpg|800px|thumb|right|Team UCSF]] <br />
|}<br />
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== '''Introduction to the 2009 UCSF IGEM Team''' ==<br />
{|border = "0"<br />
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== '''Team Members''' ==<br />
<br />
{| width=100% align="center" border="0" cellpading="20' cellspacing="20"<br />
|'''Students:'''<br />
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*'''High School Student''': [[Allen Cai]]<br />
*'''High School Student''': [[Alex Smith]]<br />
*'''High School Student''': [[Edna Miao]]<br />
*'''High School Student''': [[Ethan Chan]]<br />
*'''High School Student''': [[Eric Wong]]<br />
*'''High School Student''': [[Jackie Tam]]<br />
*'''High School Student''': [[Ryan Liang]]<br />
<br />
<br />
*'''Undergrad Student''': [[Cathy Liu]]<br />
*'''Undergrad Student''': [[Ryan Quan]]<br />
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*'''International Undergrad Student''': [[Hansi Liu]]<br />
*'''International Undergrad Student''': [[Katja Kolar]]<br />
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|'''Buddies:'''<br />
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*''' Scientific Director''': [[Ben Rhau]]<br />
*''' Scientific Director''': [[Oliver Hoeller]]<br />
<br />
*''' Buddy''': [[Aynur Tasdemir]]<br />
*''' Buddy''': [[Jason Park]] <br />
*''' Buddy''': [[Andrew Houk]] <br />
*''' Buddy''': [[Delquin Gong]] <br />
*''' Buddy''': [[Arthur Millius]] <br />
*''' Buddy''': [[David Pincus]]<br />
*''' Buddy''': [[Bethany Simmons]]<br />
*''' Teacher''': [[Saber Khan]]<br />
<br />
<br />
*''' Program Director''': [[Raquel Gomes]]<br />
*''' Advisor''': [[Wendell Lim]]<br />
*''' Advisor''': [[Orion Weiner]]<br />
*''' Advisor''': [[James Onuffer]]<br />
<br />
*''' Teacher at Lincoln High School''': [[George Cachianes]]<br />
*''' Teacher at Lincoln High School''': [[Julie Reis]]<br />
|}<br />
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Students<br />
Image:Allen_C.jpg|Allen Cai<br />
Image:Alex_Smith.jpg|Alex Smith<br />
Image:Edna_Miao.jpg|Edna Miao<br />
Image:Ethan_C.jpg|Ethan Chan<br />
Image:Eric_W.jpg|Eric Wong<br />
Image:Jackie_T.jpg|Jackie Tam<br />
Image:Ryan_L.jpg|Ryan Liang<br />
<br />
Image:Cathy_Liu.jpg|Cathy Liu<br />
Image:Ryan_Q.jpg|Ryan Quan<br />
Image:Hansi_L.jpg|Hansi Liu<br />
Image:Katja_K.jpg|Katja Kolar<br />
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Buddies<br />
Image:Wendell.JPG|Wendell Lim<br />
Image:Orion_weiner.gif|Orion Weiner<br />
Image:.|James Onuffer<br />
<br />
Image:.|Raquel Gomes<br />
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Image:Ben_R.gif|Ben Rhau<br />
Image:Oliver_H.gif|Oliver Hoeller<br />
<br />
Image:AYNUR.JPG|Aynur Tasdemir<br />
Image:Jason_P.jpg|Jason Park<br />
Image:Andrew_H.gif|Andrew Houk<br />
Image:Delquin_G.gif|Delquin Gong<br />
Image:Arthur_M.gif|Arthur Millius<br />
Image:Bethany_s.png|Bethany Simmons<br />
Image:Saber_Khan.jpg|Saber Khan<br />
</gallery></div>Prmagomeshttp://2009.igem.org/Team:UCSF/PAYLOADTeam:UCSF/PAYLOAD2009-10-21T18:13:12Z<p>Prmagomes: </p>
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<h1 align="left">Making Engineered Cells carry a PAYLOAD</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>Once a cellular robot has detected and traveled to a site of disease in the human body, it should be able to perform a useful therapeutic task. One simple but very useful task that we envision our cells performing in the body is the targeted delivery of drugs or signaling molecules to diseased areas. This would be useful in the delivery of all sorts of substances to targeted areas in the body. Some examples include: 1) cases where a drug is toxic and off-target side effects are dose-limiting (e.g. chemotherapeutics used for cancer), 2) cases where precise delivery of a contrast agent to diseased areas would aid in diagnosis (e.g. sites of cancer metastasis), 3) cases where localized delivery of a specific bioactive molecule to a site would be therapeutically beneficial (e.g. delivery of specific growth factors to areas of wound healing and regeneration). </p><br />
<blockquote>&nbsp;</blockquote><br />
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<p>&nbsp;</p><br />
<p>Toward the end of engineering our cellular robots to carry useful cargos, we designed and began testing a generalizable scheme for tethering fluorescent polymer microspheres (&quot;beads&quot;) to the outside of our cells. </p><br />
<h3>Approach</h3><br />
<p>We designed a modular and generalizable scheme for attaching fluorescent polymer microspheres to the outside of our cells. To make the approach modular, we thought of our strategy as having three main interchangeable parts: 1) Bead attachment part, 2) Linker, 3) Cell attachment part. By ensuring that we make the parts interchangeable, we made the approach generalizable. For example, changing out the cell attachment part for a different one would allow us to attach beads to a different type of cell. Likewise, we could use different types of linkers including photocleavable or enzymatically cleavable ones. Changing out the bead attachment part could allow us to attach not only beads but other cargos to cells. </p><br />
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[[Image:cellbeads_scheme.jpg|400px|thumb|center|'''Modular and generalizable scheme for attaching beads to cells''']]<br />
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<p>&nbsp;</p><br />
<p>Some important early considerations for us were therefore: 1) Best to use a scheme that does not require much (or any) genetic manipulation, so that we can use our scheme quickly and easily with any cell or cell type, 2) Use well-established chemistries and reagents, 3) Be able to swap and interchange modular parts.</p><br />
<p>For our pilot experiments this summer, we used the simplest possible parts that we could think of. For cellular attachment, we used the jack bean lectin protein <a href="http://en.wikipedia.org/wiki/Concanavalin_A">Concanavalin A (ConA)</a>, which binds mainly to internal and nonreducing terminal alpha-mannosyl groups. We left the linker out in some cases, though in others we used<a href="http://en.wikipedia.org/wiki/Polyethylene_glycol"> polyethylene glycol (PEG)</a> linkers. For bead attachment, we used either streptavidin-biotin chemistry, passive adsorption of protein on the surface of polystyrene beads, or <a href="http://en.wikipedia.org/wiki/Carbodiimide">carbodiimide chemistry</a> with the crosslinker EDC.</p><br />
<p>To observe the cells, we coated glass-bottomed <a href="http://www.nuncbrand.com/en/page.aspx?ID=235">Lab-Tek II Chambered Coverglass slides</a> with fibronectin, adhered cells to the slide, and then incubated with varying concentrations of modified beads followed by a wash step. We then visualized the cells using time-lapse microscopy. </p><br />
<h3>Results</h3><br />
<p>We have been able to observe our cells exhibiting motility while tethered to &quot;cargos&quot; of fluorescent beads. Below are two representative movies, each taken using fluorescent beads with ConA (no linker). Frame rate is 15 seconds (real time) per frame. </p><br />
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<body><br />
<p>&nbsp;</p><br />
<p>Here are some stillshots from the movies. </p><br />
<p>&nbsp;</p><br />
</body><br />
</html><br />
[[Image:cellbead1_enh.jpg]]<br />
[[Image:cellbead2_enh.jpg]]<br />
[[Image:cellbead3_enh.jpg]]<br />
<br />
<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
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<script src="Scripts/AC_RunActiveContent.js" type="text/javascript"></script><br />
</head><br />
<body><br />
<br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that <strong>we can tether simple fluorescent bead &quot;cargos&quot; to our cells</strong>. We are using a simple, generalizable, and modular scheme to attach these cargos, and we will continue to try different attachment and linker parts to improve on our initial result. Some things we are working on include efficiency and robustness of attachment and internalization of beads by cells.</p><br />
<p>We would also like to explore the ability of cells to carry larger bead cargos and different types of cargos (non-bead). In addition, we would like to study the physical limitations of these cells in carrying cargo. For example, it would be particular interesting to determine whether the cells can carry carry cargos through complex environments such as three-dimensional hydrogel environments, endothelial cell monolayers, and more. </p><br />
<p>&nbsp;</p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/Future_Applications">NEXT - Future Applications</a></strong></p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html><br />
<br />
<br />
<br />
<br />
<br />
{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/PAYLOADTeam:UCSF/PAYLOAD2009-10-21T18:12:00Z<p>Prmagomes: </p>
<hr />
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<body><br />
<h1 align="left">Making Engineered Cells carry a PAYLOAD</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>Once a cellular robot has detected and traveled to a site of disease in the human body, it should be able to perform a useful therapeutic task. One simple but very useful task that we envision our cells performing in the body is the targeted delivery of drugs or signaling molecules to diseased areas. This would be useful in the delivery of all sorts of substances to targeted areas in the body. Some examples include: 1) cases where a drug is toxic and off-target side effects are dose-limiting (e.g. chemotherapeutics used for cancer), 2) cases where precise delivery of a contrast agent to diseased areas would aid in diagnosis (e.g. sites of cancer metastasis), 3) cases where localized delivery of a specific bioactive molecule to a site would be therapeutically beneficial (e.g. delivery of specific growth factors to areas of wound healing and regeneration). </p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html><br />
[[Image:cellbead.jpg|400px|center]]<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /><br />
<title>Untitled Document</title><br />
<style type="text/css"><br />
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<script src="Scripts/AC_RunActiveContent.js" type="text/javascript"></script><br />
</head><br />
<body><br />
<p>&nbsp;</p><br />
<p>Toward the end of engineering our cellular robots to carry useful cargos, we designed and began testing a generalizable scheme for tethering fluorescent polymer microspheres (&quot;beads&quot;) to the outside of our cells. </p><br />
<h3>Approach</h3><br />
<p>We designed a modular and generalizable scheme for attaching fluorescent polymer microspheres to the outside of our cells. To make the approach modular, we thought of our strategy as having three main interchangeable parts: 1) Bead attachment part, 2) Linker, 3) Cell attachment part. By ensuring that we make the parts interchangeable, we made the approach generalizable. For example, changing out the cell attachment part for a different one would allow us to attach beads to a different type of cell. Likewise, we could use different types of linkers including photocleavable or enzymatically cleavable ones. Changing out the bead attachment part could allow us to attach not only beads but other cargos to cells. </p><br />
</body><br />
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[[Image:cellbeads_scheme.jpg|400px|thumb|center|'''Modular and generalizable scheme for attaching beads to cells''']]<br />
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<script src="Scripts/AC_RunActiveContent.js" type="text/javascript"></script><br />
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<body><br />
<p>&nbsp;</p><br />
<p>Some important early considerations for us were therefore: 1) Best to use a scheme that does not require much (or any) genetic manipulation, so that we can use our scheme quickly and easily with any cell or cell type, 2) Use well-established chemistries and reagents, 3) Be able to swap and interchange modular parts.</p><br />
<p>For our pilot experiments this summer, we used the simplest possible parts that we could think of. For cellular attachment, we used the jack bean lectin protein <a href="http://en.wikipedia.org/wiki/Concanavalin_A">Concanavalin A (ConA)</a>, which binds mainly to internal and nonreducing terminal alpha-mannosyl groups. We left the linker out in some cases, though in others we used<a href="http://en.wikipedia.org/wiki/Polyethylene_glycol"> polyethylene glycol (PEG)</a> linkers. For bead attachment, we used either streptavidin-biotin chemistry, passive adsorption of protein on the surface of polystyrene beads, or <a href="http://en.wikipedia.org/wiki/Carbodiimide">carbodiimide chemistry</a> with the crosslinker EDC.</p><br />
<p>To observe the cells, we coated glass-bottomed <a href="http://www.nuncbrand.com/en/page.aspx?ID=235">Lab-Tek II Chambered Coverglass slides</a> with fibronectin, adhered cells to the slide, and then incubated with varying concentrations of modified beads followed by a wash step. We then visualized the cells using time-lapse microscopy. </p><br />
<h3>Results</h3><br />
<p>We have been able to observe our cells exhibiting motility while tethered to &quot;cargos&quot; of fluorescent beads. Below are two representative movies, each taken using fluorescent beads with ConA (no linker). Frame rate is 15 seconds (real time) per frame. </p><br />
</body><br />
</html><br />
<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/t3a3g538hf4&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/t3a3g538hf4&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object></html><br />
<br />
<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/8RtyKD5-Y_0&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/8RtyKD5-Y_0&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object></html><br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
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<title>Untitled Document</title><br />
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</style><br />
<script src="Scripts/AC_ActiveX.js" type="text/javascript"></script><br />
<script src="Scripts/AC_RunActiveContent.js" type="text/javascript"></script><br />
</head><br />
<body><br />
<p>&nbsp;</p><br />
<p>Here are some stillshots from the movies. </p><br />
<p>&nbsp;</p><br />
</body><br />
</html><br />
[[Image:cellbead1_enh.jpg]]<br />
[[Image:cellbead2_enh.jpg]]<br />
[[Image:cellbead3_enh.jpg]]<br />
<br />
<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /><br />
<title>Untitled Document</title><br />
<style type="text/css"><br />
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body {<br />
margin-left: 20px;<br />
margin-right: 20px;<br />
width: 900px;<br />
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--><br />
</style><br />
<script src="Scripts/AC_ActiveX.js" type="text/javascript"></script><br />
<script src="Scripts/AC_RunActiveContent.js" type="text/javascript"></script><br />
</head><br />
<body><br />
<br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that <strong>we can tether simple fluorescent bead &quot;cargos&quot; to our cells</strong>. We are using a simple, generalizable, and modular scheme to attach these cargos, and we will continue to try different attachment and linker parts to improve on our initial result. Some things we are working on include efficiency and robustness of attachment and internalization of beads by cells.</p><br />
<p>We would also like to explore the ability of cells to carry larger bead cargos and different types of cargos (non-bead). In addition, we would like to study the physical limitations of these cells in carrying cargo. For example, it would be particular interesting to determine whether the cells can carry carry cargos through complex environments such as three-dimensional hydrogel environments, endothelial cell monolayers, and more. </p><br />
<p>&nbsp;</p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/SPEED">NEXT - Future Applications</a></strong></p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html><br />
<br />
<br />
<br />
<br />
<br />
{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/PAYLOADTeam:UCSF/PAYLOAD2009-10-21T18:11:28Z<p>Prmagomes: </p>
<hr />
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</head><br />
<body><br />
<h1 align="left">Making Engineered Cells carry a PAYLOAD</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>Once a cellular robot has detected and traveled to a site of disease in the human body, it should be able to perform a useful therapeutic task. One simple but very useful task that we envision our cells performing in the body is the targeted delivery of drugs or signaling molecules to diseased areas. This would be useful in the delivery of all sorts of substances to targeted areas in the body. Some examples include: 1) cases where a drug is toxic and off-target side effects are dose-limiting (e.g. chemotherapeutics used for cancer), 2) cases where precise delivery of a contrast agent to diseased areas would aid in diagnosis (e.g. sites of cancer metastasis), 3) cases where localized delivery of a specific bioactive molecule to a site would be therapeutically beneficial (e.g. delivery of specific growth factors to areas of wound healing and regeneration). </p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html><br />
[[Image:cellbead.jpg|400px|center]]<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /><br />
<title>Untitled Document</title><br />
<style type="text/css"><br />
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margin-right: 20px;<br />
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</style><br />
<script src="Scripts/AC_ActiveX.js" type="text/javascript"></script><br />
<script src="Scripts/AC_RunActiveContent.js" type="text/javascript"></script><br />
</head><br />
<body><br />
<p>&nbsp;</p><br />
<p>Toward the end of engineering our cellular robots to carry useful cargos, we designed and began testing a generalizable scheme for tethering fluorescent polymer microspheres (&quot;beads&quot;) to the outside of our cells. </p><br />
<h3>Approach</h3><br />
<p>We designed a modular and generalizable scheme for attaching fluorescent polymer microspheres to the outside of our cells. To make the approach modular, we thought of our strategy as having three main interchangeable parts: 1) Bead attachment part, 2) Linker, 3) Cell attachment part. By ensuring that we make the parts interchangeable, we made the approach generalizable. For example, changing out the cell attachment part for a different one would allow us to attach beads to a different type of cell. Likewise, we could use different types of linkers including photocleavable or enzymatically cleavable ones. Changing out the bead attachment part could allow us to attach not only beads but other cargos to cells. </p><br />
</body><br />
</html><br />
[[Image:cellbeads_scheme.jpg|400px|thumb|center|'''Modular and generalizable scheme for attaching beads to cells''']]<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
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<script src="Scripts/AC_RunActiveContent.js" type="text/javascript"></script><br />
</head><br />
<body><br />
<p>&nbsp;</p><br />
<p>Some important early considerations for us were therefore: 1) Best to use a scheme that does not require much (or any) genetic manipulation, so that we can use our scheme quickly and easily with any cell or cell type, 2) Use well-established chemistries and reagents, 3) Be able to swap and interchange modular parts.</p><br />
<p>For our pilot experiments this summer, we used the simplest possible parts that we could think of. For cellular attachment, we used the jack bean lectin protein <a href="http://en.wikipedia.org/wiki/Concanavalin_A">Concanavalin A (ConA)</a>, which binds mainly to internal and nonreducing terminal alpha-mannosyl groups. We left the linker out in some cases, though in others we used<a href="http://en.wikipedia.org/wiki/Polyethylene_glycol"> polyethylene glycol (PEG)</a> linkers. For bead attachment, we used either streptavidin-biotin chemistry, passive adsorption of protein on the surface of polystyrene beads, or <a href="http://en.wikipedia.org/wiki/Carbodiimide">carbodiimide chemistry</a> with the crosslinker EDC.</p><br />
<p>To observe the cells, we coated glass-bottomed <a href="http://www.nuncbrand.com/en/page.aspx?ID=235">Lab-Tek II Chambered Coverglass slides</a> with fibronectin, adhered cells to the slide, and then incubated with varying concentrations of modified beads followed by a wash step. We then visualized the cells using time-lapse microscopy. </p><br />
<h3>Results</h3><br />
<p>We have been able to observe our cells exhibiting motility while tethered to &quot;cargos&quot; of fluorescent beads. Below are two representative movies, each taken using fluorescent beads with ConA (no linker). Frame rate is 15 seconds (real time) per frame. </p><br />
</body><br />
</html><br />
<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/t3a3g538hf4&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/t3a3g538hf4&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object></html><br />
<br />
<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/8RtyKD5-Y_0&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/8RtyKD5-Y_0&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object></html><br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
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<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /><br />
<title>Untitled Document</title><br />
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<script src="Scripts/AC_ActiveX.js" type="text/javascript"></script><br />
<script src="Scripts/AC_RunActiveContent.js" type="text/javascript"></script><br />
</head><br />
<body><br />
<p>&nbsp;</p><br />
<p>Here are some stillshots from the movies. </p><br />
<p>&nbsp;</p><br />
</body><br />
</html><br />
[[Image:cellbead1_enh.jpg]]<br />
[[Image:cellbead2_enh.jpg]]<br />
[[Image:cellbead3_enh.jpg]]<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /><br />
<title>Untitled Document</title><br />
<style type="text/css"><br />
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margin-left: 20px;<br />
margin-right: 20px;<br />
width: 900px;<br />
}<br />
--><br />
</style><br />
<script src="Scripts/AC_ActiveX.js" type="text/javascript"></script><br />
<script src="Scripts/AC_RunActiveContent.js" type="text/javascript"></script><br />
</head><br />
<body><br />
<br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that <strong>we can tether simple fluorescent bead &quot;cargos&quot; to our cells</strong>. We are using a simple, generalizable, and modular scheme to attach these cargos, and we will continue to try different attachment and linker parts to improve on our initial result. Some things we are working on include efficiency and robustness of attachment and internalization of beads by cells.</p><br />
<p>We would also like to explore the ability of cells to carry larger bead cargos and different types of cargos (non-bead). In addition, we would like to study the physical limitations of these cells in carrying cargo. For example, it would be particular interesting to determine whether the cells can carry carry cargos through complex environments such as three-dimensional hydrogel environments, endothelial cell monolayers, and more. </p><br />
<p>&nbsp;</p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/SPEED">NEXT - Future Applications</a></strong></p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html><br />
<br />
<br />
<br />
<br />
<br />
{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/PAYLOADTeam:UCSF/PAYLOAD2009-10-21T18:07:48Z<p>Prmagomes: </p>
<hr />
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<script src="Scripts/AC_RunActiveContent.js" type="text/javascript"></script><br />
</head><br />
<body><br />
<h1 align="left">Making Engineered Cells carry a PAYLOAD</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>Once a cellular robot has detected and traveled to a site of disease in the human body, it should be able to perform a useful therapeutic task. One simple but very useful task that we envision our cells performing in the body is the targeted delivery of drugs or signaling molecules to diseased areas. This would be useful in the delivery of all sorts of substances to targeted areas in the body. Some examples include: 1) cases where a drug is toxic and off-target side effects are dose-limiting (e.g. chemotherapeutics used for cancer), 2) cases where precise delivery of a contrast agent to diseased areas would aid in diagnosis (e.g. sites of cancer metastasis), 3) cases where localized delivery of a specific bioactive molecule to a site would be therapeutically beneficial (e.g. delivery of specific growth factors to areas of wound healing and regeneration). </p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html><br />
[[Image:cellbead.jpg|400px|center]]<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /><br />
<title>Untitled Document</title><br />
<style type="text/css"><br />
<!--<br />
body {<br />
margin-left: 20px;<br />
margin-right: 20px;<br />
width: 900px;<br />
}<br />
--><br />
</style><br />
<script src="Scripts/AC_ActiveX.js" type="text/javascript"></script><br />
<script src="Scripts/AC_RunActiveContent.js" type="text/javascript"></script><br />
</head><br />
<body><br />
<p>&nbsp;</p><br />
<p>Toward the end of engineering our cellular robots to carry useful cargos, we designed and began testing a generalizable scheme for tethering fluorescent polymer microspheres (&quot;beads&quot;) to the outside of our cells. </p><br />
<h3>Approach</h3><br />
<p>We designed a modular and generalizable scheme for attaching fluorescent polymer microspheres to the outside of our cells. To make the approach modular, we thought of our strategy as having three main interchangeable parts: 1) Bead attachment part, 2) Linker, 3) Cell attachment part. By ensuring that we make the parts interchangeable, we made the approach generalizable. For example, changing out the cell attachment part for a different one would allow us to attach beads to a different type of cell. Likewise, we could use different types of linkers including photocleavable or enzymatically cleavable ones. Changing out the bead attachment part could allow us to attach not only beads but other cargos to cells. </p><br />
<br />
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<p>&nbsp;</p><br />
<p>Some important early considerations for us were therefore: 1) Best to use a scheme that does not require much (or any) genetic manipulation, so that we can use our scheme quickly and easily with any cell or cell type, 2) Use well-established chemistries and reagents, 3) Be able to swap and interchange modular parts.</p><br />
<p>For our pilot experiments this summer, we used the simplest possible parts that we could think of. For cellular attachment, we used the jack bean lectin protein <a href="http://en.wikipedia.org/wiki/Concanavalin_A">Concanavalin A (ConA)</a>, which binds mainly to internal and nonreducing terminal alpha-mannosyl groups. We left the linker out in some cases, though in others we used<a href="http://en.wikipedia.org/wiki/Polyethylene_glycol"> polyethylene glycol (PEG)</a> linkers. For bead attachment, we used either streptavidin-biotin chemistry, passive adsorption of protein on the surface of polystyrene beads, or <a href="http://en.wikipedia.org/wiki/Carbodiimide">carbodiimide chemistry</a> with the crosslinker EDC.</p><br />
<p>To observe the cells, we coated glass-bottomed <a href="http://www.nuncbrand.com/en/page.aspx?ID=235">Lab-Tek II Chambered Coverglass slides</a> with fibronectin, adhered cells to the slide, and then incubated with varying concentrations of modified beads followed by a wash step. We then visualized the cells using time-lapse microscopy. </p><br />
<h3>Results</h3><br />
<p>We have been able to observe our cells exhibiting motility while tethered to &quot;cargos&quot; of fluorescent beads. Below are two representative movies, each taken using fluorescent beads with ConA (no linker). Frame rate is 15 seconds (real time) per frame. </p><br />
<p>&nbsp;</p><br />
<p>Here are some stillshots from the movies. </p><br />
<p>&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that <strong>we can tether simple fluorescent bead &quot;cargos&quot; to our cells</strong>. We are using a simple, generalizable, and modular scheme to attach these cargos, and we will continue to try different attachment and linker parts to improve on our initial result. Some things we are working on include efficiency and robustness of attachment and internalization of beads by cells.</p><br />
<p>We would also like to explore the ability of cells to carry larger bead cargos and different types of cargos (non-bead). In addition, we would like to study the physical limitations of these cells in carrying cargo. For example, it would be particular interesting to determine whether the cells can carry carry cargos through complex environments such as three-dimensional hydrogel environments, endothelial cell monolayers, and more. </p><br />
<p>&nbsp;</p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/SPEED">NEXT - Future Applications</a></strong></p><br />
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!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/PAYLOADTeam:UCSF/PAYLOAD2009-10-21T17:51:45Z<p>Prmagomes: </p>
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<h1 align="left">Making Engineered Cells carry a PAYLOAD</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>Once a cellular robot has detected and traveled to a site of disease in the human body, it should be able to perform a useful therapeutic task. One simple but very useful task that we envision our cells performing in the body is the targeted delivery of drugs or signaling molecules to diseased areas. This would be useful in the delivery of all sorts of substances to targeted areas in the body. Some examples include: 1) cases where a drug is toxic and off-target side effects are dose-limiting (e.g. chemotherapeutics used for cancer), 2) cases where precise delivery of a contrast agent to diseased areas would aid in diagnosis (e.g. sites of cancer metastasis), 3) cases where localized delivery of a specific bioactive molecule to a site would be therapeutically beneficial (e.g. delivery of specific growth factors to areas of wound healing and regeneration). </p><br />
<p>&nbsp;</p><br />
<p>Toward the end of engineering our cellular robots to carry useful cargos, we designed and began testing a generalizable scheme for tethering fluorescent polymer microspheres (&quot;beads&quot;) to the outside of our cells. </p><br />
<h3>Approach</h3><br />
<p>We designed a modular and generalizable scheme for attaching fluorescent polymer microspheres to the outside of our cells. To make the approach modular, we thought of our strategy as having three main interchangeable parts: 1) Bead attachment part, 2) Linker, 3) Cell attachment part. By ensuring that we make the parts interchangeable, we made the approach generalizable. For example, changing out the cell attachment part for a different one would allow us to attach beads to a different type of cell. Likewise, we could use different types of linkers including photocleavable or enzymatically cleavable ones. Changing out the bead attachment part could allow us to attach not only beads but other cargos to cells. </p><br />
<p>&nbsp;</p><br />
<p>Some important early considerations for us were therefore: 1) Best to use a scheme that does not require much (or any) genetic manipulation, so that we can use our scheme quickly and easily with any cell or cell type, 2) Use well-established chemistries and reagents, 3) Be able to swap and interchange modular parts.</p><br />
<p>For our pilot experiments this summer, we used the simplest possible parts that we could think of. For cellular attachment, we used the jack bean lectin protein <a href="http://en.wikipedia.org/wiki/Concanavalin_A">Concanavalin A (ConA)</a>, which binds mainly to internal and nonreducing terminal alpha-mannosyl groups. We left the linker out in some cases, though in others we used<a href="http://en.wikipedia.org/wiki/Polyethylene_glycol"> polyethylene glycol (PEG)</a> linkers. For bead attachment, we used either streptavidin-biotin chemistry, passive adsorption of protein on the surface of polystyrene beads, or <a href="http://en.wikipedia.org/wiki/Carbodiimide">carbodiimide chemistry</a> with the crosslinker EDC.</p><br />
<p>To observe the cells, we coated glass-bottomed <a href="http://www.nuncbrand.com/en/page.aspx?ID=235">Lab-Tek II Chambered Coverglass slides</a> with fibronectin, adhered cells to the slide, and then incubated with varying concentrations of modified beads followed by a wash step. We then visualized the cells using time-lapse microscopy. </p><br />
<h3>Results</h3><br />
<p>We have been able to observe our cells exhibiting motility while tethered to &quot;cargos&quot; of fluorescent beads. Below are two representative movies, each taken using fluorescent beads with ConA (no linker). Frame rate is 15 seconds (real time) per frame. </p><br />
<p>&nbsp;</p><br />
<p>Here are some stillshots from the movies. </p><br />
<p>&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that <strong>we can tether simple fluorescent bead &quot;cargos&quot; to our cells</strong>. We are using a simple, generalizable, and modular scheme to attach these cargos, and we will continue to try different attachment and linker parts to improve on our initial result. Some things we are working on include efficiency and robustness of attachment and internalization of beads by cells.</p><br />
<p>We would also like to explore the ability of cells to carry larger bead cargos and different types of cargos (non-bead). In addition, we would like to study the physical limitations of these cells in carrying cargo. For example, it would be particular interesting to determine whether the cells can carry carry cargos through complex environments such as three-dimensional hydrogel environments, endothelial cell monolayers, and more. </p><br />
<p>&nbsp;</p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/SPEED">NEXT - Future Applications</a></strong></p><br />
<p align="left">&nbsp;</p><br />
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!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/SPEEDTeam:UCSF/SPEED2009-10-21T17:43:41Z<p>Prmagomes: </p>
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<h1 align="left">Engineering SPEED: Creating Synthetic Brakes and Accelerators</h1><br />
<p align="left">&nbsp;</p><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>Just like we have control over speed in a car – we can brake or accelerate – it would be useful to engineer such behavior into our cellular nanorobots. Just think about it: We could <strong>speed cells up</strong> so that they reach their targets faster and <strong>stop them</strong> once they have arrived or do not behave properly.</p><br />
<p>&nbsp;</p><br />
<h3>Background</h3><br />
<p>For these experiments we chose Dictyostelium discoideum cells to test our prototypical brakes and accelerators quickly. We expect that our brakes and accelerators can be used in a plug and play fashion because Dicty’s way of movement is very similar to a neutrophil’s:</p><br />
<p>When a receptor binds chemoattractant, it induces the conversion of PhosphatidylInositol(4,5)bisphosphate (PIP2) to PhosphatidylInositol(3,4,5)trisphosphate (PIP3) (two signaling lipids in the plasma membrane) at the front of our cells. In a <strong>positive feedback loop</strong> PIP3 triggers the formation of more PIP3 at the front while similarly PIP2 leads to more PIP2 production at the sides and rear of the cell. This system sets the axis of polarity of the cell. The PIP3 patch at the front aligns the actin network and accordingly functions as a ‘turbo boost’ pushing the cell forward. </p><br />
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[[Image:BACKGROUND1.jpg|350px|thumb|center|'''Polarized distribution of PIP3 and PIP2:''' A patch of PIP3 is localized at the front of a cell while PIP2 is at the back. Feedback loops are used to establish and maintain this polarized distribution. ]]<br />
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<h3>Approach</h3><br />
<p>Inspired by nature we tried to build accelerators and brakes by introducing our own synthetic protein based feedback loops. We designed feedback elements by fusing localization and catalytic domains involved in PIP3 production and degradation to modulate localization and level of PIP3 and PIP2 in the cell.</p><br />
<p>Here is an example of a positive feedback loop: a PIP3 binding localization domain fused to a PIP3 producing catalytic domain could produce more PIP3 where there is already PIP3- at the front. This might strengthen polarity and accelerate a cell. </p><br />
<p>Similarly a PIP3 binding domain fused to a PIP2 producing catalytic domain could put PIP2 in place of PIP3 and thereby act as a negative feedback loop.</p><br />
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[[Image:feedbackloops.jpg|600px|thumb|center|'''Synthetic protein based feedback loops:''' <br />
On the left: '''a positive feedback loop''' made of a PIP3 binding domain fused to a PIP3 generating enzyme.<br />
On the right: '''a negative feedback loop''' made of a PIP3 binding domain fused to a PIP2 generating enzyme.]]<br />
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<h3>Results</h3><br />
<p>Over the summer we assembled more than 100 fusions of localization and catalytic domains and screened whether they work. How? We measured the effect our constructs have on motility of Dictyostelium cells: stronger polarity should make cells faster while weaker polarity ought to slow them down!</p><br />
<p><a href="https://2009.igem.org/Matrix_of_Fusion_Constructs">Here is an overview of all feedback loops we screened</a> and the effect they had on the speed of cells. We used automated cell tracking on more than 196 hours worth of movies (note: one movie is 10 minutes!) and identified strains that moved faster or slower at a very stringent statistical cutoff (p&lt;0.0001).</p><br />
<p>This way we were able to identify <strong>7 brakes and 1 accelerator!</strong></p><br />
<p>Check out the movie of one of our strong brakes (<strong><a href="http://www.youtube.com/watch?v=_9od33Nx06Y">BBa_K209066: PTEN-RasC dominant active (da)</a></strong>) compared to <strong><a href="http://www.youtube.com/watch?v=Vtdtf8-zSRs">wildtype</a></strong>.</p><br />
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[[Image:HO230.jpg|400px|thumb|right|'''PTEN fused to RasC da (PIP2 binding - PIP3 generating); speed: 3.5 um/min''' Cells were plated in buffer and basic motile behavior was recorded for 10 minutes taking 1 picture every 15 seconds.]] [[Image:wt.jpg|400px|thumb|left|'''wildtype; speed: 5.9 um/min'''<br />
Cells were plated in buffer and basic motile behavior was recorded for 10 minutes taking 1 picture every 15 seconds.]]<br />
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<p>This is indeed an effect of fusing the particular localization to the catalytic domain as neither of them alone has such a strong effect (see details).<br /><br />
</p><br />
<p>We hypothesize that this construct acts as a <strong>negative feedback loop on PIP2</strong> - (generating PIP3 where PIP2 should be) thereby confusing the cell with multiple fronts: </p><br />
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<h3>Summary and Outlook</h3><br />
<p>We have screened more than 100 synthetic feedback elements for their ability to accelerate or slow down speed of cell motility. We have isolated a hand full of functional elements. Now we need to confirm the mechanism of action of these elements. In the future we would like to make them inducible by a signal from outside – like a stoplight! </p><br />
<p>&nbsp;</p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/PAYLOAD">NEXT - PAYLOAD</a></strong></p><br />
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!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationPart2Team:UCSF/NavigationPart22009-10-21T17:40:25Z<p>Prmagomes: </p>
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<h1 align="left">Engineering NAVIGATION: Tuning Engineered Cells Sensitivity towards New Signals</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>In our experiments, we could control exactly how much ligand was presented to our cells. In &quot;real-life,&quot; however, we would want our cellular robots to be able to respond to a variety of signal strengths: from very low to very high. To accomplish this, we would want to be able to control the sensitivity of our receptors, or how the receptor's output changes when the measured quantity of ligand changes.</p><br />
<h3>Approach</h3><br />
<p>We felt that one key determinant of sensitivity would be the number of receptors present at the plasma membrane of the cell. Therefore, we measured the migration response of a receptor (delta Opioid receptor) whose recycling behavior could be engineered by fusing different recycling interaction modules to the C terminus of the GPCR. We tested a number of such receptor-module fusions for migration response and compared them to receptor alone. The primary assay here was again the Boyden chamber (transwell) assay.</p><br />
<p>&nbsp;</p><br />
<h3>Results</h3><br />
<p>We found that virtually any protein domain/module known to alter the recycling of delta Opioid receptor (DOR) affected cellular migration to a low concentration of ligand (1 nM DADLE). Below, we show two examples of such domains. The actin binding domain from alpha-Actinin-1 (ACTN1ABD) appears to potentiate cellular migration when compared to the wild-type receptor. On the other hand, cellular response is inhibited at this concentration when DOR is bound to a domain of EBP50 (ERMbd) that binds to ERM (ezrin/radixin/moesin) family of proteins. </p><br />
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[[Image:Sensitivity.png|300px|thumb|center|Recycling modules affect sensitivity of DOR]]<br />
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<h3>Summary and Outlook</h3><br />
<p>We have shown that we can tune the sensitivity of a receptor both up and down by fusing it to different recycling modules. Next, we would like to measure recycling directly, and determine whether changes in recycling are necessary for this difference in chemotactic response. All experiments in HL-60 cells would benefit the generation of stable cell lines, which would allow us to more precisely quantify these new behaviors. </p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/SPEED">NEXT - SPEED</a></strong></p><br />
<p align="left">&nbsp;</p><br />
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</html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationPart2Team:UCSF/NavigationPart22009-10-21T17:37:45Z<p>Prmagomes: </p>
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<h1 align="left">Engineering NAVIGATION: Tuning Engineered Cells Sensitivity towards New Signals</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>In our experiments, we could control exactly how much ligand was presented to our cells. In &quot;real-life,&quot; however, we would want our cellular robots to be able to respond to a variety of signal strengths: from very low to very high. To accomplish this, we would want to be able to control the sensitivity of our receptors, or how the receptor's output changes when the measured quantity of ligand changes.</p><br />
<h3>Approach</h3><br />
<p>We felt that one key determinant of sensitivity would be the number of receptors present at the plasma membrane of the cell. Therefore, we measured the migration response of a receptor (delta Opioid receptor) whose recycling behavior could be engineered by fusing different recycling interaction modules to the C terminus of the GPCR. We tested a number of such receptor-module fusions for migration response and compared them to receptor alone. The primary assay here was again the Boyden chamber (transwell) assay.</p><br />
<p>&nbsp;</p><br />
<h3>Results</h3><br />
<p>We found that virtually any protein domain/module known to alter the recycling of delta Opioid receptor (DOR) affected cellular migration to a low concentration of ligand (1 nM DADLE). Below, we show two examples of such domains. The actin binding domain from alpha-Actinin-1 (ACTN1ABD) appears to potentiate cellular migration when compared to the wild-type receptor. On the other hand, cellular response is inhibited at this concentration when DOR is bound to a domain of EBP50 (ERMbd) that binds to ERM (ezrin/radixin/moesin) family of proteins. </p><br />
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[[Image:Sensitivity.png|300px|thumb|center|Recycling modules affect sensitivity of DOR]]<br />
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<h3>Summary and Outlook</h3><br />
<p>We have shown that we can tune the sensitivity of a receptor both up and down by fusing it to different recycling modules. Next, we would like to measure recycling directly, and determine whether changes in recycling are necessary for this difference in chemotactic response. All experiments in HL-60 cells would benefit the generation of stable cell lines, which would allow us to more precisely quantify these new behaviors. </p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/SPEED">NEXT - SPEED</a></strong></p><br />
<p align="left">&nbsp;</p><br />
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</html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationPart2Team:UCSF/NavigationPart22009-10-21T17:36:20Z<p>Prmagomes: </p>
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<h1 align="left">Engineering NAVIGATION: Tuning Engineered Cells Sensitivity towards New Signals</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>In our experiments, we could control exactly how much ligand was presented to our cells. In &quot;real-life,&quot; however, we would want our cellular robots to be able to respond to a variety of signal strengths: from very low to very high. To accomplish this, we would want to be able to control the sensitivity of our receptors, or how the receptor's output changes when the measured quantity of ligand changes.</p><br />
<h3>Approach</h3><br />
<p>We felt that one key determinant of sensitivity would be the number of receptors present at the plasma membrane of the cell. Therefore, we measured the migration response of a receptor (delta Opioid receptor) whose recycling behavior could be engineered by fusing different recycling interaction modules to the C terminus of the GPCR. We tested a number of such receptor-module fusions for migration response and compared them to receptor alone. The primary assay here was again the Boyden chamber (transwell) assay.</p><br />
<p>&nbsp;</p><br />
<h3>Results</h3><br />
<p>We found that virtually any protein domain/module known to alter the recycling of delta Opioid receptor (DOR) affected cellular migration to a low concentration of ligand (1 nM DADLE). Below, we show two examples of such domains. The actin binding domain from alpha-Actinin-1 (ACTN1ABD) appears to potentiate cellular migration when compared to the wild-type receptor. On the other hand, cellular response is inhibited at this concentration when DOR is bound to a domain of EBP50 (ERMbd) that binds to ERM (ezrin/radixin/moesin) family of proteins. </p><br />
<p>&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that we can tune the sensitivity of a receptor both up and down by fusing it to different recycling modules. Next, we would like to measure recycling directly, and determine whether changes in recycling are necessary for this difference in chemotactic response. All experiments in HL-60 cells would benefit the generation of stable cell lines, which would allow us to more precisely quantify these new behaviors. </p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/SPEED">NEXT - SPEED</a></strong></p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationPart2Team:UCSF/NavigationPart22009-10-21T17:35:38Z<p>Prmagomes: </p>
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<h1 align="left">Engineering NAVIGATION: Tuning Cell sensitivity towards new signals</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>In our experiments, we could control exactly how much ligand was presented to our cells. In &quot;real-life,&quot; however, we would want our cellular robots to be able to respond to a variety of signal strengths: from very low to very high. To accomplish this, we would want to be able to control the sensitivity of our receptors, or how the receptor's output changes when the measured quantity of ligand changes.</p><br />
<h3>Approach</h3><br />
<p>We felt that one key determinant of sensitivity would be the number of receptors present at the plasma membrane of the cell. Therefore, we measured the migration response of a receptor (delta Opioid receptor) whose recycling behavior could be engineered by fusing different recycling interaction modules to the C terminus of the GPCR. We tested a number of such receptor-module fusions for migration response and compared them to receptor alone. The primary assay here was again the Boyden chamber (transwell) assay.</p><br />
<p>&nbsp;</p><br />
<h3>Results</h3><br />
<p>We found that virtually any protein domain/module known to alter the recycling of delta Opioid receptor (DOR) affected cellular migration to a low concentration of ligand (1 nM DADLE). Below, we show two examples of such domains. The actin binding domain from alpha-Actinin-1 (ACTN1ABD) appears to potentiate cellular migration when compared to the wild-type receptor. On the other hand, cellular response is inhibited at this concentration when DOR is bound to a domain of EBP50 (ERMbd) that binds to ERM (ezrin/radixin/moesin) family of proteins. </p><br />
<p>&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that we can tune the sensitivity of a receptor both up and down by fusing it to different recycling modules. Next, we would like to measure recycling directly, and determine whether changes in recycling are necessary for this difference in chemotactic response. All experiments in HL-60 cells would benefit the generation of stable cell lines, which would allow us to more precisely quantify these new behaviors. </p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/SPEED">NEXT - SPEED</a></strong></p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/SPEEDTeam:UCSF/SPEED2009-10-21T17:23:54Z<p>Prmagomes: </p>
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<h1 align="left">Engineering SPEED: Creating Synthetic Brakes and Accelerators</h1><br />
<p align="left">&nbsp;</p><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>Just like we have control over speed in a car – we can brake or accelerate – it would be useful to engineer such behavior into our cellular nanorobots. Just think about it: We could <strong>speed cells up</strong> so that they reach their targets faster and <strong>stop them</strong> once they have arrived or do not behave properly.</p><br />
<p>&nbsp;</p><br />
<h3>Background</h3><br />
<p>For these experiments we chose Dictyostelium discoideum cells to test our prototypical brakes and accelerators quickly. We expect that our brakes and accelerators can be used in a plug and play fashion because Dicty’s way of movement is very similar to a neutrophil’s:</p><br />
<p>When a receptor binds chemoattractant, it induces the conversion of PhosphatidylInositol(4,5)bisphosphate (PIP2) to PhosphatidylInositol(3,4,5)trisphosphate (PIP3) (two signaling lipids in the plasma membrane) at the front of our cells. In a <strong>positive feedback loop</strong> PIP3 triggers the formation of more PIP3 at the front while similarly PIP2 leads to more PIP2 production at the sides and rear of the cell. This system sets the axis of polarity of the cell. The PIP3 patch at the front aligns the actin network and accordingly functions as a ‘turbo boost’ pushing the cell forward. </p><br />
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[[Image:BACKGROUND1.jpg|350px|thumb|center|'''Polarized distribution of PIP3 and PIP2:''' A patch of PIP3 is localized at the front of a cell while PIP2 is at the back. Feedback loops are used to establish and maintain this polarized distribution. ]]<br />
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<h3>Approach</h3><br />
<p>Inspired by nature we tried to build accelerators and brakes by introducing our own synthetic protein based feedback loops. We designed feedback elements by fusing localization and catalytic domains involved in PIP3 production and degradation to modulate localization and level of PIP3 and PIP2 in the cell.</p><br />
<p>Here is an example of a positive feedback loop: a PIP3 binding localization domain fused to a PIP3 producing catalytic domain could produce more PIP3 where there is already PIP3- at the front. This might strengthen polarity and accelerate a cell. </p><br />
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[[Image:feedbackloops.jpg|600px|thumb|center|'''Synthetic protein based feedback loops:''' <br />
On the left: '''a positive feedback loop''' made of a PIP3 binding domain fused to a PIP3 generating enzyme.<br />
On the right: '''a negative feedback loop''' made of a PIP3 binding domain fused to a PIP2 generating enzyme.]]<br />
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<h3>Results</h3><br />
<p>Over the summer we assembled more than 100 fusions of localization and catalytic domains and screened whether they work. How? We measured the effect our constructs have on motility of Dictyostelium cells: stronger polarity should make cells faster while weaker polarity ought to slow them down!</p><br />
<p><a href="https://2009.igem.org/Matrix_of_Fusion_Constructs">Here</a> is an overview of all feedback loops we screened and the effect they had on the speed of cells. We used automated cell tracking on more than 196 hours worth of movies (note: one movie is 10 minutes!) and identified strains that moved faster or slower at a very stringent statistical cutoff (p&lt;0.0001).</p><br />
<p>This way we were able to identify <strong>7 brakes and 1 accelerator!</strong></p><br />
<p>Check out the movie of one of our strong brakes (<strong><a href="http://www.youtube.com/watch?v=_9od33Nx06Y">PTEN-RasC dominant active (da)</a></strong>) compared to <strong><a href="http://www.youtube.com/watch?v=Vtdtf8-zSRs">wildtype</a></strong>.</p><br />
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[[Image:HO230.jpg|400px|thumb|right|'''PTEN fused to RasC da (PIP2 binding - PIP3 generating); speed: 3.5 um/min''' Cells were plated in buffer and basic motile behavior was recorded for 10 minutes taking 1 picture every 15 seconds.]] [[Image:wt.jpg|400px|thumb|left|'''wildtype; speed: 5.9 um/min'''<br />
Cells were plated in buffer and basic motile behavior was recorded for 10 minutes taking 1 picture every 15 seconds.]]<br />
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<p>This is indeed an effect of fusing the particular localization to the catalytic domain as neither of them alone has such a strong effect (see details).<br /><br />
</p><br />
<p>We hypothesize that this construct acts as a <strong>negative feedback loop on PIP2</strong> - (generating PIP3 where PIP2 should be) thereby confusing the cell with multiple fronts: </p><br />
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<h3>Summary and Outlook</h3><br />
<p>We have screened more than 100 synthetic feedback elements for their ability to accelerate or slow down speed of cell motility. We have isolated a hand full of functional elements. Now we need to confirm the mechanism of action of these elements. In the future we would like to make them inducible by a signal from outside – like a stoplight! </p><br />
<p>&nbsp;</p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/NavigationPart2">NEXT - PAYLOAD</a></strong></p><br />
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!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/SPEEDTeam:UCSF/SPEED2009-10-21T17:06:01Z<p>Prmagomes: </p>
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<h1 align="left">Engineering SPEED: Creating Synthetic Brakes and Accelerators</h1><br />
<p align="left">&nbsp;</p><br />
</body><br />
</html><br />
=== Motivation: ''Why is this useful?'' ===<br />
Just like we have control over speed in a car – we can brake or accelerate – it would be useful to engineer such behavior into our cellular nanorobots.<br />
Just think about it: We could '''speed cells up''' so that they reach their targets faster and '''stop them''' once they have arrived or do not behave properly.<br />
<br />
=== Background: ===<br />
For these experiments we chose ''Dictyostelium discoideum'' cells to test our prototypical brakes and accelerators quickly. We expect that our brakes and accelerators can be used in a plug and play fashion because Dicty’s way of movement is very similar to a neutrophil’s:<br />
<br />
When a receptor binds chemoattractant, it induces the conversion of PhosphatidylInositol(4,5)bisphosphate (PIP2) to PhosphatidylInositol(3,4,5)trisphosphate (PIP3) (two signaling lipids in the plasma membrane) at the front of our cells. In a '''positive feedback loop''' PIP3 triggers the formation of more PIP3 at the front while similarly PIP2 leads to more PIP2 production at the sides and rear of the cell. This system sets the axis of polarity of the cell. The PIP3 patch at the front aligns the actin network and accordingly functions as a ‘turbo boost’ pushing the cell forward.<br />
<br />
<br />
[[Image:BACKGROUND1.jpg|350px|thumb|center|'''Polarized distribution of PIP3 and PIP2:''' A patch of PIP3 is localized at the front of a cell while PIP2 is at the back. Feedback loops are used to establish and maintain this polarized distribution. ]]<br />
<br />
=== Approach: ===<br />
Inspired by nature we tried to build accelerators and brakes by introducing our own synthetic protein based feedback loops. We designed feedback elements by fusing localization and catalytic domains involved in PIP3 production and degradation to modulate localization and level of PIP3 and PIP2 in the cell. <br />
<br />
Here is an example of a positive feedback loop: a PIP3 binding localization domain fused to a PIP3 producing catalytic domain could produce more PIP3 where there is already PIP3- at the front. This might strengthen polarity and accelerate a cell.<br />
<br />
[[Image:feedbackloops.jpg|600px|thumb|center|'''Synthetic protein based feedback loops:''' <br />
On the left: '''a positive feedback loop''' made of a PIP3 binding domain fused to a PIP3 generating enzyme.<br />
On the right: '''a negative feedback loop''' made of a PIP3 binding domain fused to a PIP2 generating enzyme.]]<br />
<br />
=== Results: ===<br />
Over the summer we assembled more than 100 fusions of localization and catalytic domains and screened whether they work. How? We measured the effect our constructs have on motility of ''Dictyostelium'' cells: stronger polarity should make cells faster while weaker polarity ought to slow them down!<br />
<br />
[[Matrix of Fusion Constructs|Here]] is an overview of all feedback loops we screened and the effect they had on the speed of cells. <br />
We used automated cell tracking on more than 196 hours worth of movies (note: one movie is 10 minutes!) and identified strains that moved faster or slower at a very stringent statistical cutoff (p<0.0001). <br />
<br />
<br />
This way we were able to identify '''7 brakes and 1 accelerator!'''<br />
<br />
Check out the movie of '''one of our strong brakes (PTEN-RasC dominant active (da))[http://www.youtube.com/watch?v=_9od33Nx06Y] compared to wildtype [http://www.youtube.com/watch?v=Vtdtf8-zSRs].''' <br />
<br />
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[[Image:HO230.jpg|400px|thumb|right|'''PTEN fused to RasC da (PIP2 binding - PIP3 generating); speed: 3.5 um/min''' Cells were plated in buffer and basic motile behavior was recorded for 10 minutes taking 1 picture every 15 seconds.]] [[Image:wt.jpg|400px|thumb|left|'''wildtype; speed: 5.9 um/min'''<br />
Cells were plated in buffer and basic motile behavior was recorded for 10 minutes taking 1 picture every 15 seconds.]]<br />
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This is indeed an effect of fusing the particular localization to the catalytic domain as neither of them alone has such a strong effect ([[Further Results|see details]]).<br />
<br />
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We hypothesize that this construct acts as a '''negative feedback loop on PIP2''' - (generating PIP3 where PIP2 should be) thereby confusing the cell with multiple fronts:<br />
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[[Image:brake3.jpg|500px|center]]<br />
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=== Summary and outlook: ===<br />
We have screened more than 100 synthetic feedback elements for their ability to accelerate or slow down speed of cell motility. We have isolated a hand full of functional elements. Now we need to confirm the mechanism of action of these elements. In the future we would like to make them inducible by a signal from outside – like a stoplight!<br />
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<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/NavigationPart2">NEXT - PAYLOAD</a></strong></p><br />
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!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/SPEEDTeam:UCSF/SPEED2009-10-21T17:00:05Z<p>Prmagomes: </p>
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<h1 align="left">Engineering SPEED: Creating Synthetic Brakes and Accelerators</h1><br />
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|}</div>Prmagomeshttp://2009.igem.org/Team:UCSFTeam:UCSF2009-10-21T16:55:56Z<p>Prmagomes: </p>
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<div><!--- The Mission, Experiments ---><br />
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<p align="center"><img src="https://static.igem.org/mediawiki/2009/7/7e/Wiki_2009CellBots.jpg" width="276" height="258" align="middle" /></p><br />
<p align="center" class="style2">Biological Detection and Delivery Systems</p><br />
<blockquote><br />
<br><br />
<h2 align="left">Engineering the Movement of Cellular Robots</h2><br />
<p align="left">Some eukaryotic cells, such as white blood cells, have the amazing ability to sense specific external chemical signals and move toward those signals. This behavior, known as chemotaxis, is a fundamental biological process crucial to such diverse functions as development, wound healing and immune response. In our project, we used a synthetic biology approach to manipulate signaling pathways that mediate chemotaxis in two model organisms:<br> HL-60 (neutrophil-like) cells and the slime mold, Dictyostelium discoideum. </p> <br />
<br />
<p align="left">In doing so, <strong>we have demonstrated that we can regulate both the navigation and speed of our cells, as well as harness their ability to carry a payload.</strong></p><br />
<br />
<p align="left">Through our manipulations, we hope to better understand how these systems work, and eventually to build or reprogram cells that can perform useful tasks. Imagine, for example, therapeutic nanorobots that could home to a directed site in the body and execute complex, user-defined functions (e.g., kill tumors, deliver drugs, guide stem cell migration and differentiation). Alternatively, imagine bioremediation nanorobots that could find and retrieve toxic substances. Such cellular robots could be revolutionary biotechnological tools.</p><br />
<p align="right"><a href="https://2009.igem.org/Team:UCSF/Background">More...</a></p><br />
<p align="right">&nbsp;</p><br />
<table width="870" border="0" cellpadding="3"><br />
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<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/0/02/Wiki_2009project.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>BUILDING CELL-BOTS</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Background">Introduction</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Navigation">Step 1 - Engineering NAVIGATION</a></h4><br />
<ul><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:UCSF/Navigation">Inserting New Sensors</a></li><br />
<li><a href="https://2009.igem.org/Team:UCSF/NavigationPart2">Tuning Sensor Sensitivity</a></li><br />
</ul><br />
</ul><br />
<h4><a href="https://2009.igem.org/Team:UCSF/SPEED">Step 2 - Engineering SPEED</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/PAYLOAD">Step 3 - Carrying a PAYLOAD</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Future Applications">Our Vision for the Future</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
</tr><br />
</table><br />
<table width="870" border="0" cellpadding="3"><br />
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<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/d/db/Wiki_2009Team.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>OUR TEAM</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Team">Team Members</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Notebook">Notebooks</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Our_summer_experience">Summer Experience</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Human Practices">Human Practices</a></h4><br />
<h4><a href="http://dspace.mit.edu/handle/1721.1/46721">NEW BIOBRICK Standard RFC28 - Aar1 Cloning System</a></h4><br />
<h4><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=UCSF">Parts submitted to the Registry</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Gold Medal Requisites">GOLD MEDAL Requisites</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
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</table><br />
<br><br></br></br><br />
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'''UCSF iGEM 2009 is sponsored by...'''<br />
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<html><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/8/85/Wiki_2009Sponsors.jpg" width="690" height="419" align="middle" /></p></html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/SPEEDTeam:UCSF/SPEED2009-10-21T16:54:18Z<p>Prmagomes: </p>
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<h1 align="left">Engineering SPEED: Creating Synthetic Brakes and Accelerators</h1><br />
<p align="left">&nbsp;</p><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>Just like we have control over speed in a car – we can brake or accelerate – it would be useful to engineer such behavior into our cellular nanorobots. Just think about it: We could <strong>speed cells up</strong> so that they reach their targets faster and <strong>stop them</strong> once they have arrived or do not behave properly.</p><br />
<p>&nbsp;</p><br />
<h3>Background</h3><br />
<p>For these experiments we chose Dictyostelium discoideum cells to test our prototypical brakes and accelerators quickly. We expect that our brakes and accelerators can be used in a plug and play fashion because Dicty’s way of movement is very similar to a neutrophil’s:</p><br />
<p>When a receptor binds chemoattractant, it induces the conversion of PhosphatidylInositol(4,5)bisphosphate (PIP2) to PhosphatidylInositol(3,4,5)trisphosphate (PIP3) (two signaling lipids in the plasma membrane) at the front of our cells. In a <strong>positive feedback loop</strong> PIP3 triggers the formation of more PIP3 at the front while similarly PIP2 leads to more PIP2 production at the sides and rear of the cell. This system sets the axis of polarity of the cell. The PIP3 patch at the front aligns the actin network and accordingly functions as a ‘turbo boost’ pushing the cell forward. </p><br />
<blockquote>&nbsp;</blockquote><br />
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[[Image:BACKGROUND1.jpg|350px|thumb|center|'''Polarized distribution of PIP3 and PIP2:''' A patch of PIP3 is localized at the front of a cell while PIP2 is at the back. Feedback loops are used to establish and maintain this polarized distribution. ]]<br />
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</p><br />
<h3>Approach</h3><br />
<p>Inspired by nature we tried to build accelerators and brakes by introducing our own synthetic protein based feedback loops. We designed feedback elements by fusing localization and catalytic domains involved in PIP3 production and degradation to modulate localization and level of PIP3 and PIP2 in the cell.</p><br />
<p>Here is an example of a positive feedback loop: a PIP3 binding localization domain fused to a PIP3 producing catalytic domain could produce more PIP3 where there is already PIP3- at the front. This might strengthen polarity and accelerate a cell. </p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
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[[Image:feedbackloops.jpg|600px|thumb|center|'''Synthetic protein based feedback loops:''' <br />
On the left: '''a positive feedback loop''' made of a PIP3 binding domain fused to a PIP3 generating enzyme.<br />
On the right: '''a negative feedback loop''' made of a PIP3 binding domain fused to a PIP2 generating enzyme.]]<br />
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<h3>Results</h3><br />
<p>Over the summer we assembled more than 100 fusions of localization and catalytic domains and screened whether they work. How? We measured the effect our constructs have on motility of Dictyostelium cells: stronger polarity should make cells faster while weaker polarity ought to slow them down!</p><br />
<p><a href="https://2009.igem.org/Matrix_of_Fusion_Constructs">Here</a> is an overview of all feedback loops we screened and the effect they had on the speed of cells. We used automated cell tracking on more than 196 hours worth of movies (note: one movie is 10 minutes!) and identified strains that moved faster or slower at a very stringent statistical cutoff (p&lt;0.0001).</p><br />
<p>This way we were able to identify <strong>7 brakes and 1 accelerator!</strong></p><br />
<p>Check out the movie of one of our strong brakes (<strong><a href="http://www.youtube.com/watch?v=_9od33Nx06Y">PTEN-RasC dominant active (da)</a></strong>) compared to <strong><a href="http://www.youtube.com/watch?v=Vtdtf8-zSRs">wildtype</a></strong>.</p><br />
<blockquote>&nbsp;</blockquote><br />
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[[Image:HO230.jpg|400px|thumb|right|'''PTEN fused to RasC da (PIP2 binding - PIP3 generating); speed: 3.5 um/min''' Cells were plated in buffer and basic motile behavior was recorded for 10 minutes taking 1 picture every 15 seconds.]] [[Image:wt.jpg|400px|thumb|left|'''wildtype; speed: 5.9 um/min'''<br />
Cells were plated in buffer and basic motile behavior was recorded for 10 minutes taking 1 picture every 15 seconds.]]<br />
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<blockquote>&nbsp;</blockquote><br />
<p>This is indeed an effect of fusing the particular localization to the catalytic domain as neither of them alone has such a strong effect (see details).<br /><br />
</p><br />
<p>We hypothesize that this construct acts as a <strong>negative feedback loop on PIP2</strong> - (generating PIP3 where PIP2 should be) thereby confusing the cell with multiple fronts: </p><br />
<h3>Summary and Outlook</h3><br />
<p>We have screened more than 100 synthetic feedback elements for their ability to accelerate or slow down speed of cell motility. We have isolated a hand full of functional elements. Now we need to confirm the mechanism of action of these elements. In the future we would like to make them inducible by a signal from outside – like a stoplight! </p><br />
<p>&nbsp;</p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/NavigationPart2">NEXT - PAYLOAD</a></strong></p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
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<br />
<br />
<br />
{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/SPEEDTeam:UCSF/SPEED2009-10-21T16:46:00Z<p>Prmagomes: </p>
<hr />
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</style></head><br />
<body><br />
<br />
<h1 align="left">Engineering SPEED: Creating Synthetic Brakes and Accelerators</h1><br />
<p align="left">&nbsp;</p><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>Just like we have control over speed in a car – we can brake or accelerate – it would be useful to engineer such behavior into our cellular nanorobots. Just think about it: We could <strong>speed cells up</strong> so that they reach their targets faster and <strong>stop them</strong> once they have arrived or do not behave properly.</p><br />
<p>&nbsp;</p><br />
<h3>Background</h3><br />
<p>For these experiments we chose Dictyostelium discoideum cells to test our prototypical brakes and accelerators quickly. We expect that our brakes and accelerators can be used in a plug and play fashion because Dicty’s way of movement is very similar to a neutrophil’s:</p><br />
<p>When a receptor binds chemoattractant, it induces the conversion of PhosphatidylInositol(4,5)bisphosphate (PIP2) to PhosphatidylInositol(3,4,5)trisphosphate (PIP3) (two signaling lipids in the plasma membrane) at the front of our cells. In a <strong>positive feedback loop</strong> PIP3 triggers the formation of more PIP3 at the front while similarly PIP2 leads to more PIP2 production at the sides and rear of the cell. This system sets the axis of polarity of the cell. The PIP3 patch at the front aligns the actin network and accordingly functions as a ‘turbo boost’ pushing the cell forward. </p><br />
<p><br /><br />
</p><br />
<h3>Approach</h3><br />
<p>Inspired by nature we tried to build accelerators and brakes by introducing our own synthetic protein based feedback loops. We designed feedback elements by fusing localization and catalytic domains involved in PIP3 production and degradation to modulate localization and level of PIP3 and PIP2 in the cell.</p><br />
<p>Here is an example of a positive feedback loop: a PIP3 binding localization domain fused to a PIP3 producing catalytic domain could produce more PIP3 where there is already PIP3- at the front. This might strengthen polarity and accelerate a cell. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Results</h3><br />
<p>Over the summer we assembled more than 100 fusions of localization and catalytic domains and screened whether they work. How? We measured the effect our constructs have on motility of Dictyostelium cells: stronger polarity should make cells faster while weaker polarity ought to slow them down!</p><br />
<p><a href="https://2009.igem.org/Matrix_of_Fusion_Constructs">Here</a> is an overview of all feedback loops we screened and the effect they had on the speed of cells. We used automated cell tracking on more than 196 hours worth of movies (note: one movie is 10 minutes!) and identified strains that moved faster or slower at a very stringent statistical cutoff (p&lt;0.0001).</p><br />
<p>This way we were able to identify <strong>7 brakes and 1 accelerator!</strong></p><br />
<p>Check out the movie of one of our strong brakes (<strong><a href="http://www.youtube.com/watch?v=_9od33Nx06Y">PTEN-RasC dominant active (da)</a></strong>) compared to <strong><a href="http://www.youtube.com/watch?v=Vtdtf8-zSRs">wildtype</a></strong>.</p><br />
<p>This is indeed an effect of fusing the particular localization to the catalytic domain as neither of them alone has such a strong effect (see details).<br /><br />
</p><br />
<p>We hypothesize that this construct acts as a <strong>negative feedback loop on PIP2</strong> - (generating PIP3 where PIP2 should be) thereby confusing the cell with multiple fronts: </p><br />
<h3>Summary and Outlook</h3><br />
<p>We have screened more than 100 synthetic feedback elements for their ability to accelerate or slow down speed of cell motility. We have isolated a hand full of functional elements. Now we need to confirm the mechanism of action of these elements. In the future we would like to make them inducible by a signal from outside – like a stoplight! </p><br />
<p>&nbsp;</p><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/NavigationPart2">NEXT - PAYLOAD</a></strong></p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
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{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSFTeam:UCSF2009-10-21T16:42:11Z<p>Prmagomes: </p>
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<div><!--- The Mission, Experiments ---><br />
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<p align="center" class="style2">Biological Detection and Delivery Systems</p><br />
<blockquote><br />
<br><br />
<h2 align="left">Engineering the Movement of Cellular Robots</h2><br />
<p align="left">Some eukaryotic cells, such as white blood cells, have the amazing ability to sense specific external chemical signals and move toward those signals. This behavior, known as chemotaxis, is a fundamental biological process crucial to such diverse functions as development, wound healing and immune response. In our project, we used a synthetic biology approach to manipulate signaling pathways that mediate chemotaxis in two model organisms:<br> HL-60 (neutrophil-like) cells and the slime mold, Dictyostelium discoideum. </p> <br />
<br />
<p align="left">In doing so, <strong>we have demonstrated that we can regulate both the navigation and speed of our cells, as well as harness their ability to carry a payload.</strong></p><br />
<br />
<p align="left">Through our manipulations, we hope to better understand how these systems work, and eventually to build or reprogram cells that can perform useful tasks. Imagine, for example, therapeutic nanorobots that could home to a directed site in the body and execute complex, user-defined functions (e.g., kill tumors, deliver drugs, guide stem cell migration and differentiation). Alternatively, imagine bioremediation nanorobots that could find and retrieve toxic substances. Such cellular robots could be revolutionary biotechnological tools.</p><br />
<p align="right"><a href="https://2009.igem.org/Team:UCSF/Background">More...</a></p><br />
<p align="right">&nbsp;</p><br />
<table width="870" border="0" cellpadding="3"><br />
<tr><br />
<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/0/02/Wiki_2009project.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>BUILDING CELL-BOTS</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Background">Introduction</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Navigation">Step 1 - Engineering NAVIGATION</a></h4><br />
<ul><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:UCSF/Navigation">Inserting New Sensors</a></li><br />
<li><a href="https://2009.igem.org/Team:UCSF/NavigationPart2">Tuning Sensor Sensitivity</a></li><br />
</ul><br />
</ul><br />
<h4><a href="https://2009.igem.org/Team:UCSF/SPEED">Step 2 - Engineering SPEED</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/PAYLOAD">Step 3 - Carrying a PAYLOAD</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Future Applications">Our Vision for the Future</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
</tr><br />
</table><br />
<table width="870" border="0" cellpadding="3"><br />
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<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/d/db/Wiki_2009Team.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>OUR TEAM</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Team">Team Members</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Our_summer_experience">Summer Experience</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Notebook">Notebooks</a></h4><br />
<h4><a href="http://dspace.mit.edu/handle/1721.1/46721">NEW BIOBRICK Standard RFC28 - Aar1 Cloning System</a></h4><br />
<h4><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=UCSF">Parts submitted to the Registry</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Gold Medal Requisites">GOLD MEDAL Requisites</a></h4><br />
</blockquote><br />
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<br />
'''UCSF iGEM 2009 is sponsored by...'''<br />
<br />
<br />
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<html><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/8/85/Wiki_2009Sponsors.jpg" width="690" height="419" align="middle" /></p></html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationTeam:UCSF/Navigation2009-10-21T16:07:52Z<p>Prmagomes: </p>
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<h1 align="left">Engineering NAVIGATION: Rewiring the cell to move towards new Targets</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>We envision a cellular robot that could travel to practically any site in the human body. This would provide a flexible platform that could be used for a variety of therapeutic tasks. The first step toward achieving this goal is to broaden the range of possible chemotactic targets for our cells. Ideally, we could connect virtually any input to chemotaxis in a generalized way.</p><br />
<p>&nbsp;</p><br />
<h3>Approach</h3><br />
<p>Neutrophils (a type of white blood cells) sense most of their chemotactic signals through G protein-coupled receptors (GPCRs). The spectrum of chemical signals to which these cells respond is therefore determined, at least in part, by the set of GPCRs they express. Can this spectrum be broadened arbitrarily by the introduction of new GPCRs? We tested this idea by transiently expressing 23 exogenous GPCRs in HL-60 (neutrophil-like) cells.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/6/67/New_targets.jpg" width="400" height="189" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="left">These cells were then tested for their ability to migrate toward ligands for the new GPCRs in multiwell Boyden chamber assays. We measured the fold change in % of cells migrating toward the new ligand (with vs without added GPCR) at the peak response. We refer to this ratio as the &quot;Migration Index.&quot; For receptors that appeared to activate a migration response (Migration Index &gt; 3), we also conducted time-lapse microscopy to determine whether the cell movement was directed toward the gradient of ligand. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Results</h3><br />
<p>6 of the GPCRs we transiently expressed in our cells resulted in a Migration Index &gt; 3.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/a/ac/Fold_Changes.png" width="600" height="284" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>Here, we show an example of one of these receptors (M3/M2 chimera) mediating directional migration up a stable, linear gradient of ligand. Transfected cells are fluorescent, and the concentration of ligand increases in the direction corresponding to the top of the image:</p><br />
<p><a href="https://static.igem.org/mediawiki/2009/a/a9/EZT-qt.mov">Video</a></p><br />
<object width="560" height="340"><param name="movie" value="http://www.youtube.com/v/rB0QQlfa7f4&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/rB0QQlfa7f4&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="560" height="340"></embed></object><br />
<p>To directly compare these cells to those transfected with empty vector, we plotted center-zeroed tracks of individuals cells in each treatment. Qualitatively, cells expressing the chimera tend to move more directly toward the source of ligand. </p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/e/eb/Traces.png" width="800" height="264" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p><strong>Common characteristics of chemotaxis receptors</strong>: All 6 receptors we identified couple to the Gi signaling pathway. The behavior of the M3/M2 chimera, however, suggests that it may be possible to convert receptors with different coupling specificities into chemotaxis receptors. To generate this chimera, the third intracellular loop (i3) from the M3 muscarinic acetylcholine receptor (Gq coupled) was exchanged with that of M2 muscarinic receptor (Gi coupled). It has previously been shown that this chimera now couples to Gi.</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/3/36/Chimera.png" width="493" height="319" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p align="left">Why the i3 loop allows the M3/M2 chimeric receptor to signal to the cell's chemotaxis machinery remains a question for further study. However, the possibility exists that more Gq-coupled (and possibly Gs-coupled) receptors could be converted in this way, thus dramatically increasing the number of potential chemotaxis targets. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that we can program our cells to migrate to new chemical signals by expressing exogenous GPCRs. One of these GPCRs, a chimeric protein, suggests that there may be a way to convert even more GPCRs into chemotaxis receptors. In the future, we are interested in understanding more about why certain receptors mediate chemotaxis while others do not. It would also be interesting to go back to the receptors that did not work, and confirm that they are functional and signaling to other known pathways. </p><br />
<br></br><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/NavigationPart2">NEXT - Tuning receptor sensitivity</a></strong></p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html><br />
<br />
<br />
{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationTeam:UCSF/Navigation2009-10-21T16:07:08Z<p>Prmagomes: </p>
<hr />
<div><html xmlns="http://www.w3.org/1999/xhtml"><br />
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<br></br><br />
<h1 align="left">Engineering NAVIGATION: Rewiring the cell to move towards new Targets</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>We envision a cellular robot that could travel to practically any site in the human body. This would provide a flexible platform that could be used for a variety of therapeutic tasks. The first step toward achieving this goal is to broaden the range of possible chemotactic targets for our cells. Ideally, we could connect virtually any input to chemotaxis in a generalized way.</p><br />
<p>&nbsp;</p><br />
<h3>Approach</h3><br />
<p>Neutrophils (a type of white blood cells) sense most of their chemotactic signals through G protein-coupled receptors (GPCRs). The spectrum of chemical signals to which these cells respond is therefore determined, at least in part, by the set of GPCRs they express. Can this spectrum be broadened arbitrarily by the introduction of new GPCRs? We tested this idea by transiently expressing 23 exogenous GPCRs in HL-60 (neutrophil-like) cells.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/6/67/New_targets.jpg" width="400" height="189" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="left">These cells were then tested for their ability to migrate toward ligands for the new GPCRs in multiwell Boyden chamber assays. We measured the fold change in % of cells migrating toward the new ligand (with vs without added GPCR) at the peak response. We refer to this ratio as the &quot;Migration Index.&quot; For receptors that appeared to activate a migration response (Migration Index &gt; 3), we also conducted time-lapse microscopy to determine whether the cell movement was directed toward the gradient of ligand. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Results</h3><br />
<p>6 of the GPCRs we transiently expressed in our cells resulted in a Migration Index &gt; 3.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/a/ac/Fold_Changes.png" width="600" height="284" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>Here, we show an example of one of these receptors (M3/M2 chimera) mediating directional migration up a stable, linear gradient of ligand. Transfected cells are fluorescent, and the concentration of ligand increases in the direction corresponding to the top of the image:</p><br />
<p><a href="https://static.igem.org/mediawiki/2009/a/a9/EZT-qt.mov">Video</a></p><br />
<object width="560" height="340"><param name="movie" value="http://www.youtube.com/v/rB0QQlfa7f4&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/rB0QQlfa7f4&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="560" height="340"></embed></object><br />
<p>To directly compare these cells to those transfected with empty vector, we plotted center-zeroed tracks of individuals cells in each treatment. Qualitatively, cells expressing the chimera tend to move more directly toward the source of ligand. </p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/e/eb/Traces.png" width="800" height="264" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p><strong>Common characteristics of chemotaxis receptors</strong>: All 6 receptors we identified couple to the Gi signaling pathway. The behavior of the M3/M2 chimera, however, suggests that it may be possible to convert receptors with different coupling specificities into chemotaxis receptors. To generate this chimera, the third intracellular loop (i3) from the M3 muscarinic acetylcholine receptor (Gq coupled) was exchanged with that of M2 muscarinic receptor (Gi coupled). It has previously been shown that this chimera now couples to Gi.</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/3/36/Chimera.png" width="493" height="319" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p align="left">Why the i3 loop allows the M3/M2 chimeric receptor to signal to the cell's chemotaxis machinery remains a question for further study. However, the possibility exists that more Gq-coupled (and possibly Gs-coupled) receptors could be converted in this way, thus dramatically increasing the number of potential chemotaxis targets. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that we can program our cells to migrate to new chemical signals by expressing exogenous GPCRs. One of these GPCRs, a chimeric protein, suggests that there may be a way to convert even more GPCRs into chemotaxis receptors. In the future, we are interested in understanding more about why certain receptors mediate chemotaxis while others do not. It would also be interesting to go back to the receptors that did not work, and confirm that they are functional and signaling to other known pathways. </p><br />
<br></br><br />
<p align="right"><strong><a href="https://2009.igem.org/Team:UCSF/NavigationPart2">NAVIGATION PART 2 - Tuning receptor sensitivity</a></strong></p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html><br />
<br />
<br />
{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSFTeam:UCSF2009-10-21T16:04:08Z<p>Prmagomes: </p>
<hr />
<div><!--- The Mission, Experiments ---><br />
<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
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<p align="center"><img src="https://static.igem.org/mediawiki/2009/7/7e/Wiki_2009CellBots.jpg" width="276" height="258" align="middle" /></p><br />
<p align="center" class="style2">Biological Detection and Delivery Systems</p><br />
<blockquote><br />
<br><br />
<h2 align="left">Engineering the Movement of Cellular Robots</h2><br />
<p align="left">Some eukaryotic cells, such as white blood cells, have the amazing<br />
ability to sense specific external chemical signals, and move toward<br />
those signals. This behavior, known as chemotaxis, is a fundamental<br />
biological process crucial to such diverse functions as development,<br />
wound healing and immune response. Our project focuses on using a<br />
synthetic biology approach to manipulate signaling pathways that mediate<br />
chemotaxis in two model organisms: HL-60 (neutrophil-like) cells and the<br />
slime mold, Dictyostelium discoideum. We are attempting to reprogram<br />
the movements that the cells undergo by altering the guidance and<br />
movement machinery of these cells in a modular way. For example, can we<br />
make cells move faster? Slower? Can we steer them to migrate toward<br />
new signals?<br />
<br />
Through our manipulations, we hope to better understand how these<br />
systems work, and eventually to build or reprogram cells that can<br />
perform useful tasks. Imagine, for example, therapeutic nanorobots that<br />
could home to a directed site in the body and execute complex,<br />
user-defined functions (e.g., kill tumors, deliver drugs, guide stem<br />
cell migration and differentiation). Alternatively, imagine<br />
bioremediation nanorobots that could find and retrieve toxic<br />
substances. Such cellular robots could be revolutionary<br />
biotechnological tools.</p><br />
<p align="right"><a href="https://2009.igem.org/Team:UCSF/Background">More...</a></p><br />
<p align="right">&nbsp;</p><br />
<table width="870" border="0" cellpadding="3"><br />
<tr><br />
<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/0/02/Wiki_2009project.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>BUILDING CELL-BOTS</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Navigation">Step 1 - Engineering NAVIGATION</a></h4><br />
<ul><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:UCSF/Navigation">Inserting New Sensors</a></li><br />
<li><a href="https://2009.igem.org/Team:UCSF/NavigationPart2">Tuning Sensor Sensitivity</a></li><br />
</ul><br />
</ul><br />
<h4><a href="https://2009.igem.org/Team:UCSF/SPEED">Step 2 - Engineering SPEED</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/PAYLOAD">Step 3 - Carrying a PAYLOAD</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Future Applications">Our Vision for the Future</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
</tr><br />
</table><br />
<table width="870" border="0" cellpadding="3"><br />
<tr><br />
<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/d/db/Wiki_2009Team.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>OUR TEAM</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Team">Team Members</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Our_summer_experience">Summer Experience</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Notebook">Notebooks</a></h4><br />
<h4><a href="http://dspace.mit.edu/handle/1721.1/46721">NEW BIOBRICK Standard RFC28 - Aar1 Cloning System</a></h4><br />
<h4><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=UCSF">Parts submitted to the Registry</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Gold Medal Requisites">GOLD MEDAL Requisites</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
</tr><br />
</table><br />
<br><br></br></br><br />
</blockquote><br />
</body><br />
</html><br />
<br />
<br />
'''UCSF iGEM 2009 is sponsored by...'''<br />
<br />
<br />
<br />
<html><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/8/85/Wiki_2009Sponsors.jpg" width="690" height="419" align="middle" /></p></html></div>Prmagomeshttp://2009.igem.org/Team:UCSFTeam:UCSF2009-10-21T16:03:17Z<p>Prmagomes: </p>
<hr />
<div><!--- The Mission, Experiments ---><br />
<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
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font-size: 30px;<br />
font-weight: bold;<br />
}<br />
--><br />
</style></head><br />
<body><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/7/7e/Wiki_2009CellBots.jpg" width="276" height="258" align="middle" /></p><br />
<p align="center" class="style2">Biological Detection and Delivery Systems</p><br />
<blockquote><br />
<br><br />
<h2 align="left">Engineering the Movement of Cellular Robots</h2><br />
<p align="left">Some eukaryotic cells, such as white blood cells, have the amazing<br />
ability to sense specific external chemical signals, and move toward<br />
those signals. This behavior, known as chemotaxis, is a fundamental<br />
biological process crucial to such diverse functions as development,<br />
wound healing and immune response. Our project focuses on using a<br />
synthetic biology approach to manipulate signaling pathways that mediate<br />
chemotaxis in two model organisms: HL-60 (neutrophil-like) cells and the<br />
slime mold, Dictyostelium discoideum. We are attempting to reprogram<br />
the movements that the cells undergo by altering the guidance and<br />
movement machinery of these cells in a modular way. For example, can we<br />
make cells move faster? Slower? Can we steer them to migrate toward<br />
new signals?<br />
<br />
Through our manipulations, we hope to better understand how these<br />
systems work, and eventually to build or reprogram cells that can<br />
perform useful tasks. Imagine, for example, therapeutic nanorobots that<br />
could home to a directed site in the body and execute complex,<br />
user-defined functions (e.g., kill tumors, deliver drugs, guide stem<br />
cell migration and differentiation). Alternatively, imagine<br />
bioremediation nanorobots that could find and retrieve toxic<br />
substances. Such cellular robots could be revolutionary<br />
biotechnological tools.</p><br />
<p align="right"><a href="https://2009.igem.org/Team:UCSF/Background">More...</a></p><br />
<p align="right">&nbsp;</p><br />
<table width="870" border="0" cellpadding="3"><br />
<tr><br />
<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/0/02/Wiki_2009project.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>BUILDING CELL-BOTS</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Navigation">Step 1 - Engineering NAVIGATION</a></h4><br />
<ul><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:UCSF/Navigation">Inserting New Sensors</a></li><br />
<li><a href="https://2009.igem.org/Team:UCSF/Project">Tuning Sensor Sensitivity</a></li><br />
</ul><br />
</ul><br />
<h4><a href="https://2009.igem.org/Team:UCSF/SPEED">Step 2 - Engineering SPEED</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/PAYLOAD">Step 3 - Carrying a PAYLOAD</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Future Applications">Our Vision for the Future</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
</tr><br />
</table><br />
<table width="870" border="0" cellpadding="3"><br />
<tr><br />
<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/d/db/Wiki_2009Team.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>OUR TEAM</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Team">Team Members</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Our_summer_experience">Summer Experience</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Notebook">Notebooks</a></h4><br />
<h4><a href="http://dspace.mit.edu/handle/1721.1/46721">NEW BIOBRICK Standard RFC28 - Aar1 Cloning System</a></h4><br />
<h4><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=UCSF">Parts submitted to the Registry</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Gold Medal Requisites">GOLD MEDAL Requisites</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
</tr><br />
</table><br />
<br><br></br></br><br />
</blockquote><br />
</body><br />
</html><br />
<br />
<br />
'''UCSF iGEM 2009 is sponsored by...'''<br />
<br />
<br />
<br />
<html><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/8/85/Wiki_2009Sponsors.jpg" width="690" height="419" align="middle" /></p></html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/BackgroundTeam:UCSF/Background2009-10-21T15:59:55Z<p>Prmagomes: </p>
<hr />
<div>Our team was inspired by the use of robots which perform tasks that would otherwise be difficult to accomplish. For example, the Mars rovers allow us to make observations and conduct physical experiments in harsh, remote locations otherwise inaccessible to humans. In many ways, the human body represents unexplored territory on a different scale. For example, we may understand many aspects of human disease. However, certain markers of disease are extremely difficult to detect (e.g., primary tumors) and treatment of disease can be hampered by the impracticality of performing invasive surgeries.<br />
<br />
<br />
The "holy grail" of nanomedicine would be to develop microscopic robots that could travel anywhere in the body and perform complex, user-defined tasks. Such devices would have several key advantages over traditional, small-molecule therapies:<br />
<br />
* They could home to specific locations in the body (minimize off-target effects)<br />
* They could make decisions based on their external environment<br />
* They could perform more complicated functions<br />
<br />
<br />
While the idea of microscopic, therapeutic robots may seem far-fetched, there are examples of such machines in nature. For example, neutrophils (a type of white blood cell) are capable of <br />
<br />
* Detecting and homing to a wide range of chemical signals, at times localizing to very specific sites of inflammation<br />
* Triggering a variety of different pathways (extravasation, phagocytosis, apoptosis) in response to external signals<br />
* Navigating through different types of barriers (endothelial tissue, blood-brain barrier, etc)<br />
<br />
<br />
Taking cues from nature, we were interested in harnessing or hijacking the function of complicated, natural cellular robots such as neutrophils to perform therapeutically useful tasks. In other words, we wanted to use neutrophils (or similarly motile cells) as a chassis for engineering. Minimally, we wanted to control how and when these cells move. Over the course of the summer, were were able to:<br />
<br />
* Control the '''NAVIGATION''' system of our cells (alter what the cells pursue, how strong a signal they require)<br />
* Control the '''SPEED''' of our cells (engineer accelerators and brakes)<br />
* Deliver a '''PAYLOAD''' with our cells<br />
<br />
<br />
Our efforts toward these goals are described in the remainder of this site. Throughout our project, we worked with two model organisms: HL-60 (neutrophil-like) cells and ''Dictyostelium discoideum'', a soil-dwelling amoeba that feeds on bacteria. Both cell types are common models for the study of chemotaxis (directed migration toward chemical signals), and each has its distinct advantages with respect to studying specific questions.<br />
<br />
<br />
<br />
{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationTeam:UCSF/Navigation2009-10-21T15:57:43Z<p>Prmagomes: </p>
<hr />
<div><html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /><br />
<title>Untitled Document</title><br />
<style type="text/css"><br />
<!--<br />
body {<br />
margin-left: 20px;<br />
margin-right: 20px;<br />
width: 900px;<br />
}<br />
--><br />
</style></head><br />
<body><br />
<br></br><br />
<h1 align="left">Engineering NAVIGATION: Rewiring the cell to move towards new Targets</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>We envision a cellular robot that could travel to practically any site in the human body. This would provide a flexible platform that could be used for a variety of therapeutic tasks. The first step toward achieving this goal is to broaden the range of possible chemotactic targets for our cells. Ideally, we could connect virtually any input to chemotaxis in a generalized way.</p><br />
<p>&nbsp;</p><br />
<h3>Approach</h3><br />
<p>Neutrophils (a type of white blood cells) sense most of their chemotactic signals through G protein-coupled receptors (GPCRs). The spectrum of chemical signals to which these cells respond is therefore determined, at least in part, by the set of GPCRs they express. Can this spectrum be broadened arbitrarily by the introduction of new GPCRs? We tested this idea by transiently expressing 23 exogenous GPCRs in HL-60 (neutrophil-like) cells.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/6/67/New_targets.jpg" width="400" height="189" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="left">These cells were then tested for their ability to migrate toward ligands for the new GPCRs in multiwell Boyden chamber assays. We measured the fold change in % of cells migrating toward the new ligand (with vs without added GPCR) at the peak response. We refer to this ratio as the &quot;Migration Index.&quot; For receptors that appeared to activate a migration response (Migration Index &gt; 3), we also conducted time-lapse microscopy to determine whether the cell movement was directed toward the gradient of ligand. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Results</h3><br />
<p>6 of the GPCRs we transiently expressed in our cells resulted in a Migration Index &gt; 3.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/a/ac/Fold_Changes.png" width="600" height="284" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>Here, we show an example of one of these receptors (M3/M2 chimera) mediating directional migration up a stable, linear gradient of ligand. Transfected cells are fluorescent, and the concentration of ligand increases in the direction corresponding to the top of the image:</p><br />
<p><a href="https://static.igem.org/mediawiki/2009/a/a9/EZT-qt.mov">Video</a></p><br />
<object width="560" height="340"><param name="movie" value="http://www.youtube.com/v/rB0QQlfa7f4&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/rB0QQlfa7f4&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="560" height="340"></embed></object><br />
<p>To directly compare these cells to those transfected with empty vector, we plotted center-zeroed tracks of individuals cells in each treatment. Qualitatively, cells expressing the chimera tend to move more directly toward the source of ligand. </p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/e/eb/Traces.png" width="800" height="264" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p><strong>Common characteristics of chemotaxis receptors</strong>: All 6 receptors we identified couple to the Gi signaling pathway. The behavior of the M3/M2 chimera, however, suggests that it may be possible to convert receptors with different coupling specificities into chemotaxis receptors. To generate this chimera, the third intracellular loop (i3) from the M3 muscarinic acetylcholine receptor (Gq coupled) was exchanged with that of M2 muscarinic receptor (Gi coupled). It has previously been shown that this chimera now couples to Gi.</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/3/36/Chimera.png" width="493" height="319" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p align="left">Why the i3 loop allows the M3/M2 chimeric receptor to signal to the cell's chemotaxis machinery remains a question for further study. However, the possibility exists that more Gq-coupled (and possibly Gs-coupled) receptors could be converted in this way, thus dramatically increasing the number of potential chemotaxis targets. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that we can program our cells to migrate to new chemical signals by expressing exogenous GPCRs. One of these GPCRs, a chimeric protein, suggests that there may be a way to convert even more GPCRs into chemotaxis receptors. In the future, we are interested in understanding more about why certain receptors mediate chemotaxis while others do not. It would also be interesting to go back to the receptors that did not work, and confirm that they are functional and signaling to other known pathways. </p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html><br />
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{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSFTeam:UCSF2009-10-21T02:11:37Z<p>Prmagomes: </p>
<hr />
<div><!--- The Mission, Experiments ---><br />
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<p align="center"><img src="https://static.igem.org/mediawiki/2009/7/7e/Wiki_2009CellBots.jpg" width="276" height="258" align="middle" /></p><br />
<p align="center" class="style2">Biological Detection and Delivery Systems</p><br />
<blockquote><br />
<br><br />
<h2 align="left">Engineering the Movement of Cellular Robots</h2><br />
<p align="left">Some eukaryotic cells, such as white blood cells, have the amazing<br />
ability to sense specific external chemical signals, and move toward<br />
those signals. This behavior, known as chemotaxis, is a fundamental<br />
biological process crucial to such diverse functions as development,<br />
wound healing and immune response. Our project focuses on using a<br />
synthetic biology approach to manipulate signaling pathways that mediate<br />
chemotaxis in two model organisms: HL-60 (neutrophil-like) cells and the<br />
slime mold, Dictyostelium discoideum. We are attempting to reprogram<br />
the movements that the cells undergo by altering the guidance and<br />
movement machinery of these cells in a modular way. For example, can we<br />
make cells move faster? Slower? Can we steer them to migrate toward<br />
new signals?<br />
<br />
Through our manipulations, we hope to better understand how these<br />
systems work, and eventually to build or reprogram cells that can<br />
perform useful tasks. Imagine, for example, therapeutic nanorobots that<br />
could home to a directed site in the body and execute complex,<br />
user-defined functions (e.g., kill tumors, deliver drugs, guide stem<br />
cell migration and differentiation). Alternatively, imagine<br />
bioremediation nanorobots that could find and retrieve toxic<br />
substances. Such cellular robots could be revolutionary<br />
biotechnological tools.</p><br />
<p align="right"><a href="https://2009.igem.org/Team:UCSF/Background">More...</a></p><br />
<p align="right">&nbsp;</p><br />
<table width="870" border="0" cellpadding="3"><br />
<tr><br />
<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/0/02/Wiki_2009project.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>BUILDING CELL-BOTS</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Navigation">Step 1 - Engineering NAVIGATION</a></h4><br />
<ul><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:UCSF/Project">Inserting New Sensors</a></li><br />
<li><a href="https://2009.igem.org/Team:UCSF/Project">Tuning Sensor Sensitivity</a></li><br />
</ul><br />
</ul><br />
<h4><a href="https://2009.igem.org/Team:UCSF/SPEED">Step 2 - Engineering SPEED</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/PAYLOAD">Step 3 - Carrying a PAYLOAD</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Future Applications">Our Vision for the Future</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
</tr><br />
</table><br />
<table width="870" border="0" cellpadding="3"><br />
<tr><br />
<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/d/db/Wiki_2009Team.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>OUR TEAM</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Team">Team Members</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Our_summer_experience">Summer Experience</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Notebook">Notebooks</a></h4><br />
<h4><a href="http://dspace.mit.edu/handle/1721.1/46721">NEW BIOBRICK Standard RFC28 - Aar1 Cloning System</a></h4><br />
<h4><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=UCSF">Parts submitted to the Registry</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Gold Medal Requisites">GOLD MEDAL Requisites</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
</tr><br />
</table><br />
<br><br></br></br><br />
</blockquote><br />
</body><br />
</html><br />
<br />
<br />
'''UCSF iGEM 2009 is sponsored by...'''<br />
<br />
<br />
<br />
<html><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/8/85/Wiki_2009Sponsors.jpg" width="690" height="419" align="middle" /></p></html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationTeam:UCSF/Navigation2009-10-21T02:08:57Z<p>Prmagomes: </p>
<hr />
<div><html xmlns="http://www.w3.org/1999/xhtml"><br />
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margin-right: 20px;<br />
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<body><br />
<br></br><br />
<h1 align="left">Engineering NAVIGATION: Rewiring the cell to move towards new Targets</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>We envision a cellular robot that could travel to practically any site in the human body. This would provide a flexible platform that could be used for a variety of therapeutic tasks. The first step toward achieving this goal is to broaden the range of possible chemotactic targets for our cells. Ideally, we could connect virtually any input to chemotaxis in a generalized way.</p><br />
<p>&nbsp;</p><br />
<h3>Approach</h3><br />
<p>Neutrophils (a type of white blood cells) sense most of their chemotactic signals through G protein-coupled receptors (GPCRs). The spectrum of chemical signals to which these cells respond is therefore determined, at least in part, by the set of GPCRs they express. Can this spectrum be broadened arbitrarily by the introduction of new GPCRs? We tested this idea by transiently expressing 23 exogenous GPCRs in HL-60 (neutrophil-like) cells.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/6/67/New_targets.jpg" width="400" height="189" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="left">These cells were then tested for their ability to migrate toward ligands for the new GPCRs in multiwell Boyden chamber assays. We measured the fold change in % of cells migrating toward the new ligand (with vs without added GPCR) at the peak response. We refer to this ratio as the &quot;Migration Index.&quot; For receptors that appeared to activate a migration response (Migration Index &gt; 3), we also conducted time-lapse microscopy to determine whether the cell movement was directed toward the gradient of ligand. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Results</h3><br />
<p>6 of the GPCRs we transiently expressed in our cells resulted in a Migration Index &gt; 3.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/a/ac/Fold_Changes.png" width="600" height="284" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>Here, we show an example of one of these receptors (M3/M2 chimera) mediating directional migration up a stable, linear gradient of ligand. Transfected cells are fluorescent, and the concentration of ligand increases in the direction corresponding to the top of the image:</p><br />
<p><a href="https://static.igem.org/mediawiki/2009/a/a9/EZT-qt.mov">Video</a></p><br />
<p>To directly compare these cells to those transfected with empty vector, we plotted center-zeroed tracks of individuals cells in each treatment. Qualitatively, cells expressing the chimera tend to move more directly toward the source of ligand. </p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/e/eb/Traces.png" width="800" height="264" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p><strong>Common characteristics of chemotaxis receptors</strong>: All 6 receptors we identified couple to the Gi signaling pathway. The behavior of the M3/M2 chimera, however, suggests that it may be possible to convert receptors with different coupling specificities into chemotaxis receptors. To generate this chimera, the third intracellular loop (i3) from the M3 muscarinic acetylcholine receptor (Gq coupled) was exchanged with that of M2 muscarinic receptor (Gi coupled). It has previously been shown that this chimera now couples to Gi.</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/3/36/Chimera.png" width="493" height="319" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p align="left">Why the i3 loop allows the M3/M2 chimeric receptor to signal to the cell's chemotaxis machinery remains a question for further study. However, the possibility exists that more Gq-coupled (and possibly Gs-coupled) receptors could be converted in this way, thus dramatically increasing the number of potential chemotaxis targets. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that we can program our cells to migrate to new chemical signals by expressing exogenous GPCRs. One of these GPCRs, a chimeric protein, suggests that there may be a way to convert even more GPCRs into chemotaxis receptors. In the future, we are interested in understanding more about why certain receptors mediate chemotaxis while others do not. It would also be interesting to go back to the receptors that did not work, and confirm that they are functional and signaling to other known pathways. </p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html><br />
<br />
<br />
{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationTeam:UCSF/Navigation2009-10-21T01:55:08Z<p>Prmagomes: </p>
<hr />
<div><html xmlns="http://www.w3.org/1999/xhtml"><br />
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<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /><br />
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<style type="text/css"><br />
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body {<br />
margin-left: 20px;<br />
margin-right: 20px;<br />
width: 900px;<br />
}<br />
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</style></head><br />
<body><br />
<br></br><br />
<h1 align="left">Engineering NAVIGATION: Rewiring the cell to move towards new Targets</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>We envision a cellular robot that could travel to practically any site in the human body. This would provide a flexible platform that could be used for a variety of therapeutic tasks. The first step toward achieving this goal is to broaden the range of possible chemotactic targets for our cells. Ideally, we could connect virtually any input to chemotaxis in a generalized way.</p><br />
<p>&nbsp;</p><br />
<h3>Approach</h3><br />
<p>Neutrophils (a type of white blood cells) sense most of their chemotactic signals through G protein-coupled receptors (GPCRs). The spectrum of chemical signals to which these cells respond is therefore determined, at least in part, by the set of GPCRs they express. Can this spectrum be broadened arbitrarily by the introduction of new GPCRs? We tested this idea by transiently expressing 23 exogenous GPCRs in HL-60 (neutrophil-like) cells.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/6/67/New_targets.jpg" width="400" height="189" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="left">These cells were then tested for their ability to migrate toward ligands for the new GPCRs in multiwell Boyden chamber assays. We measured the fold change in % of cells migrating toward the new ligand (with vs without added GPCR) at the peak response. We refer to this ratio as the &quot;Migration Index.&quot; For receptors that appeared to activate a migration response (Migration Index &gt; 3), we also conducted time-lapse microscopy to determine whether the cell movement was directed toward the gradient of ligand. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Results</h3><br />
<p>6 of the GPCRs we transiently expressed in our cells resulted in a Migration Index &gt; 3.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/a/ac/Fold_Changes.png" width="600" height="284" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>Here, we show an example of one of these receptors (M3/M2 chimera) mediating directional migration up a stable, linear gradient of ligand. Transfected cells are fluorescent, and the concentration of ligand increases in the direction corresponding to the top of the image:</p><br />
<p><a href="https://static.igem.org/mediawiki/2009/a/a9/EZT-qt.mov">Video</a></p><br />
<p>To directly compare these cells to those transfected with empty vector, we plotted center-zeroed tracks of individuals cells in each treatment. Qualitatively, cells expressing the chimera tend to move more directly toward the source of ligand. </p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/e/eb/Traces.png" width="800" height="264" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p><strong>Common characteristics of chemotaxis receptors</strong>: All 6 receptors we identified couple to the Gi signaling pathway. The behavior of the M3/M2 chimera, however, suggests that it may be possible to convert receptors with different coupling specificities into chemotaxis receptors. To generate this chimera, the third intracellular loop (i3) from the M3 muscarinic acetylcholine receptor (Gq coupled) was exchanged with that of M2 muscarinic receptor (Gi coupled). It has previously been shown that this chimera now couples to Gi.</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/3/36/Chimera.png" width="493" height="319" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p align="left">Why the i3 loop allows the M3/M2 chimeric receptor to signal to the cell's chemotaxis machinery remains a question for further study. However, the possibility exists that more Gq-coupled (and possibly Gs-coupled) receptors could be converted in this way, thus dramatically increasing the number of potential chemotaxis targets. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that we can program our cells to migrate to new chemical signals by expressing exogenous GPCRs. One of these GPCRs, a chimeric protein, suggests that there may be a way to convert even more GPCRs into chemotaxis receptors. In the future, we are interested in understanding more about why certain receptors mediate chemotaxis while others do not. It would also be interesting to go back to the receptors that did not work, and confirm that they are functional and signaling to other known pathways. </p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationTeam:UCSF/Navigation2009-10-21T01:53:27Z<p>Prmagomes: </p>
<hr />
<div><html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /><br />
<title>Untitled Document</title><br />
<style type="text/css"><br />
<!--<br />
body {<br />
margin-left: 20px;<br />
margin-right: 20px;<br />
width: 900px;<br />
}<br />
--><br />
</style></head><br />
<body><br />
<br></br><br />
<h1 align="left">Engineering NAVIGATION: Rewiring the cell to move towards new Targets</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>We envision a cellular robot that could travel to practically any site in the human body. This would provide a flexible platform that could be used for a variety of therapeutic tasks. The first step toward achieving this goal is to broaden the range of possible chemotactic targets for our cells. Ideally, we could connect virtually any input to chemotaxis in a generalized way.</p><br />
<p>&nbsp;</p><br />
<h3>Approach</h3><br />
<p>Neutrophils (a type of white blood cells) sense most of their chemotactic signals through G protein-coupled receptors (GPCRs). The spectrum of chemical signals to which these cells respond is therefore determined, at least in part, by the set of GPCRs they express. Can this spectrum be broadened arbitrarily by the introduction of new GPCRs? We tested this idea by transiently expressing 23 exogenous GPCRs in HL-60 (neutrophil-like) cells.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/6/67/New_targets.jpg" width="400" height="189" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="left">These cells were then tested for their ability to migrate toward ligands for the new GPCRs in multiwell Boyden chamber assays. We measured the fold change in % of cells migrating toward the new ligand (with vs without added GPCR) at the peak response. We refer to this ratio as the &quot;Migration Index.&quot; For receptors that appeared to activate a migration response (Migration Index &gt; 3), we also conducted time-lapse microscopy to determine whether the cell movement was directed toward the gradient of ligand. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Results</h3><br />
<p>6 of the GPCRs we transiently expressed in our cells resulted in a Migration Index &gt; 3.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/a/ac/Fold_Changes.png" width="600" height="284" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>Here, we show an example of one of these receptors (M3/M2 chimera) mediating directional migration up a stable, linear gradient of ligand. Transfected cells are fluorescent, and the concentration of ligand increases in the direction corresponding to the top of the image:</p><br />
<p><a href="https://static.igem.org/mediawiki/2009/a/a9/EZT-qt.mov">Video</a></p><br />
<p>To directly compare these cells to those transfected with empty vector, we plotted center-zeroed tracks of individuals cells in each treatment. Qualitatively, cells expressing the chimera tend to move more directly toward the source of ligand. </p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/e/eb/Traces.png" width="800" height="264" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p><strong>Common characteristics of chemotaxis receptors</strong>: All 6 receptors we identified couple to the Gi signaling pathway. The behavior of the M3/M2 chimera, however, suggests that it may be possible to convert receptors with different coupling specificities into chemotaxis receptors. To generate this chimera, the third intracellular loop (i3) from the M3 muscarinic acetylcholine receptor (Gq coupled) was exchanged with that of M2 muscarinic receptor (Gi coupled). It has previously been shown that this chimera now couples to Gi.</p><br />
<br />
<p align="left">Why the i3 loop allows the M3/M2 chimeric receptor to signal to the cell's chemotaxis machinery remains a question for further study. However, the possibility exists that more Gq-coupled (and possibly Gs-coupled) receptors could be converted in this way, thus dramatically increasing the number of potential chemotaxis targets. </p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/3/36/Chimera.png" width="493" height="319" /><br /><br />
</p><br />
<p align="left">&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that we can program our cells to migrate to new chemical signals by expressing exogenous GPCRs. One of these GPCRs, a chimeric protein, suggests that there may be a way to convert even more GPCRs into chemotaxis receptors. In the future, we are interested in understanding more about why certain receptors mediate chemotaxis while others do not. It would also be interesting to go back to the receptors that did not work, and confirm that they are functional and signaling to other known pathways. </p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationTeam:UCSF/Navigation2009-10-21T01:50:14Z<p>Prmagomes: </p>
<hr />
<div><html xmlns="http://www.w3.org/1999/xhtml"><br />
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<br></br><br />
<h1 align="left">Engineering NAVIGATION: Rewiring the cell to move towards new Targets</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>We envision a cellular robot that could travel to practically any site in the human body. This would provide a flexible platform that could be used for a variety of therapeutic tasks. The first step toward achieving this goal is to broaden the range of possible chemotactic targets for our cells. Ideally, we could connect virtually any input to chemotaxis in a generalized way.</p><br />
<p>&nbsp;</p><br />
<h3>Approach</h3><br />
<p>Neutrophils (a type of white blood cells) sense most of their chemotactic signals through G protein-coupled receptors (GPCRs). The spectrum of chemical signals to which these cells respond is therefore determined, at least in part, by the set of GPCRs they express. Can this spectrum be broadened arbitrarily by the introduction of new GPCRs? We tested this idea by transiently expressing 23 exogenous GPCRs in HL-60 (neutrophil-like) cells.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/6/67/New_targets.jpg" width="400" height="189" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="left">These cells were then tested for their ability to migrate toward ligands for the new GPCRs in multiwell Boyden chamber assays. We measured the fold change in % of cells migrating toward the new ligand (with vs without added GPCR) at the peak response. We refer to this ratio as the &quot;Migration Index.&quot; For receptors that appeared to activate a migration response (Migration Index &gt; 3), we also conducted time-lapse microscopy to determine whether the cell movement was directed toward the gradient of ligand. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Results</h3><br />
<p>6 of the GPCRs we transiently expressed in our cells resulted in a Migration Index &gt; 3.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/a/ac/Fold_Changes.png" width="600" height="284" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>Here, we show an example of one of these receptors (M3/M2 chimera) mediating directional migration up a stable, linear gradient of ligand. Transfected cells are fluorescent, and the concentration of ligand increases in the direction corresponding to the top of the image:</p><br />
<p><a href="https://static.igem.org/mediawiki/2009/a/a9/EZT-qt.mov">Video</a></p><br />
<p>To directly compare these cells to those transfected with empty vector, we plotted center-zeroed tracks of individuals cells in each treatment. Qualitatively, cells expressing the chimera tend to move more directly toward the source of ligand. </p><br />
<p>&nbsp;</p><br />
<p><strong>Common characteristics of chemotaxis receptors</strong>: All 6 receptors we identified couple to the Gi signaling pathway. The behavior of the M3/M2 chimera, however, suggests that it may be possible to convert receptors with different coupling specificities into chemotaxis receptors. To generate this chimera, the third intracellular loop (i3) from the M3 muscarinic acetylcholine receptor (Gq coupled) was exchanged with that of M2 muscarinic receptor (Gi coupled). It has previously been shown that this chimera now couples to Gi.</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/e/eb/Traces.png" width="800" height="264" /><br /><br />
</p><br />
<p align="left">Why the i3 loop allows the M3/M2 chimeric receptor to signal to the cell's chemotaxis machinery remains a question for further study. However, the possibility exists that more Gq-coupled (and possibly Gs-coupled) receptors could be converted in this way, thus dramatically increasing the number of potential chemotaxis targets. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that we can program our cells to migrate to new chemical signals by expressing exogenous GPCRs. One of these GPCRs, a chimeric protein, suggests that there may be a way to convert even more GPCRs into chemotaxis receptors. In the future, we are interested in understanding more about why certain receptors mediate chemotaxis while others do not. It would also be interesting to go back to the receptors that did not work, and confirm that they are functional and signaling to other known pathways. </p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationTeam:UCSF/Navigation2009-10-21T01:43:47Z<p>Prmagomes: </p>
<hr />
<div><html xmlns="http://www.w3.org/1999/xhtml"><br />
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<body><br />
<br></br><br />
<h1 align="left">Engineering NAVIGATION: Rewiring the cell to move towards new Targets</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>We envision a cellular robot that could travel to practically any site in the human body. This would provide a flexible platform that could be used for a variety of therapeutic tasks. The first step toward achieving this goal is to broaden the range of possible chemotactic targets for our cells. Ideally, we could connect virtually any input to chemotaxis in a generalized way.</p><br />
<p>&nbsp;</p><br />
<h3>Approach</h3><br />
<p>Neutrophils (a type of white blood cells) sense most of their chemotactic signals through G protein-coupled receptors (GPCRs). The spectrum of chemical signals to which these cells respond is therefore determined, at least in part, by the set of GPCRs they express. Can this spectrum be broadened arbitrarily by the introduction of new GPCRs? We tested this idea by transiently expressing 23 exogenous GPCRs in HL-60 (neutrophil-like) cells.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/6/67/New_targets.jpg" width="400" height="189" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="left">These cells were then tested for their ability to migrate toward ligands for the new GPCRs in multiwell Boyden chamber assays. We measured the fold change in % of cells migrating toward the new ligand (with vs without added GPCR) at the peak response. We refer to this ratio as the &quot;Migration Index.&quot; For receptors that appeared to activate a migration response (Migration Index &gt; 3), we also conducted time-lapse microscopy to determine whether the cell movement was directed toward the gradient of ligand. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Results</h3><br />
<p>6 of the GPCRs we transiently expressed in our cells resulted in a Migration Index &gt; 3.</p><br />
<p>&nbsp;</p><br />
<p>Here, we show an example of one of these receptors (M3/M2 chimera) mediating directional migration up a stable, linear gradient of ligand. Transfected cells are fluorescent, and the concentration of ligand increases in the direction corresponding to the top of the image:</p><br />
<p><a href="https://static.igem.org/mediawiki/2009/a/a9/EZT-qt.mov">Video</a></p><br />
<p>To directly compare these cells to those transfected with empty vector, we plotted center-zeroed tracks of individuals cells in each treatment. Qualitatively, cells expressing the chimera tend to move more directly toward the source of ligand. </p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/a/ac/Fold_Changes.png" width="600" height="284" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>Common characteristics of chemotaxis receptors: All 6 receptors we identified couple to the Gi signaling pathway. The behavior of the M3/M2 chimera, however, suggests that it may be possible to convert receptors with different coupling specificities into chemotaxis receptors. To generate this chimera, the third intracellular loop (i3) from the M3 muscarinic acetylcholine receptor (Gq coupled) was exchanged with that of M2 muscarinic receptor (Gi coupled). It has previously been shown that this chimera now couples to Gi.</p><br />
<p align="left">image place holder</p><br />
<p align="left">Why the i3 loop allows the M3/M2 chimeric receptor to signal to the cell's chemotaxis machinery remains a question for further study. However, the possibility exists that more Gq-coupled (and possibly Gs-coupled) receptors could be converted in this way, thus dramatically increasing the number of potential chemotaxis targets. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that we can program our cells to migrate to new chemical signals by expressing exogenous GPCRs. One of these GPCRs, a chimeric protein, suggests that there may be a way to convert even more GPCRs into chemotaxis receptors. In the future, we are interested in understanding more about why certain receptors mediate chemotaxis while others do not. It would also be interesting to go back to the receptors that did not work, and confirm that they are functional and signaling to other known pathways. </p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationTeam:UCSF/Navigation2009-10-21T01:38:38Z<p>Prmagomes: </p>
<hr />
<div><html xmlns="http://www.w3.org/1999/xhtml"><br />
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</style></head><br />
<body><br />
<br></br><br />
<h1 align="left">Engineering NAVIGATION: Rewiring the cell to move towards new Targets</h1><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>We envision a cellular robot that could travel to practically any site in the human body. This would provide a flexible platform that could be used for a variety of therapeutic tasks. The first step toward achieving this goal is to broaden the range of possible chemotactic targets for our cells. Ideally, we could connect virtually any input to chemotaxis in a generalized way.</p><br />
<p>&nbsp;</p><br />
<h3>Approach</h3><br />
<p>Neutrophils (a type of white blood cells) sense most of their chemotactic signals through G protein-coupled receptors (GPCRs). The spectrum of chemical signals to which these cells respond is therefore determined, at least in part, by the set of GPCRs they express. Can this spectrum be broadened arbitrarily by the introduction of new GPCRs? We tested this idea by transiently expressing 23 exogenous GPCRs in HL-60 (neutrophil-like) cells.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/6/67/New_targets.jpg" width="400" height="189" /><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p align="left">These cells were then tested for their ability to migrate toward ligands for the new GPCRs in multiwell Boyden chamber assays. We measured the fold change in % of cells migrating toward the new ligand (with vs without added GPCR) at the peak response. We refer to this ratio as the &quot;Migration Index.&quot; For receptors that appeared to activate a migration response (Migration Index &gt; 3), we also conducted time-lapse microscopy to determine whether the cell movement was directed toward the gradient of ligand. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Results</h3><br />
<p>6 of the GPCRs we transiently expressed in our cells resulted in a Migration Index &gt; 3.</p><br />
<p>&nbsp;</p><br />
<p>Here, we show an example of one of these receptors (M3/M2 chimera) mediating directional migration up a stable, linear gradient of ligand. Transfected cells are fluorescent, and the concentration of ligand increases in the direction corresponding to the top of the image:</p><br />
<p><a href="https://static.igem.org/mediawiki/2009/a/a9/EZT-qt.mov">Video</a></p><br />
<object width="560" height="340"><param name="movie" value="https://static.igem.org/mediawiki/2009/a/a9/EZT-qt.mov"></param></object><br />
<p>To directly compare these cells to those transfected with empty vector, we plotted center-zeroed tracks of individuals cells in each treatment. Qualitatively, cells expressing the chimera tend to move more directly toward the source of ligand. </p><br />
<p>Image place holder</p><br />
<p>&nbsp;</p><br />
<p>Common characteristics of chemotaxis receptors: All 6 receptors we identified couple to the Gi signaling pathway. The behavior of the M3/M2 chimera, however, suggests that it may be possible to convert receptors with different coupling specificities into chemotaxis receptors. To generate this chimera, the third intracellular loop (i3) from the M3 muscarinic acetylcholine receptor (Gq coupled) was exchanged with that of M2 muscarinic receptor (Gi coupled). It has previously been shown that this chimera now couples to Gi.</p><br />
<p align="left">image place holder</p><br />
<p align="left">Why the i3 loop allows the M3/M2 chimeric receptor to signal to the cell's chemotaxis machinery remains a question for further study. However, the possibility exists that more Gq-coupled (and possibly Gs-coupled) receptors could be converted in this way, thus dramatically increasing the number of potential chemotaxis targets. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that we can program our cells to migrate to new chemical signals by expressing exogenous GPCRs. One of these GPCRs, a chimeric protein, suggests that there may be a way to convert even more GPCRs into chemotaxis receptors. In the future, we are interested in understanding more about why certain receptors mediate chemotaxis while others do not. It would also be interesting to go back to the receptors that did not work, and confirm that they are functional and signaling to other known pathways. </p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationTeam:UCSF/Navigation2009-10-21T01:25:44Z<p>Prmagomes: </p>
<hr />
<div><html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /><br />
<title>Untitled Document</title><br />
<style type="text/css"><br />
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body {<br />
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margin-right: 20px;<br />
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</style></head><br />
<body><br />
<br></br><br />
<h1 align="left">Engineering NAVIGATION: Rewiring the cell to move towards new Targets</h1><br />
<p align="left">&nbsp;</p><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>We envision a cellular robot that could travel to practically any site in the human body. This would provide a flexible platform that could be used for a variety of therapeutic tasks. The first step toward achieving this goal is to broaden the range of possible chemotactic targets for our cells. Ideally, we could connect virtually any input to chemotaxis in a generalized way.<br /><br />
</p><br />
<p>&nbsp;</p><br />
<h3>Approach</h3><br />
<p>Neutrophils (a type of white blood cells) sense most of their chemotactic signals through G protein-coupled receptors (GPCRs). The spectrum of chemical signals to which these cells respond is therefore determined, at least in part, by the set of GPCRs they express. Can this spectrum be broadened arbitrarily by the introduction of new GPCRs? We tested this idea by transiently expressing 23 exogenous GPCRs in HL-60 (neutrophil-like) cells.</p><br />
<p align="center"><img src="wiki_UCSF2009.jpg" width="276" height="258" /><br /><br />
</p><br />
<p align="left">These cells were then tested for their ability to migrate toward ligands for the new GPCRs in multiwell Boyden chamber assays. We measured the fold change in % of cells migrating toward the new ligand (with vs without added GPCR) at the peak response. We refer to this ratio as the &quot;Migration Index.&quot; For receptors that appeared to activate a migration response (Migration Index &gt; 3), we also conducted time-lapse microscopy to determine whether the cell movement was directed toward the gradient of ligand. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Results</h3><br />
<p>6 of the GPCRs we transiently expressed in our cells resulted in a Migration Index &gt; 3.</p><br />
<p>&nbsp;</p><br />
<p>Here, we show an example of one of these receptors (M3/M2 chimera) mediating directional migration up a stable, linear gradient of ligand. Transfected cells are fluorescent, and the concentration of ligand increases in the direction corresponding to the top of the image:</p><br />
<p><a href="http://www.youtube.com/watch?v=rB0QQlfa7f4&amp;feature=player_embedded">Video</a></p><br />
<object width="560" height="340"><param name="movie" value="http://www.youtube.com/v/rB0QQlfa7f4&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/rB0QQlfa7f4&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="560" height="340"></embed></object><br />
<p>To directly compare these cells to those transfected with empty vector, we plotted center-zeroed tracks of individuals cells in each treatment. Qualitatively, cells expressing the chimera tend to move more directly toward the source of ligand. </p><br />
<p>Image place holder</p><br />
<p>&nbsp;</p><br />
<p>Common characteristics of chemotaxis receptors: All 6 receptors we identified couple to the Gi signaling pathway. The behavior of the M3/M2 chimera, however, suggests that it may be possible to convert receptors with different coupling specificities into chemotaxis receptors. To generate this chimera, the third intracellular loop (i3) from the M3 muscarinic acetylcholine receptor (Gq coupled) was exchanged with that of M2 muscarinic receptor (Gi coupled). It has previously been shown that this chimera now couples to Gi.</p><br />
<p align="left">image place holder</p><br />
<p align="left">Why the i3 loop allows the M3/M2 chimeric receptor to signal to the cell's chemotaxis machinery remains a question for further study. However, the possibility exists that more Gq-coupled (and possibly Gs-coupled) receptors could be converted in this way, thus dramatically increasing the number of potential chemotaxis targets. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that we can program our cells to migrate to new chemical signals by expressing exogenous GPCRs. One of these GPCRs, a chimeric protein, suggests that there may be a way to convert even more GPCRs into chemotaxis receptors. In the future, we are interested in understanding more about why certain receptors mediate chemotaxis while others do not. It would also be interesting to go back to the receptors that did not work, and confirm that they are functional and signaling to other known pathways. </p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationTeam:UCSF/Navigation2009-10-21T01:24:11Z<p>Prmagomes: </p>
<hr />
<div><html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /><br />
<title>Untitled Document</title><br />
<style type="text/css"><br />
<!--<br />
body {<br />
margin-left: 20px;<br />
margin-right: 20px;<br />
width: 900px;<br />
}<br />
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</style></head><br />
<body><br />
<br />
<h1 align="left">Engineering NAVIGATION: Rewiring the cell to move towards new Targets</h1><br />
<p align="left">&nbsp;</p><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>We envision a cellular robot that could travel to practically any site in the human body. This would provide a flexible platform that could be used for a variety of therapeutic tasks. The first step toward achieving this goal is to broaden the range of possible chemotactic targets for our cells. Ideally, we could connect virtually any input to chemotaxis in a generalized way.<br /><br />
</p><br />
<p>&nbsp;</p><br />
<h3>Approach</h3><br />
<p>Neutrophils (a type of white blood cells) sense most of their chemotactic signals through G protein-coupled receptors (GPCRs). The spectrum of chemical signals to which these cells respond is therefore determined, at least in part, by the set of GPCRs they express. Can this spectrum be broadened arbitrarily by the introduction of new GPCRs? We tested this idea by transiently expressing 23 exogenous GPCRs in HL-60 (neutrophil-like) cells.</p><br />
<p align="center"><img src="wiki_UCSF2009.jpg" width="276" height="258" /><br /><br />
</p><br />
<p align="left">These cells were then tested for their ability to migrate toward ligands for the new GPCRs in multiwell Boyden chamber assays. We measured the fold change in % of cells migrating toward the new ligand (with vs without added GPCR) at the peak response. We refer to this ratio as the &quot;Migration Index.&quot; For receptors that appeared to activate a migration response (Migration Index &gt; 3), we also conducted time-lapse microscopy to determine whether the cell movement was directed toward the gradient of ligand. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Results</h3><br />
<p>6 of the GPCRs we transiently expressed in our cells resulted in a Migration Index &gt; 3.</p><br />
<p>&nbsp;</p><br />
<p>Here, we show an example of one of these receptors (M3/M2 chimera) mediating directional migration up a stable, linear gradient of ligand. Transfected cells are fluorescent, and the concentration of ligand increases in the direction corresponding to the top of the image:</p><br />
<p><a href="http://www.youtube.com/watch?v=rB0QQlfa7f4&amp;feature=player_embedded">Video</a></p><br />
<object width="560" height="340"><param name="movie" value="http://www.youtube.com/v/rB0QQlfa7f4&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/rB0QQlfa7f4&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="560" height="340"></embed></object><br />
<p>To directly compare these cells to those transfected with empty vector, we plotted center-zeroed tracks of individuals cells in each treatment. Qualitatively, cells expressing the chimera tend to move more directly toward the source of ligand. </p><br />
<p>Image place holder</p><br />
<p>&nbsp;</p><br />
<p>Common characteristics of chemotaxis receptors: All 6 receptors we identified couple to the Gi signaling pathway. The behavior of the M3/M2 chimera, however, suggests that it may be possible to convert receptors with different coupling specificities into chemotaxis receptors. To generate this chimera, the third intracellular loop (i3) from the M3 muscarinic acetylcholine receptor (Gq coupled) was exchanged with that of M2 muscarinic receptor (Gi coupled). It has previously been shown that this chimera now couples to Gi.</p><br />
<p align="left">image place holder</p><br />
<p align="left">Why the i3 loop allows the M3/M2 chimeric receptor to signal to the cell's chemotaxis machinery remains a question for further study. However, the possibility exists that more Gq-coupled (and possibly Gs-coupled) receptors could be converted in this way, thus dramatically increasing the number of potential chemotaxis targets. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that we can program our cells to migrate to new chemical signals by expressing exogenous GPCRs. One of these GPCRs, a chimeric protein, suggests that there may be a way to convert even more GPCRs into chemotaxis receptors. In the future, we are interested in understanding more about why certain receptors mediate chemotaxis while others do not. It would also be interesting to go back to the receptors that did not work, and confirm that they are functional and signaling to other known pathways. </p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/NavigationTeam:UCSF/Navigation2009-10-21T01:23:42Z<p>Prmagomes: New page: <!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> <html xmlns="http://www.w3.org/1999/xhtml"> <head> <meta http-equi...</p>
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<body><br />
<br />
<h1 align="left">Engineering NAVIGATION: Rewiring the cell to move towards new Targets</h1><br />
<p align="left">&nbsp;</p><br />
<h3>Motivation: <em>Why is this useful?</em></h3><br />
<p>We envision a cellular robot that could travel to practically any site in the human body. This would provide a flexible platform that could be used for a variety of therapeutic tasks. The first step toward achieving this goal is to broaden the range of possible chemotactic targets for our cells. Ideally, we could connect virtually any input to chemotaxis in a generalized way.<br /><br />
</p><br />
<p>&nbsp;</p><br />
<h3>Approach</h3><br />
<p>Neutrophils (a type of white blood cells) sense most of their chemotactic signals through G protein-coupled receptors (GPCRs). The spectrum of chemical signals to which these cells respond is therefore determined, at least in part, by the set of GPCRs they express. Can this spectrum be broadened arbitrarily by the introduction of new GPCRs? We tested this idea by transiently expressing 23 exogenous GPCRs in HL-60 (neutrophil-like) cells.</p><br />
<p align="center"><img src="wiki_UCSF2009.jpg" width="276" height="258" /><br /><br />
</p><br />
<p align="left">These cells were then tested for their ability to migrate toward ligands for the new GPCRs in multiwell Boyden chamber assays. We measured the fold change in % of cells migrating toward the new ligand (with vs without added GPCR) at the peak response. We refer to this ratio as the &quot;Migration Index.&quot; For receptors that appeared to activate a migration response (Migration Index &gt; 3), we also conducted time-lapse microscopy to determine whether the cell movement was directed toward the gradient of ligand. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Results</h3><br />
<p>6 of the GPCRs we transiently expressed in our cells resulted in a Migration Index &gt; 3.</p><br />
<p>&nbsp;</p><br />
<p>Here, we show an example of one of these receptors (M3/M2 chimera) mediating directional migration up a stable, linear gradient of ligand. Transfected cells are fluorescent, and the concentration of ligand increases in the direction corresponding to the top of the image:</p><br />
<p><a href="http://www.youtube.com/watch?v=rB0QQlfa7f4&amp;feature=player_embedded">Video</a></p><br />
<object width="560" height="340"><param name="movie" value="http://www.youtube.com/v/rB0QQlfa7f4&hl=en&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/rB0QQlfa7f4&hl=en&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="560" height="340"></embed></object><br />
<p>To directly compare these cells to those transfected with empty vector, we plotted center-zeroed tracks of individuals cells in each treatment. Qualitatively, cells expressing the chimera tend to move more directly toward the source of ligand. </p><br />
<p>Image place holder</p><br />
<p>&nbsp;</p><br />
<p>Common characteristics of chemotaxis receptors: All 6 receptors we identified couple to the Gi signaling pathway. The behavior of the M3/M2 chimera, however, suggests that it may be possible to convert receptors with different coupling specificities into chemotaxis receptors. To generate this chimera, the third intracellular loop (i3) from the M3 muscarinic acetylcholine receptor (Gq coupled) was exchanged with that of M2 muscarinic receptor (Gi coupled). It has previously been shown that this chimera now couples to Gi.</p><br />
<p align="left">image place holder</p><br />
<p align="left">Why the i3 loop allows the M3/M2 chimeric receptor to signal to the cell's chemotaxis machinery remains a question for further study. However, the possibility exists that more Gq-coupled (and possibly Gs-coupled) receptors could be converted in this way, thus dramatically increasing the number of potential chemotaxis targets. </p><br />
<p align="left">&nbsp;</p><br />
<h3>Summary and Outlook</h3><br />
<p>We have shown that we can program our cells to migrate to new chemical signals by expressing exogenous GPCRs. One of these GPCRs, a chimeric protein, suggests that there may be a way to convert even more GPCRs into chemotaxis receptors. In the future, we are interested in understanding more about why certain receptors mediate chemotaxis while others do not. It would also be interesting to go back to the receptors that did not work, and confirm that they are functional and signaling to other known pathways. </p><br />
<p align="left">&nbsp;</p><br />
<blockquote>&nbsp;</blockquote><br />
</body><br />
</html></div>Prmagomeshttp://2009.igem.org/Team:UCSFTeam:UCSF2009-10-21T01:23:32Z<p>Prmagomes: </p>
<hr />
<div><!--- The Mission, Experiments ---><br />
<br />
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<p align="center"><img src="https://static.igem.org/mediawiki/2009/7/7e/Wiki_2009CellBots.jpg" width="276" height="258" align="middle" /></p><br />
<p align="center" class="style2">Biological Detection and Delivery Systems</p><br />
<blockquote><br />
<br><br />
<h2 align="left">Engineering the Movement of Cellular Robots</h2><br />
<p align="left">Some eukaryotic cells, such as white blood cells, have the amazing<br />
ability to sense specific external chemical signals, and move toward<br />
those signals. This behavior, known as chemotaxis, is a fundamental<br />
biological process crucial to such diverse functions as development,<br />
wound healing and immune response. Our project focuses on using a<br />
synthetic biology approach to manipulate signaling pathways that mediate<br />
chemotaxis in two model organisms: HL-60 (neutrophil-like) cells and the<br />
slime mold, Dictyostelium discoideum. We are attempting to reprogram<br />
the movements that the cells undergo by altering the guidance and<br />
movement machinery of these cells in a modular way. For example, can we<br />
make cells move faster? Slower? Can we steer them to migrate toward<br />
new signals?<br />
<br />
Through our manipulations, we hope to better understand how these<br />
systems work, and eventually to build or reprogram cells that can<br />
perform useful tasks. Imagine, for example, therapeutic nanorobots that<br />
could home to a directed site in the body and execute complex,<br />
user-defined functions (e.g., kill tumors, deliver drugs, guide stem<br />
cell migration and differentiation). Alternatively, imagine<br />
bioremediation nanorobots that could find and retrieve toxic<br />
substances. Such cellular robots could be revolutionary<br />
biotechnological tools.</p><br />
<p align="right"><a href="https://2009.igem.org/Team:UCSF/Background">More...</a></p><br />
<p align="right">&nbsp;</p><br />
<table width="870" border="0" cellpadding="3"><br />
<tr><br />
<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/0/02/Wiki_2009project.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>BUILDING CELL-BOTS</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Navigation">Step 1 - Engineering NAVIGATION</a></h4><br />
<ul><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:UCSF/Project">New Sensors</a></li><br />
<li><a href="https://2009.igem.org/Team:UCSF/Project">Sensor Sensitivity</a></li><br />
</ul><br />
</ul><br />
<h4><a href="https://2009.igem.org/Team:UCSF/SPEED">Step 2 - Engineering SPEED</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/PAYLOAD">Step 3 - Carrying a PAYLOAD</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Future Applications">Our Vision of the Future</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
</tr><br />
</table><br />
<table width="870" border="0" cellpadding="3"><br />
<tr><br />
<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/d/db/Wiki_2009Team.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>OUR TEAM</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Team">Team Members</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Our_summer_experience">Summer Experience</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Notebook">Notebooks</a></h4><br />
<h4><a href="http://dspace.mit.edu/handle/1721.1/46721">NEW BIOBRICK Standard RFC28 - Aar1 Cloning System</a></h4><br />
<h4><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=UCSF">Parts submitted to the Registry</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Gold Medal Requisites">GOLD MEDAL Requisites</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
</tr><br />
</table><br />
<br><br></br></br><br />
</blockquote><br />
</body><br />
</html><br />
<br />
<br />
'''UCSF iGEM 2009 is sponsored by...'''<br />
<br />
<br />
<br />
<html><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/8/85/Wiki_2009Sponsors.jpg" width="690" height="419" align="middle" /></p></html></div>Prmagomeshttp://2009.igem.org/Team:UCSFTeam:UCSF2009-10-21T01:02:30Z<p>Prmagomes: </p>
<hr />
<div><!--- The Mission, Experiments ---><br />
<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /><br />
<title>Untitled Document</title><br />
<style type="text/css"><br />
<!--<br />
body {<br />
margin-left: 20px;<br />
margin-right: 20px;<br />
width: 900px;<br />
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.style2 {<br />
font-size: 30px;<br />
font-weight: bold;<br />
}<br />
--><br />
</style></head><br />
<body><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/7/7e/Wiki_2009CellBots.jpg" width="276" height="258" align="middle" /></p><br />
<p align="center" class="style2">Biological Detection and Delivery Systems</p><br />
<blockquote><br />
<br><br />
<h2 align="left">Engineering the Movement of Cellular Robots</h2><br />
<p align="left">Some eukaryotic cells, such as white blood cells, have the amazing<br />
ability to sense specific external chemical signals, and move toward<br />
those signals. This behavior, known as chemotaxis, is a fundamental<br />
biological process crucial to such diverse functions as development,<br />
wound healing and immune response. Our project focuses on using a<br />
synthetic biology approach to manipulate signaling pathways that mediate<br />
chemotaxis in two model organisms: HL-60 (neutrophil-like) cells and the<br />
slime mold, Dictyostelium discoideum. We are attempting to reprogram<br />
the movements that the cells undergo by altering the guidance and<br />
movement machinery of these cells in a modular way. For example, can we<br />
make cells move faster? Slower? Can we steer them to migrate toward<br />
new signals?<br />
<br />
Through our manipulations, we hope to better understand how these<br />
systems work, and eventually to build or reprogram cells that can<br />
perform useful tasks. Imagine, for example, therapeutic nanorobots that<br />
could home to a directed site in the body and execute complex,<br />
user-defined functions (e.g., kill tumors, deliver drugs, guide stem<br />
cell migration and differentiation). Alternatively, imagine<br />
bioremediation nanorobots that could find and retrieve toxic<br />
substances. Such cellular robots could be revolutionary<br />
biotechnological tools.</p><br />
<p align="right"><a href="https://2009.igem.org/Team:UCSF/Background">More...</a></p><br />
<p align="right">&nbsp;</p><br />
<table width="870" border="0" cellpadding="3"><br />
<tr><br />
<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/0/02/Wiki_2009project.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>BUILDING CELL-BOTS</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Project">Step 1 - Engineering NAVIGATION</a></h4><br />
<ul><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:UCSF/Project">New Sensors</a></li><br />
<li><a href="https://2009.igem.org/Team:UCSF/Project">Sensor Sensitivity</a></li><br />
</ul><br />
</ul><br />
<h4><a href="https://2009.igem.org/Team:UCSF/SPEED">Step 2 - Engineering SPEED</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/PAYLOAD">Step 3 - Carrying a PAYLOAD</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Future Applications">Our Vision of the Future</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
</tr><br />
</table><br />
<table width="870" border="0" cellpadding="3"><br />
<tr><br />
<td width="202" height="208"><img src="https://static.igem.org/mediawiki/2009/d/db/Wiki_2009Team.jpg" alt="" width="240" height="224" /></td><br />
<td width="650"><h3>&nbsp;</h3><br />
<blockquote><br />
<h3>OUR TEAM</h3><br />
<blockquote><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Team">Team Members</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Our_summer_experience">Summer Experience</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Notebook">Notebooks</a></h4><br />
<h4><a href="http://dspace.mit.edu/handle/1721.1/46721">NEW BIOBRICK Standard RFC28 - Aar1 Cloning System</a></h4><br />
<h4><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=UCSF">Parts submitted to the Registry</a></h4><br />
<h4><a href="https://2009.igem.org/Team:UCSF/Gold Medal Requisites">GOLD MEDAL Requisites</a></h4><br />
</blockquote><br />
</blockquote><br />
<p>&nbsp;</p></td><br />
</tr><br />
</table><br />
<br><br></br></br><br />
</blockquote><br />
</body><br />
</html><br />
<br />
<br />
'''UCSF iGEM 2009 is sponsored by...'''<br />
<br />
<br />
<br />
<html><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2009/8/85/Wiki_2009Sponsors.jpg" width="690" height="419" align="middle" /></p></html></div>Prmagomeshttp://2009.igem.org/Team:UCSF/Gold_Medal_RequisitesTeam:UCSF/Gold Medal Requisites2009-10-21T00:56:40Z<p>Prmagomes: New page: {| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center" !align="center"|Home !align="ce...</p>
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!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
!align="center"|[[Team:UCSF/Human Practices|Human Practices]]<br />
|}</div>Prmagomes