http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Ryanliang&year=&month=2010.igem.org - User contributions [en]2024-03-29T14:30:09ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:UCSF/TeamTeam:UCSF/Team2010-11-16T22:38:16Z<p>Ryanliang: /* Buddies */</p>
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Our team is composed of 7 students from Lincoln High School, 3 first year students from SF City College and 1 undergraduate from Peking University.<br />
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===Students===<br />
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<center>Images Courtesy of June Park</center><br />
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<h4 style="color:black; font-weight:bold;">Carmen Zhou</h4><br />
My name is Carmen Zhou and I will soon be entering UCSD as a freshman molecular biology major. The seeding of my interest in biotechnology began when I first observed my transformed plate of fluorescing bacteria under a black light. A silly seed, I know, but having been able to see an actual result of one of my experiments first hand was something quite thrilling. This single seed grew as my knowledge of the causes and effects of diseases expanded, which made biology seem more dynamic, disgusting, and like it was begging to be changed. I guess that is where I stand today-on an open field full of possibilities to reverse such diseases.<br />
And that is where iGEM comes in. Although I initially joined iGEM as a means to get to learn more about the techniques bioengineers use and to just get an idea of how research is conducted, I was pleased to find out that my iGEM experience was going to be one of those possibilities in the open field. The combination of health, cancer killing, new techniques, and silly mentors just surpassed my expectations and made this summer unforgettable. I can now see myself zooming through lab work with confidence and even landing a research position as an undergraduate!<br />
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<h4 style="color:black; font-weight:bold;">Lianna Fung</h4><br />
I just recently graduated from Abraham Lincoln High School and am now attending UCSD. I have always been interested in science and the limitless possibilities it can yield. Biotechnology in particular caught my interest after taking a course on it in high school. It fascinated me that we had reached a point where we could modify and improve upon the biology of organisms for specific purposes. This fascination caused me to seek out more opportunities to learn and improve my experience in the field. This led me to my desire to participate in iGEM. iGEM seemed a perfect combination of what I wanted and more. It was a chance to work with others as a team in a welcoming environment. It also gave me the chance to learn new skills that I would normally not be exposed to until further down in my education.<br />
My iGEM experience was interesting. It could be tiring and frustrating at times, but it was also very fun and rewarding. iGEM also creates a sort of independence in people that isn’t as easy to find in the classroom. Overall, the iGEM experience was wonderful and well worth the long hours spent on it. As a bonus, I got to meet some very interesting people as well and make some good friends too.<br />
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<h4 style="color:black; font-weight:bold;">Connor Grant</h4><br />
My name is Connor Grant and I just graduated from Abraham Lincoln High School and will be a freshman at UCSD next year. I almost didn't join iGEM because it conflicted with soccer, but in the end I decided (with some encouragement from my biotech teacher) to spend the summer in the lab at UCSF. iGEM was a great way to learn lab techniques that are used very often in company and University research all over the world. It was good to get experience working in a lab and interacting with post docs and PHD students. It was a good experience over all and I'm glad I did it.<br />
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<h4 style="color:black; font-weight:bold;">Hannah Yan</h4><br />
I recently graduated from Abraham Lincoln High School of San Francisco and am attending Barnard College. For me, iGEM was a chance for me to see if I wanted get into the field of science and to do something awesome during the summer. It really has been a great experience, with its ups and downs. Despite all the failures I have encountered while doing my cloning and minipreps, the ecstatic feeling I get when something worked made everything worthwhile. During iGEM, I was able to use the skills I had learned in biotechnology class and I learned a few new ones as well. I think my experience at iGEM will help me a lot when I decide to pursue the field of science in the future. My summer has been extremely academic, but being able to work with friends and on such a great project has been a lot of fun.<br />
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<h4 style="color:black; font-weight:bold;">John Elam</h4><br />
Hey, I'm John Elam from this year's UCSF iGEM team. I was born and raised in San Francisco and am currently attending UC Davis, majoring in molecular biology and biochemistry. I joined iGEM because I knew it would be a great chance to actually do some real lab work before college, and also because I really liked the basic idea for the project that got pitched to us in May. As of right now a four year degree from Davis is all that I am certain about; as for medical school or graduate school, I'd certainly like to go but you never know what will happen in the future. I played football for three years in high school but currently have no plans to play in college. I'm looking forward to presenting at the Jamboree and hope to learn a lot while I'm there.<br />
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<h4 style="color:black; font-weight:bold;">Crystal Liu</h4><br />
Hello! I'm Crystal Liu, and I am currently an undergraduate at UC Davis majoring in Biochemistry and Molecular Biology. I first heard about the UCSF iGEM team as a freshman in high school, and decided that it was an experience I really wanted to be a part of at the end of my senior year. Four years later, I made it onto the team! I definitely had one of the best summers of my life. Working in a lab really opened my eyes to the world of research and helped me understand firsthand why progress and results aren't immediate. In addition to labwork, I am extremely grateful to have met all the amazing people from the Lim Lab & CPL, as well as everyone else who contributed their impressive skills and knowledge to the 2010 UCSF iGEM team. :) On a side note, I love expensive chocolate.<br />
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<h4 style="color:black; font-weight:bold;">Sam Zorn</h4><br />
Although am still a senior at Abraham Lincoln High School, I am an active participant on the iGEM team. Lab work has been my passion for the last few years of my life. When I was picked for the iGEM team, I was ecstatic. Since that day I have been committed to working as hard as I could to make our project successful. iGEM has given me a real world career experience that has helped me decide on my academic path. The field of synthetic biology has appealed to me greatly, and this summer that I spent at iGEM has more than fulfilled my expectations. I hope that my summer here will not only help me get into college, but also help kick start my career as a bench researcher. When i'm neither working in the labs nor at school, I enjoy getting out and being active. My favorite sports are soccer, and parkour, although I also like to run track, swim, and spar in various martial arts.<br />
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===International Student===<br />
<h4 style="color:black; font-weight:bold;">Min Lin</h4><br />
I'm Min Lin from China; I just got my Bachelor degree in Biological Science from Peking University. This is my second year in iGEM; I'm also in Peking University iGEM 2009 team. I think I've learned a lot in iGEM, and the knowledge will be really useful to me in the future. I like our project this year, because I feel that medicine is a very promising field for synthetic biology applications, although we are still at very first steps. I've been enjoying the nice environment and weather in San Francisco during the summer. And it is really an unforgettable experience to work in iGEM in the Cell Propulsion Lab.<br />
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===Super Buddies===<br />
<h4 style="color:black; font-weight:bold;">Ryan Liang</h4><br />
This is my second year of iGEM and I must say that I am excited to do this for a second round! I am a student in City College of San Francisco with an intent on transferring to UC Davis. Early on I had a passion for the arts and pursued it up until high school where I was introduced to biotechnology and science as an industry. This is when I realized that science is more than just reading and memorization - it is life and the most basic fundamentals of each and every one of us. iGEM allowed me to bring forth my passion for creativity in correlation with synthetic biology. iGEM has encapsulated synthetic biology into such a fun and innovative experience that getting the opportunity to participate once again is truly a blessing.<br />
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<h4 style="color:black; font-weight:bold;">Ethan Chan</h4><br />
IGEM has always been such a great opportunity for all the teams to come up with project ideas are that out of the ordinary. The idea of developing a project that has never been done before is the most exciting part for me. I believe that the work done by iGEM teams have impacted the field so much that it will only get better. I am currently working towards attaining a Biotechnology degree at City College of San Francisco. I am hoping to transfer to UC Davis afterwards. After finding out UCSF's project for the 2010 year, I knew I wanted to come back. Throughout this summer, I have learned to work with mammalian cells. Before the iGEM experience, I was only familiar with prokaryotic cells. iGEM has expanded my skills and knowledge in so many ways. All the skills that i have learned will definitely help me in the future. With all these great pharmaceuticals that are being produced, the field of synthetic biology have proven its effectiveness and its future applications. I am looking forward to being a part of this field in the near future.<br />
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<h4 style="color:black; font-weight:bold;">Eric Wong</h4><br />
Good Afternoon, my name is Eric Wong and i am a graduate of Abraham Lincoln High School class of 2009. I am current pursuing my education in CCSF hoping to transfer into the UC system with the intended major of Molecular Cell Development. I was apart of the iGem team last year and in the time span of 4 months i have learned so many various things from running Assays, Cloning, Troubleshooting and analyzing Experiments and Data. I enjoyed the experience so much so that i decided to come back this year as a mentor for this year's team. I view this and last years iGem experience as something more than just a competition, I has given me the necessary education experience to prepare me for this field of study and allow me to learn about different project from various teams.<br><br><br />
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===UCSF iGEM Program===<br />
<h4 style="color:black; font-weight:bold;">Raquel Gomes</h4><br />
I run the UCSF iGEM program for the last three years. I really enjoy designing all the educational components of the Program but especially the 2-week bootcamp. I love teaching and having the iGEM students around all summer. I will miss all their craziness.<br />
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===Advisors===<br />
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<h4 style="color:black; font-weight:bold;">James Onuffer</h4><br />
This is my second year of involvement and the second year the Cell Propulsion Lab has hosted the team. It was quite an experience to set up the program this year, especially since this is a subject area that we had not begun working on in the Cell Propulsion Lab. The immune response to cancer is a challenging topic to take on (especially coupled with synthetic biology) and required an intensive two week bootcamp to teach the students basic concepts and endogenous systems/parts that they should be aware of. We challenged the students to come up with designs for synthetic cytotoxicity logic gates and increasing the cytotoxic response. It was quite rewarding to see them propose various devices during their team challenge and to see them take charge of getting them prioritized, made , and tested. Things did not always go smoothly, after all this goes with the territory. They had to learn to be organized, think on their feet, and be problem solvers.......quite a growth opportunity that I’m sure will stay with them for the rest of their lives. <br />
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<h4 style="color:black; font-weight:bold;">Wendell Lim</h4><br />
I have had a lab at UCSF for 15 years. The most rewarding thing is working with bright, open-minded young scientists and seeing them develop. Its great to see that most of the iGEM kids that we have worked with have continued to be excited about science and synthetic biology.<br />
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===Buddies===<br />
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<h4 style="color:black; font-weight:bold;">Jason Park</h4><br />
I am a 5th year MD/PhD student in the UCSF School of Medicine and the UCSF / UC Berkeley Joint Graduate Group in Bioengineering. I'm originally from the Los Angeles area and went to college at MIT. I've been in the Cell Propulsion Lab for about two years and I am co-advised by Dr. Wendell Lim and Dr. Bruce Conklin. I became a buddy for iGEM because I thought it would be fun working with bright, motivated high school and undergraduate students for the summer and I knew I would get good experience learning to be a better mentor. (Also, I had been interested in participating in iGEM as an undergraduate at MIT but never ended up doing it!) The best thing about being part of iGEM this summer has been working with and teaching students - together going through the learning process of doing lab research with all of its ups and downs.<br />
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<h4 style="color:black; font-weight:bold;">Chia-Yung Wu</h4><br />
I am a transplant from MIT, where I participated in iGEM as a graduate advisor for the MIT team in 2008 and 2009. This summer, I joined the Lim/Cell Propulsion labs as a postdoc. It has been a great pleasure working with the UCSF team.<br />
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<h4 style="color:black; font-weight:bold;">Russell Gordley</h4><br />
I majored in Biochemistry at Swarthmore College, and performed my graduate studies with Carlos Barbas at the Scripps Research Institute in La Jolla, CA. I am interested in using synthetic biology to understand and enhance the evolvability of biological systems.<br />
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After my freshman year in college, I took a summer internship with Jim Stivers at the National Institute of Standards and Technology. The practice of research turned out to be far more complex and interesting than I could have imagined--an intellectual marathon whose path is revealed with each new data set and disproven hypothesis. I am happy to participate in a program that immerses high school students in the research process. For anyone interested in a life of science, this is a great time to join the pursuit!<br />
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<h4 style="color:black; font-weight:bold;">Jesse Zalatan</h4><br />
I got started in science research with a summer internship after my junior year in high school and I was hooked! I majored in Biochemical Sciences at Harvard and went on to get my Ph.D. in Chemistry at Stanford, where I studied how enzymes speed up chemical reactions that are otherwise incredibly slow. My current research at UCSF is focused on cell signaling, where enzymes play an important role. I am trying to understand how signaling enzymes maintain specificity for the correct targets and avoid signaling mixups. Being introduced to science research in high school inspired me to pursue science in college and beyond, and I got involved with the iGEM team to help share that experience with new students.<br />
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<h4 style="color:black; font-weight:bold;">Wilson Wong</h4><br />
My name is Wilson Wong and I am a postdoc at Wendell Lim's lab. I got my undergraduate degree in chemical engineering at UC Berkeley and graduate degree in chemical engineering at UCLA. I am currently working on applying synthetic biology to rewire human T-cell receptor signaling dynamics. I am interested in helping iGEM because I have always enjoy teaching and mentoring. My iGEM experience has been great. I am very happy with the progress they've made so far and I have a wonderful time working with them.<br />
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<h4 style="color:black; font-weight:bold;">Krista McNally</h4><br />
I am an Associate Specialist at UCSF and handle the Tissue Culture needs for the Cell Propulsion Laboratory. Before coming to UCSF, I was a Research Associate at a Medical Device company performing immunohistochemisty, histology, and thermal imaging. This is the first time I’ve encountered the iGEM program, and it was a great experience to see these young people work so hard as a team to accomplish a meaningful project. During the course of the summer, I gave a lecture to the Team on Tissue Culture and relevant Biosafety concerns, trained some of the students individually to do tissue culture work, and assisted with their use of the Flow Cytometer. I was really impressed with their commitment and diligence during the summer, and enjoyed having so many upbeat and energetic people in the lab. <br />
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<h4 style="color:black; font-weight:bold;">Silinda Neou</h4><br />
I majored in Biochemistry from Cal State East Bay and now work at UCSF in the Cell Propulsion Lab. I love scientific research and have been chasing a career in research ever since my first internship at a microbiology lab. This summer I got the pleasure of interacting with the iGEM students and watching their overall scientific development. They are a great bunch of kids.<br />
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<h4 style="color:black; font-weight:bold;">Jared Toettcher</h4><br />
http://limlab.ucsf.edu/<br />
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<h4 style="color:black; font-weight:bold;">Stacy Fang</h4><br />
http://limlab.ucsf.edu/<br />
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<h4 style="color:black; font-weight:bold;">Samantha Liang</h4><br />
I’m currently a second-year graduate student at UCSF in the Biochemistry and Molecular Biology Program, where I am a member of Zev Gartner’s lab. My research involves using chemical techniques to make protein therapeutics more specific and effective. During my undergrad, I majored in Bioengineering at UC Berkeley and worked in Chris Anderson’s lab for 3 years. At Berkeley, I was on the iGEM team in 2006 and loved it so much that I joined again in 2007. I think that iGEM is a unique opportunity for students to conduct research in a fun team context, and also gives them to skills to perform research independently in the future. Because of this, I wanted to stay involved in iGEM during grad school, and that’s why I’m a buddy for the awesome UCSF team this year!<br />
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===Instructors===<br />
'''Special thanks to the following instructors for the seminars you provided during our bootcamp!'''<br><br />
<h4 style="color:black; font-weight:bold;">Derek Wong</h4><br />
Taunton Lab<br><br />
http://cmp.ucsf.edu/faculty/pdb_show.html?id=taunton<br><br />
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<h4 style="color:black; font-weight:bold;">Dan Hostetter</h4><br />
Craik Lab<br><br />
http://www.craiklab.ucsf.edu<br><br />
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<h4 style="color:black; font-weight:bold;">David Pincus</h4><br />
El-Samad Lab<br><br />
http://biochemistry.ucsf.edu/labs/elsamad/people/people.html<br><br />
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<h4 style="color:black; font-weight:bold;">Reid Williams</h4><br />
Lim Lab<br><br />
http://limlab.ucsf.edu/<br><br />
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{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/PartsTeam:UCSF/Parts2010-11-15T22:29:17Z<p>Ryanliang: </p>
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<h3 style="font-weight:bold;">Parts submitted to the Registry</h3><br />
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<groupparts>iGEM010 UCSF</groupparts><br />
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<p>During the summer we built more than 60 devices under the BioBrick standard <a href="https://2009.igem.org/Judging/Variance/UCSF" target="_blank">RFC28</a> (see more info <a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf" target="_blank">here</a>), which was developed by the UCSF team in 2009 and was approved by the Registry of Parts. However, this year the Registry does not accept RFC28. Due to time constrains, we could not move all of these parts into pSB1C3 and submit them as we had planned.</p><br><br />
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{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSFTeam:UCSF2010-11-15T22:27:02Z<p>Ryanliang: /* Project Description */</p>
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===Project Description===<br />
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Killer cells of the immune system identify cancer and pathogen-infected cells and kill them. These potent killers travel throughout the body, recognizing proteins and other molecules on the surface of cells. In order to differentiate between healthy and diseased cells, killer cells use a variety of receptors, which bind to specific ligands on the target cells’ surface. If the target cell is deemed potentially dangerous, the killer cell grips the target cell tightly and creates an immunological synapse at the site of adhesion. Within this immunological synapse, the killer cell releases cytotoxic granules to kill the target cell without harming nearby cells, triggering a directed apoptotic response.<br />
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Our team will focus on improving killer cells’ specificity and killing efficiency towards cancerous target cells. By using tools of synthetic biology, we hope to create powerful killing bio-machines to fight cancer. Our newly engineered synthetic devices would have the potential to enhance current adoptive cell-based immunotherapy for cancer patients.<br />
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<h3 style="color:black;">TEAM</h3><br />
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<h3 style="color:black;">SPONSORS</h3><br />
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<h5 style="color:black;">Follow us on <a href="http://twitter.com/#!/iGEM_UCSF">Twitter!</a></h5><br />
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__NOTOC__</div>Ryanlianghttp://2010.igem.org/Team:UCSF/TeamTeam:UCSF/Team2010-11-15T22:25:29Z<p>Ryanliang: /* Buddies */</p>
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Our team is composed of 7 students from Lincoln High School, 3 first year students from SF City College and 1 undergraduate from Peking University.<br />
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===Students===<br />
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<center>Images Courtesy of June Park</center><br />
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<h4 style="color:black; font-weight:bold;">Carmen Zhou</h4><br />
My name is Carmen Zhou and I will soon be entering UCSD as a freshman molecular biology major. The seeding of my interest in biotechnology began when I first observed my transformed plate of fluorescing bacteria under a black light. A silly seed, I know, but having been able to see an actual result of one of my experiments first hand was something quite thrilling. This single seed grew as my knowledge of the causes and effects of diseases expanded, which made biology seem more dynamic, disgusting, and like it was begging to be changed. I guess that is where I stand today-on an open field full of possibilities to reverse such diseases.<br />
And that is where iGEM comes in. Although I initially joined iGEM as a means to get to learn more about the techniques bioengineers use and to just get an idea of how research is conducted, I was pleased to find out that my iGEM experience was going to be one of those possibilities in the open field. The combination of health, cancer killing, new techniques, and silly mentors just surpassed my expectations and made this summer unforgettable. I can now see myself zooming through lab work with confidence and even landing a research position as an undergraduate!<br />
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<h4 style="color:black; font-weight:bold;">Lianna Fung</h4><br />
I just recently graduated from Abraham Lincoln High School and am now attending UCSD. I have always been interested in science and the limitless possibilities it can yield. Biotechnology in particular caught my interest after taking a course on it in high school. It fascinated me that we had reached a point where we could modify and improve upon the biology of organisms for specific purposes. This fascination caused me to seek out more opportunities to learn and improve my experience in the field. This led me to my desire to participate in iGEM. iGEM seemed a perfect combination of what I wanted and more. It was a chance to work with others as a team in a welcoming environment. It also gave me the chance to learn new skills that I would normally not be exposed to until further down in my education.<br />
My iGEM experience was interesting. It could be tiring and frustrating at times, but it was also very fun and rewarding. iGEM also creates a sort of independence in people that isn’t as easy to find in the classroom. Overall, the iGEM experience was wonderful and well worth the long hours spent on it. As a bonus, I got to meet some very interesting people as well and make some good friends too.<br />
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<h4 style="color:black; font-weight:bold;">Connor Grant</h4><br />
My name is Connor Grant and I just graduated from Abraham Lincoln High School and will be a freshman at UCSD next year. I almost didn't join iGEM because it conflicted with soccer, but in the end I decided (with some encouragement from my biotech teacher) to spend the summer in the lab at UCSF. iGEM was a great way to learn lab techniques that are used very often in company and University research all over the world. It was good to get experience working in a lab and interacting with post docs and PHD students. It was a good experience over all and I'm glad I did it.<br />
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<h4 style="color:black; font-weight:bold;">Hannah Yan</h4><br />
I recently graduated from Abraham Lincoln High School of San Francisco and am attending Barnard College. For me, iGEM was a chance for me to see if I wanted get into the field of science and to do something awesome during the summer. It really has been a great experience, with its ups and downs. Despite all the failures I have encountered while doing my cloning and minipreps, the ecstatic feeling I get when something worked made everything worthwhile. During iGEM, I was able to use the skills I had learned in biotechnology class and I learned a few new ones as well. I think my experience at iGEM will help me a lot when I decide to pursue the field of science in the future. My summer has been extremely academic, but being able to work with friends and on such a great project has been a lot of fun.<br />
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<h4 style="color:black; font-weight:bold;">John Elam</h4><br />
Hey, I'm John Elam from this year's UCSF iGEM team. I was born and raised in San Francisco and am currently attending UC Davis, majoring in molecular biology and biochemistry. I joined iGEM because I knew it would be a great chance to actually do some real lab work before college, and also because I really liked the basic idea for the project that got pitched to us in May. As of right now a four year degree from Davis is all that I am certain about; as for medical school or graduate school, I'd certainly like to go but you never know what will happen in the future. I played football for three years in high school but currently have no plans to play in college. I'm looking forward to presenting at the Jamboree and hope to learn a lot while I'm there.<br />
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<h4 style="color:black; font-weight:bold;">Crystal Liu</h4><br />
Hello! I'm Crystal Liu, and I am currently an undergraduate at UC Davis majoring in Biochemistry and Molecular Biology. I first heard about the UCSF iGEM team as a freshman in high school, and decided that it was an experience I really wanted to be a part of at the end of my senior year. Four years later, I made it onto the team! I definitely had one of the best summers of my life. Working in a lab really opened my eyes to the world of research and helped me understand firsthand why progress and results aren't immediate. In addition to labwork, I am extremely grateful to have met all the amazing people from the Lim Lab & CPL, as well as everyone else who contributed their impressive skills and knowledge to the 2010 UCSF iGEM team. :) On a side note, I love expensive chocolate.<br />
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<h4 style="color:black; font-weight:bold;">Sam Zorn</h4><br />
Although am still a senior at Abraham Lincoln High School, I am an active participant on the iGEM team. Lab work has been my passion for the last few years of my life. When I was picked for the iGEM team, I was ecstatic. Since that day I have been committed to working as hard as I could to make our project successful. iGEM has given me a real world career experience that has helped me decide on my academic path. The field of synthetic biology has appealed to me greatly, and this summer that I spent at iGEM has more than fulfilled my expectations. I hope that my summer here will not only help me get into college, but also help kick start my career as a bench researcher. When i'm neither working in the labs nor at school, I enjoy getting out and being active. My favorite sports are soccer, and parkour, although I also like to run track, swim, and spar in various martial arts.<br />
<br><br />
<br><br />
<br />
===International Student===<br />
<h4 style="color:black; font-weight:bold;">Min Lin</h4><br />
I'm Min Lin from China; I just got my Bachelor degree in Biological Science from Peking University. This is my second year in iGEM; I'm also in Peking University iGEM 2009 team. I think I've learned a lot in iGEM, and the knowledge will be really useful to me in the future. I like our project this year, because I feel that medicine is a very promising field for synthetic biology applications, although we are still at very first steps. I've been enjoying the nice environment and weather in San Francisco during the summer. And it is really an unforgettable experience to work in iGEM in the Cell Propulsion Lab.<br />
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===Super Buddies===<br />
<h4 style="color:black; font-weight:bold;">Ryan Liang</h4><br />
This is my second year of iGEM and I must say that I am excited to do this for a second round! I am a student in City College of San Francisco with an intent on transferring to UC Davis. Early on I had a passion for the arts and pursued it up until high school where I was introduced to biotechnology and science as an industry. This is when I realized that science is more than just reading and memorization - it is life and the most basic fundamentals of each and every one of us. iGEM allowed me to bring forth my passion for creativity in correlation with synthetic biology. iGEM has encapsulated synthetic biology into such a fun and innovative experience that getting the opportunity to participate once again is truly a blessing.<br />
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<h4 style="color:black; font-weight:bold;">Ethan Chan</h4><br />
IGEM has always been such a great opportunity for all the teams to come up with project ideas are that out of the ordinary. The idea of developing a project that has never been done before is the most exciting part for me. I believe that the work done by iGEM teams have impacted the field so much that it will only get better. I am currently working towards attaining a Biotechnology degree at City College of San Francisco. I am hoping to transfer to UC Davis afterwards. After finding out UCSF's project for the 2010 year, I knew I wanted to come back. Throughout this summer, I have learned to work with mammalian cells. Before the iGEM experience, I was only familiar with prokaryotic cells. iGEM has expanded my skills and knowledge in so many ways. All the skills that i have learned will definitely help me in the future. With all these great pharmaceuticals that are being produced, the field of synthetic biology have proven its effectiveness and its future applications. I am looking forward to being a part of this field in the near future.<br />
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<h4 style="color:black; font-weight:bold;">Eric Wong</h4><br />
Good Afternoon, my name is Eric Wong and i am a graduate of Abraham Lincoln High School class of 2009. I am current pursuing my education in CCSF hoping to transfer into the UC system with the intended major of Molecular Cell Development. I was apart of the iGem team last year and in the time span of 4 months i have learned so many various things from running Assays, Cloning, Troubleshooting and analyzing Experiments and Data. I enjoyed the experience so much so that i decided to come back this year as a mentor for this year's team. I view this and last years iGem experience as something more than just a competition, I has given me the necessary education experience to prepare me for this field of study and allow me to learn about different project from various teams.<br><br><br />
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===UCSF iGEM Program===<br />
<h4 style="color:black; font-weight:bold;">Raquel Gomes</h4><br />
I run the UCSF iGEM program for the last three years. I really enjoy designing all the educational components of the Program but especially the 2-week bootcamp. I love teaching and having the iGEM students around all summer. I will miss all their craziness.<br />
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===Advisors===<br />
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<h4 style="color:black; font-weight:bold;">James Onuffer</h4><br />
This is my second year of involvement and the second year the Cell Propulsion Lab has hosted the team. It was quite an experience to set up the program this year, especially since this is a subject area that we had not begun working on in the Cell Propulsion Lab. The immune response to cancer is a challenging topic to take on (especially coupled with synthetic biology) and required an intensive two week bootcamp to teach the students basic concepts and endogenous systems/parts that they should be aware of. We challenged the students to come up with designs for synthetic cytotoxicity logic gates and increasing the cytotoxic response. It was quite rewarding to see them propose various devices during their team challenge and to see them take charge of getting them prioritized, made , and tested. Things did not always go smoothly, after all this goes with the territory. They had to learn to be organized, think on their feet, and be problem solvers.......quite a growth opportunity that I’m sure will stay with them for the rest of their lives. <br />
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<h4 style="color:black; font-weight:bold;">Wendell Lim</h4><br />
I have had a lab at UCSF for 15 years. The most rewarding thing is working with bright, open-minded young scientists and seeing them develop. Its great to see that most of the iGEM kids that we have worked with have continued to be excited about science and synthetic biology.<br />
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===Buddies===<br />
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<h4 style="color:black; font-weight:bold;">Jason Park</h4><br />
I am a 5th year MD/PhD student in the UCSF School of Medicine and the UCSF / UC Berkeley Joint Graduate Group in Bioengineering. I'm originally from the Los Angeles area and went to college at MIT. I've been in the Cell Propulsion Lab for about two years and I am co-advised by Dr. Wendell Lim and Dr. Bruce Conklin. I became a buddy for iGEM because I thought it would be fun working with bright, motivated high school and undergraduate students for the summer and I knew I would get good experience learning to be a better mentor. (Also, I had been interested in participating in iGEM as an undergraduate at MIT but never ended up doing it!) The best thing about being part of iGEM this summer has been working with and teaching students - together going through the learning process of doing lab research with all of its ups and downs.<br />
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<h4 style="color:black; font-weight:bold;">Chia-Yung Wu</h4><br />
I am a transplant from MIT, where I participated in iGEM as a graduate advisor for the MIT team in 2008 and 2009. This summer, I joined the Lim/Cell Propulsion labs as a postdoc. It has been a great pleasure working with the UCSF team.<br />
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<h4 style="color:black; font-weight:bold;">Russell Gordley</h4><br />
I majored in Biochemistry at Swarthmore College, and performed my graduate studies with Carlos Barbas at the Scripps Research Institute in La Jolla, CA. I am interested in using synthetic biology to understand and enhance the evolvability of biological systems.<br />
<br />
After my freshman year in college, I took a summer internship with Jim Stivers at the National Institute of Standards and Technology. The practice of research turned out to be far more complex and interesting than I could have imagined--an intellectual marathon whose path is revealed with each new data set and disproven hypothesis. I am happy to participate in a program that immerses high school students in the research process. For anyone interested in a life of science, this is a great time to join the pursuit!<br />
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<h4 style="color:black; font-weight:bold;">Jesse Zalatan</h4><br />
I got started in science research with a summer internship after my junior year in high school and I was hooked! I majored in Biochemical Sciences at Harvard and went on to get my Ph.D. in Chemistry at Stanford, where I studied how enzymes speed up chemical reactions that are otherwise incredibly slow. My current research at UCSF is focused on cell signaling, where enzymes play an important role. I am trying to understand how signaling enzymes maintain specificity for the correct targets and avoid signaling mixups. Being introduced to science research in high school inspired me to pursue science in college and beyond, and I got involved with the iGEM team to help share that experience with new students.<br />
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<h4 style="color:black; font-weight:bold;">Wilson Wong</h4><br />
My name is Wilson Wong and I am a postdoc at Wendell Lim's lab. I got my undergraduate degree in chemical engineering at UC Berkeley and graduate degree in chemical engineering at UCLA. I am currently working on applying synthetic biology to rewire human T-cell receptor signaling dynamics. I am interested in helping iGEM because I have always enjoy teaching and mentoring. My iGEM experience has been great. I am very happy with the progress they've made so far and I have a wonderful time working with them.<br />
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<h4 style="color:black; font-weight:bold;">Krista McNally</h4><br />
I am an Associate Specialist at UCSF and handle the Tissue Culture needs for the Cell Propulsion Laboratory. Before coming to UCSF, I was a Research Associate at a Medical Device company performing immunohistochemisty, histology, and thermal imaging. This is the first time I’ve encountered the iGEM program, and it was a great experience to see these young people work so hard as a team to accomplish a meaningful project. During the course of the summer, I gave a lecture to the Team on Tissue Culture and relevant Biosafety concerns, trained some of the students individually to do tissue culture work, and assisted with their use of the Flow Cytometer. I was really impressed with their commitment and diligence during the summer, and enjoyed having so many upbeat and energetic people in the lab. <br />
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<h4 style="color:black; font-weight:bold;">Silinda Neou</h4><br />
I majored in Biochemistry from Cal State East Bay and now work at UCSF in the Cell Propulsion Lab. I love scientific research and have been chasing a career in research ever since my first internship at a microbiology lab. This summer I got the pleasure of interacting with the iGEM students and watching their overall scientific development. They are a great bunch of kids.<br />
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<h4 style="color:black; font-weight:bold;">Jared Toettcher</h4><br />
http://limlab.ucsf.edu/<br />
<br />
<h4 style="color:black; font-weight:bold;">Samantha Liang</h4><br />
I’m currently a second-year graduate student at UCSF in the Biochemistry and Molecular Biology Program, where I am a member of Zev Gartner’s lab. My research involves using chemical techniques to make protein therapeutics more specific and effective. During my undergrad, I majored in Bioengineering at UC Berkeley and worked in Chris Anderson’s lab for 3 years. At Berkeley, I was on the iGEM team in 2006 and loved it so much that I joined again in 2007. I think that iGEM is a unique opportunity for students to conduct research in a fun team context, and also gives them to skills to perform research independently in the future. Because of this, I wanted to stay involved in iGEM during grad school, and that’s why I’m a buddy for the awesome UCSF team this year!<br />
<br><br><br />
<br />
===Instructors===<br />
'''Special thanks to the following instructors for the seminars you provided during our bootcamp!'''<br><br />
<h4 style="color:black; font-weight:bold;">Derek Wong</h4><br />
Taunton Lab<br><br />
http://cmp.ucsf.edu/faculty/pdb_show.html?id=taunton<br><br />
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<h4 style="color:black; font-weight:bold;">Dan Hostetter</h4><br />
Craik Lab<br><br />
http://www.craiklab.ucsf.edu<br><br />
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<h4 style="color:black; font-weight:bold;">David Pincus</h4><br />
El-Samad Lab<br><br />
http://biochemistry.ucsf.edu/labs/elsamad/people/people.html<br><br />
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<h4 style="color:black; font-weight:bold;">Reid Williams</h4><br />
Lim Lab<br><br />
http://limlab.ucsf.edu/<br><br />
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<h3 style="font-weight:bold;">Killer Cell Lovin' Flyer (suggested retail price: $19.95)</h3><br />
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<a href="https://static.igem.org/mediawiki/2010/c/c2/UCSF_HAHAHAH.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/c2/UCSF_HAHAHAH.jpg" border="0" alt="Get Some Killer Cell Loving'" width="400"/></a><br />
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Not sure what this flyer means? Check out <a href="http://www.youtube.com/watch?v=qHQ9CtCv778&feature=related" target="_blank">this video</a>.<br />
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<h3 style="font-weight:bold;">Team Photo Gallery</h3><hr><br><br />
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<h3 style="font-weight:bold;">The Irresistible Frisbee & its Pouch! </h3><hr><br><br />
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<h3 style="font-weight:bold;">Killer Animations</h3><hr><br><br />
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__NOTOC__</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-28T03:37:37Z<p>Ryanliang: /* BETTER ARSENAL */</p>
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==='''Better Arsenal'''===<br />
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[[Image:UCSFnewgranuleagents-06.jpg|center|500px|Immune synapse]]<br />
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<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
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===Brief Introduction===<br />
<br /><br />
[[Image:UCSFMailaddress-05.jpg|400px]]<br />
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Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
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===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br /><br />
[[Image:UCSFGranuleloadingaddress-04.jpg|400px]]<br />
<br /><br /><br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br><br><br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
<br><br><br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|'''#'''||'''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]'''||'''Plasmid Name'''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells and started to select for cells expressing our construct for our assay by selecting with the selection agent G418. The cells used for the assay were mixed populations as the selection was not complete.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br><br><br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
<br />
'''SUMMARY OF RESULTS'''<br />
(See below for more details)<br />
colocalization stats images ready for wiki<br />
{|cellspacing="0"<br />
! Construct<br />
! GFP looks granular<br />
! Granule stain worked<br />
! GFP and granule stain appear colocalized<br />
! Location of granules<br />
! Colocalization statistics<br />
|-<br />
|GZMB-eGFP-CDMPR (56)<br />
|yes<br />
|yes<br />
|yes<br />
|Center<br />
|Pearson's Correlation: 0.831 Mander's Overlap: 0.967<br />
|-<br />
|GZMB-eGFP-LIMP (59)<br />
|yes<br />
|Kind of<br />
|Not really<br />
|Edges<br />
|Pearson's Correlation: 0.577 Mander's Overlap: 0.993<br />
|-<br />
|GZMB-eGFP-Ymotif (60)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|throughout cell, mostly central<br />
|Pearson's Correlation: 0.829 Mander's Overlap: 0.936<br />
|-<br />
|GZMB-eGFP-STP (61)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|Edges<br />
|Pearson's Correlation: 0.774 Mander's Overlap: 0.976<br />
|-<br />
|pMAX-GFP<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|-<br />
|GZMM-eGFP-GILT (63)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|GZMM-eGFP-STP (67)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|NKL-untransfected<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|}<br />
<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br><br><br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br><br><br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain.<br />
<br><br><br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br><br><br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br><br><br><br />
<br />
===Future application===<br />
<br /><br />
[[Image:UCSFnewgranuleagents-07.jpg|center|600px]]<br />
<br /><br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br><br />
<br><br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
http://www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-28T03:35:58Z<p>Ryanliang: /* Results */</p>
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<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
[[Image:UCSFnewgranuleagents-06.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br><br><br />
<br />
===Brief Introduction===<br />
<br /><br />
[[Image:UCSFMailaddress-05.jpg|400px]]<br />
<br /><br /><br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br><br><br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br /><br />
[[Image:UCSFGranuleloadingaddress-04.jpg|400px]]<br />
<br /><br /><br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br><br><br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
<br><br><br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|'''#'''||'''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]'''||'''Plasmid Name'''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells and started to select for cells expressing our construct for our assay by selecting with the selection agent G418. The cells used for the assay were mixed populations as the selection was not complete.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br><br><br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
<br />
'''SUMMARY OF RESULTS'''<br />
(See below for more details)<br />
colocalization stats images ready for wiki<br />
{|cellspacing="0"<br />
! Construct<br />
! GFP looks granular<br />
! Granule stain worked<br />
! GFP and granule stain appear colocalized<br />
! Location of granules<br />
! Colocalization statistics<br />
|-<br />
|GZMB-eGFP-CDMPR (56)<br />
|yes<br />
|yes<br />
|yes<br />
|Center<br />
|Pearson's Correlation: 0.831 Mander's Overlap: 0.967<br />
|-<br />
|GZMB-eGFP-LIMP (59)<br />
|yes<br />
|Kind of<br />
|Not really<br />
|Edges<br />
|Pearson's Correlation: 0.577 Mander's Overlap: 0.993<br />
|-<br />
|GZMB-eGFP-Ymotif (60)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|throughout cell, mostly central<br />
|Pearson's Correlation: 0.829 Mander's Overlap: 0.936<br />
|-<br />
|GZMB-eGFP-STP (61)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|Edges<br />
|Pearson's Correlation: 0.774 Mander's Overlap: 0.976<br />
|-<br />
|pMAX-GFP<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|-<br />
|GZMM-eGFP-GILT (63)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|GZMM-eGFP-STP (67)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|NKL-untransfected<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|}<br />
<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br><br><br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br><br><br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain.<br />
<br><br><br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br><br><br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br><br><br><br />
<br />
===Future application===<br />
<br /><br />
[[Image:UCSFnewgranuleagents-07.jpg|center|600px]]<br />
<br /><br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br><br />
<br><br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
http://www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-28T03:35:16Z<p>Ryanliang: /* Results */</p>
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<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
[[Image:UCSFnewgranuleagents-06.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br><br><br />
<br />
===Brief Introduction===<br />
<br /><br />
[[Image:UCSFMailaddress-05.jpg|400px]]<br />
<br /><br /><br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br><br><br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br /><br />
[[Image:UCSFGranuleloadingaddress-04.jpg|400px]]<br />
<br /><br /><br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br><br><br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
<br><br><br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|'''#'''||'''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]'''||'''Plasmid Name'''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells and started to select for cells expressing our construct for our assay by selecting with the selection agent G418. The cells used for the assay were mixed populations as the selection was not complete.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br><br><br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
<br />
'''SUMMARY OF RESULTS'''<br />
(See below for more details)<br />
colocalization stats images ready for wiki<br />
{|cellspacing="0"<br />
! Construct<br />
! GFP looks granular<br />
! Granule stain worked<br />
! GFP and granule stain appear colocalized<br />
! Location of granules<br />
! Colocalization statistics<br />
|-<br />
|GZMB-eGFP-CDMPR (56)<br />
|yes<br />
|yes<br />
|yes<br />
|Center<br />
|Pearson's Correlation: 0.831 Mander's Overlap: 0.967<br />
|-<br />
|GZMB-eGFP-LIMP (59)<br />
|yes<br />
|Kind of<br />
|Not really<br />
|Edges<br />
|Pearson's Correlation: 0.577 Mander's Overlap: 0.993<br />
|-<br />
|GZMB-eGFP-Ymotif (60)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|throughout cell, mostly central<br />
|Pearson's Correlation: 0.829 Mander's Overlap: 0.936<br />
|-<br />
|GZMB-eGFP-STP (61)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|Edges<br />
|Pearson's Correlation: 0.774 Mander's Overlap: 0.976<br />
|-<br />
|pMAX-GFP<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|-<br />
|GZMM-eGFP-GILT (63)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|GZMM-eGFP-STP (67)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|NKL-untransfected<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|}<br />
<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br><br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br><br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain.<br />
<br><br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br><br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br><br><br />
<br />
===Future application===<br />
<br /><br />
[[Image:UCSFnewgranuleagents-07.jpg|center|600px]]<br />
<br /><br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br><br />
<br><br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
http://www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-28T03:34:10Z<p>Ryanliang: /* Future application */</p>
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<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
[[Image:UCSFnewgranuleagents-06.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br><br><br />
<br />
===Brief Introduction===<br />
<br /><br />
[[Image:UCSFMailaddress-05.jpg|400px]]<br />
<br /><br /><br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br><br><br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br /><br />
[[Image:UCSFGranuleloadingaddress-04.jpg|400px]]<br />
<br /><br /><br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br><br><br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
<br><br><br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|'''#'''||'''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]'''||'''Plasmid Name'''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells and started to select for cells expressing our construct for our assay by selecting with the selection agent G418. The cells used for the assay were mixed populations as the selection was not complete.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br><br><br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
<br />
'''SUMMARY OF RESULTS'''<br />
(See below for more details)<br />
colocalization stats images ready for wiki<br />
{|cellspacing="0"<br />
! Construct<br />
! GFP looks granular<br />
! Granule stain worked<br />
! GFP and granule stain appear colocalized<br />
! Location of granules<br />
! Colocalization statistics<br />
|-<br />
|GZMB-eGFP-CDMPR (56)<br />
|yes<br />
|yes<br />
|yes<br />
|Center<br />
|Pearson's Correlation: 0.831 Mander's Overlap: 0.967<br />
|-<br />
|GZMB-eGFP-LIMP (59)<br />
|yes<br />
|Kind of<br />
|Not really<br />
|Edges<br />
|Pearson's Correlation: 0.577 Mander's Overlap: 0.993<br />
|-<br />
|GZMB-eGFP-Ymotif (60)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|throughout cell, mostly central<br />
|Pearson's Correlation: 0.829 Mander's Overlap: 0.936<br />
|-<br />
|GZMB-eGFP-STP (61)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|Edges<br />
|Pearson's Correlation: 0.774 Mander's Overlap: 0.976<br />
|-<br />
|pMAX-GFP<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|-<br />
|GZMM-eGFP-GILT (63)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|GZMM-eGFP-STP (67)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|NKL-untransfected<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|}<br />
<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
===Future application===<br />
<br /><br />
[[Image:UCSFnewgranuleagents-07.jpg|center|600px]]<br />
<br /><br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br><br />
<br><br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
http://www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-28T03:33:25Z<p>Ryanliang: /* Results */</p>
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<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
[[Image:UCSFnewgranuleagents-06.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br><br><br />
<br />
===Brief Introduction===<br />
<br /><br />
[[Image:UCSFMailaddress-05.jpg|400px]]<br />
<br /><br /><br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br><br><br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br /><br />
[[Image:UCSFGranuleloadingaddress-04.jpg|400px]]<br />
<br /><br /><br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br><br><br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
<br><br><br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|'''#'''||'''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]'''||'''Plasmid Name'''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells and started to select for cells expressing our construct for our assay by selecting with the selection agent G418. The cells used for the assay were mixed populations as the selection was not complete.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br><br><br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
<br />
'''SUMMARY OF RESULTS'''<br />
(See below for more details)<br />
colocalization stats images ready for wiki<br />
{|cellspacing="0"<br />
! Construct<br />
! GFP looks granular<br />
! Granule stain worked<br />
! GFP and granule stain appear colocalized<br />
! Location of granules<br />
! Colocalization statistics<br />
|-<br />
|GZMB-eGFP-CDMPR (56)<br />
|yes<br />
|yes<br />
|yes<br />
|Center<br />
|Pearson's Correlation: 0.831 Mander's Overlap: 0.967<br />
|-<br />
|GZMB-eGFP-LIMP (59)<br />
|yes<br />
|Kind of<br />
|Not really<br />
|Edges<br />
|Pearson's Correlation: 0.577 Mander's Overlap: 0.993<br />
|-<br />
|GZMB-eGFP-Ymotif (60)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|throughout cell, mostly central<br />
|Pearson's Correlation: 0.829 Mander's Overlap: 0.936<br />
|-<br />
|GZMB-eGFP-STP (61)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|Edges<br />
|Pearson's Correlation: 0.774 Mander's Overlap: 0.976<br />
|-<br />
|pMAX-GFP<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|-<br />
|GZMM-eGFP-GILT (63)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|GZMM-eGFP-STP (67)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|NKL-untransfected<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|}<br />
<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
===Future application===<br />
<br /><br />
[[Image:UCSFnewgranuleagents-07.jpg|center|600px]]<br />
<br /><br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
http://www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-28T03:32:34Z<p>Ryanliang: /* Results */</p>
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<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
[[Image:UCSFnewgranuleagents-06.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br><br><br />
<br />
===Brief Introduction===<br />
<br /><br />
[[Image:UCSFMailaddress-05.jpg|400px]]<br />
<br /><br /><br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br><br><br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br /><br />
[[Image:UCSFGranuleloadingaddress-04.jpg|400px]]<br />
<br /><br /><br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br><br><br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
<br><br><br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|'''#'''||'''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]'''||'''Plasmid Name'''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells and started to select for cells expressing our construct for our assay by selecting with the selection agent G418. The cells used for the assay were mixed populations as the selection was not complete.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br><br><br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
'''SUMMARY OF RESULTS'''<br />
(See below for more details)<br />
colocalization stats images ready for wiki<br />
{|cellspacing="0"<br />
! Construct<br />
! GFP looks granular<br />
! Granule stain worked<br />
! GFP and granule stain appear colocalized<br />
! Location of granules<br />
! Colocalization statistics<br />
|-<br />
|GZMB-eGFP-CDMPR (56)<br />
|yes<br />
|yes<br />
|yes<br />
|Center<br />
|Pearson's Correlation: 0.831 Mander's Overlap: 0.967<br />
|-<br />
|GZMB-eGFP-LIMP (59)<br />
|yes<br />
|Kind of<br />
|Not really<br />
|Edges<br />
|Pearson's Correlation: 0.577 Mander's Overlap: 0.993<br />
|-<br />
|GZMB-eGFP-Ymotif (60)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|throughout cell, mostly central<br />
|Pearson's Correlation: 0.829 Mander's Overlap: 0.936<br />
|-<br />
|GZMB-eGFP-STP (61)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|Edges<br />
|Pearson's Correlation: 0.774 Mander's Overlap: 0.976<br />
|-<br />
|pMAX-GFP<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|-<br />
|GZMM-eGFP-GILT (63)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|GZMM-eGFP-STP (67)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|NKL-untransfected<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|}<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
===Future application===<br />
<br /><br />
[[Image:UCSFnewgranuleagents-07.jpg|center|600px]]<br />
<br /><br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
http://www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
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{{Template:UCSF/RightStart}}<br />
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__TOC__<br />
{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-28T03:30:59Z<p>Ryanliang: /* Testing the Constructs */</p>
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<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
[[Image:UCSFnewgranuleagents-06.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br><br><br />
<br />
===Brief Introduction===<br />
<br /><br />
[[Image:UCSFMailaddress-05.jpg|400px]]<br />
<br /><br /><br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br><br><br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br /><br />
[[Image:UCSFGranuleloadingaddress-04.jpg|400px]]<br />
<br /><br /><br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br><br><br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
<br><br><br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|'''#'''||'''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]'''||'''Plasmid Name'''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells and started to select for cells expressing our construct for our assay by selecting with the selection agent G418. The cells used for the assay were mixed populations as the selection was not complete.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br><br><br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
SUMMARY OF RESULTS<br />
(See below for more details)<br />
colocalization stats images ready for wiki<br />
{|cellspacing="0"<br />
! Construct<br />
! GFP looks granular<br />
! Granule stain worked<br />
! GFP and granule stain appear colocalized<br />
! Location of granules<br />
! Colocalization statistics<br />
|-<br />
|GZMB-eGFP-CDMPR (56)<br />
|yes<br />
|yes<br />
|yes<br />
|Center<br />
|Pearson's Correlation: 0.831 Mander's Overlap: 0.967<br />
|-<br />
|GZMB-eGFP-LIMP (59)<br />
|yes<br />
|Kind of<br />
|Not really<br />
|Edges<br />
|Pearson's Correlation: 0.577 Mander's Overlap: 0.993<br />
|-<br />
|GZMB-eGFP-Ymotif (60)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|throughout cell, mostly central<br />
|Pearson's Correlation: 0.829 Mander's Overlap: 0.936<br />
|-<br />
|GZMB-eGFP-STP (61)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|Edges<br />
|Pearson's Correlation: 0.774 Mander's Overlap: 0.976<br />
|-<br />
|pMAX-GFP<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|-<br />
|GZMM-eGFP-GILT (63)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|GZMM-eGFP-STP (67)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|NKL-untransfected<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|}<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
===Future application===<br />
<br /><br />
[[Image:UCSFnewgranuleagents-07.jpg|center|600px]]<br />
<br /><br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
http://www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
<br />
__TOC__<br />
{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-28T03:30:44Z<p>Ryanliang: /* Cloning Strategy */</p>
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<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
[[Image:UCSFnewgranuleagents-06.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br><br><br />
<br />
===Brief Introduction===<br />
<br /><br />
[[Image:UCSFMailaddress-05.jpg|400px]]<br />
<br /><br /><br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br><br><br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br /><br />
[[Image:UCSFGranuleloadingaddress-04.jpg|400px]]<br />
<br /><br /><br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br><br><br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
<br><br><br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|'''#'''||'''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]'''||'''Plasmid Name'''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells and started to select for cells expressing our construct for our assay by selecting with the selection agent G418. The cells used for the assay were mixed populations as the selection was not complete.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
SUMMARY OF RESULTS<br />
(See below for more details)<br />
colocalization stats images ready for wiki<br />
{|cellspacing="0"<br />
! Construct<br />
! GFP looks granular<br />
! Granule stain worked<br />
! GFP and granule stain appear colocalized<br />
! Location of granules<br />
! Colocalization statistics<br />
|-<br />
|GZMB-eGFP-CDMPR (56)<br />
|yes<br />
|yes<br />
|yes<br />
|Center<br />
|Pearson's Correlation: 0.831 Mander's Overlap: 0.967<br />
|-<br />
|GZMB-eGFP-LIMP (59)<br />
|yes<br />
|Kind of<br />
|Not really<br />
|Edges<br />
|Pearson's Correlation: 0.577 Mander's Overlap: 0.993<br />
|-<br />
|GZMB-eGFP-Ymotif (60)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|throughout cell, mostly central<br />
|Pearson's Correlation: 0.829 Mander's Overlap: 0.936<br />
|-<br />
|GZMB-eGFP-STP (61)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|Edges<br />
|Pearson's Correlation: 0.774 Mander's Overlap: 0.976<br />
|-<br />
|pMAX-GFP<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|-<br />
|GZMM-eGFP-GILT (63)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|GZMM-eGFP-STP (67)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|NKL-untransfected<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|}<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
===Future application===<br />
<br /><br />
[[Image:UCSFnewgranuleagents-07.jpg|center|600px]]<br />
<br /><br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
http://www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-28T03:30:22Z<p>Ryanliang: /* Directing GFP to killer cells' granules by fusing "address" tags */</p>
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==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
[[Image:UCSFnewgranuleagents-06.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br><br><br />
<br />
===Brief Introduction===<br />
<br /><br />
[[Image:UCSFMailaddress-05.jpg|400px]]<br />
<br /><br /><br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br><br><br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br /><br />
[[Image:UCSFGranuleloadingaddress-04.jpg|400px]]<br />
<br /><br /><br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br><br><br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|'''#'''||'''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]'''||'''Plasmid Name'''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells and started to select for cells expressing our construct for our assay by selecting with the selection agent G418. The cells used for the assay were mixed populations as the selection was not complete.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
SUMMARY OF RESULTS<br />
(See below for more details)<br />
colocalization stats images ready for wiki<br />
{|cellspacing="0"<br />
! Construct<br />
! GFP looks granular<br />
! Granule stain worked<br />
! GFP and granule stain appear colocalized<br />
! Location of granules<br />
! Colocalization statistics<br />
|-<br />
|GZMB-eGFP-CDMPR (56)<br />
|yes<br />
|yes<br />
|yes<br />
|Center<br />
|Pearson's Correlation: 0.831 Mander's Overlap: 0.967<br />
|-<br />
|GZMB-eGFP-LIMP (59)<br />
|yes<br />
|Kind of<br />
|Not really<br />
|Edges<br />
|Pearson's Correlation: 0.577 Mander's Overlap: 0.993<br />
|-<br />
|GZMB-eGFP-Ymotif (60)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|throughout cell, mostly central<br />
|Pearson's Correlation: 0.829 Mander's Overlap: 0.936<br />
|-<br />
|GZMB-eGFP-STP (61)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|Edges<br />
|Pearson's Correlation: 0.774 Mander's Overlap: 0.976<br />
|-<br />
|pMAX-GFP<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|-<br />
|GZMM-eGFP-GILT (63)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|GZMM-eGFP-STP (67)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|NKL-untransfected<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|}<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
===Future application===<br />
<br /><br />
[[Image:UCSFnewgranuleagents-07.jpg|center|600px]]<br />
<br /><br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
http://www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-28T03:29:55Z<p>Ryanliang: /* Brief Introduction */</p>
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<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
[[Image:UCSFnewgranuleagents-06.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br><br><br />
<br />
===Brief Introduction===<br />
<br /><br />
[[Image:UCSFMailaddress-05.jpg|400px]]<br />
<br /><br /><br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br><br><br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br /><br />
[[Image:UCSFGranuleloadingaddress-04.jpg|400px]]<br />
<br /><br /><br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|'''#'''||'''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]'''||'''Plasmid Name'''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells and started to select for cells expressing our construct for our assay by selecting with the selection agent G418. The cells used for the assay were mixed populations as the selection was not complete.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
SUMMARY OF RESULTS<br />
(See below for more details)<br />
colocalization stats images ready for wiki<br />
{|cellspacing="0"<br />
! Construct<br />
! GFP looks granular<br />
! Granule stain worked<br />
! GFP and granule stain appear colocalized<br />
! Location of granules<br />
! Colocalization statistics<br />
|-<br />
|GZMB-eGFP-CDMPR (56)<br />
|yes<br />
|yes<br />
|yes<br />
|Center<br />
|Pearson's Correlation: 0.831 Mander's Overlap: 0.967<br />
|-<br />
|GZMB-eGFP-LIMP (59)<br />
|yes<br />
|Kind of<br />
|Not really<br />
|Edges<br />
|Pearson's Correlation: 0.577 Mander's Overlap: 0.993<br />
|-<br />
|GZMB-eGFP-Ymotif (60)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|throughout cell, mostly central<br />
|Pearson's Correlation: 0.829 Mander's Overlap: 0.936<br />
|-<br />
|GZMB-eGFP-STP (61)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|Edges<br />
|Pearson's Correlation: 0.774 Mander's Overlap: 0.976<br />
|-<br />
|pMAX-GFP<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|-<br />
|GZMM-eGFP-GILT (63)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|GZMM-eGFP-STP (67)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|NKL-untransfected<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|}<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
===Future application===<br />
<br /><br />
[[Image:UCSFnewgranuleagents-07.jpg|center|600px]]<br />
<br /><br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
http://www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-28T03:29:28Z<p>Ryanliang: /* BETTER ARSENAL */</p>
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==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
[[Image:UCSFnewgranuleagents-06.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br><br><br />
<br />
===Brief Introduction===<br />
<br /><br />
[[Image:UCSFMailaddress-05.jpg|400px]]<br />
<br /><br /><br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br /><br />
[[Image:UCSFGranuleloadingaddress-04.jpg|400px]]<br />
<br /><br /><br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|'''#'''||'''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]'''||'''Plasmid Name'''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells and started to select for cells expressing our construct for our assay by selecting with the selection agent G418. The cells used for the assay were mixed populations as the selection was not complete.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
SUMMARY OF RESULTS<br />
(See below for more details)<br />
colocalization stats images ready for wiki<br />
{|cellspacing="0"<br />
! Construct<br />
! GFP looks granular<br />
! Granule stain worked<br />
! GFP and granule stain appear colocalized<br />
! Location of granules<br />
! Colocalization statistics<br />
|-<br />
|GZMB-eGFP-CDMPR (56)<br />
|yes<br />
|yes<br />
|yes<br />
|Center<br />
|Pearson's Correlation: 0.831 Mander's Overlap: 0.967<br />
|-<br />
|GZMB-eGFP-LIMP (59)<br />
|yes<br />
|Kind of<br />
|Not really<br />
|Edges<br />
|Pearson's Correlation: 0.577 Mander's Overlap: 0.993<br />
|-<br />
|GZMB-eGFP-Ymotif (60)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|throughout cell, mostly central<br />
|Pearson's Correlation: 0.829 Mander's Overlap: 0.936<br />
|-<br />
|GZMB-eGFP-STP (61)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|Edges<br />
|Pearson's Correlation: 0.774 Mander's Overlap: 0.976<br />
|-<br />
|pMAX-GFP<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|-<br />
|GZMM-eGFP-GILT (63)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|GZMM-eGFP-STP (67)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|NKL-untransfected<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|}<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
===Future application===<br />
<br /><br />
[[Image:UCSFnewgranuleagents-07.jpg|center|600px]]<br />
<br /><br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
http://www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/SponsorsTeam:UCSF/Sponsors2010-10-28T03:28:44Z<p>Ryanliang: </p>
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<h3 style="font-weight:bold;">Many thanks to our Sponsors:</h3><br><br />
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<h3 style="font-weight:bold;">Also many thanks to the talented <a href="https://2010.igem.org/Team:Peking/Team/WYWang">Weiye Wang</a> from Peking University Team for drawing us such a beautiful banner!</h3></p><br />
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{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Notes/BootcampTeam:UCSF/Notes/Bootcamp2010-10-28T03:28:02Z<p>Ryanliang: /* SUMMER BOOTCAMP */</p>
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=== '''Summer Bootcamp''' ===<br />
<br />
Before the summer Bootcamp Raquel Gomes and the super buddies (students from UCSF iGEM 2009) came to Lincoln High School for three afterschool sessions where they taught us about synthetic biology, immunology, UCSF iGEM 2009 project and techniques the super buddies were learning in the lab. We also were given papers to read and discuss. As soon as our school year was over a two week bootcamp started at UCSF.<br />
<br />
<br />
<br />
===='''Week 1: June 14-18'''====<br />
<br />
We kicked off our summer with a brief introduction of the iGEM team and Lim Lab members after a tour of the UCSF campus. It didn’t take long for us to plunge right into lectures though. Each morning we would learn about a new topic that falls under the broader category of immunity and end with an assignments that test our understanding of that topic. The highlights of our lectures are organized with the help of the following handy table:<br />
<br />
<br />
'''''Seminar Topic and Highlights'''''<br />
<br />
<br />
'''Intro to Cancer and Synthetic Biology''' - By Raquel Gomes<br />
<br />
A. How cancer develops<br />
<br />
B. Synthetic Biology design<br />
<br />
C. Introduction to topics covered during bootcamp and instructors <br />
<br />
D. Main rules to work in lab<br />
<br />
<br />
'''Immune Response''' - by Raquel Gomes<br />
<br />
A. Immune cells recognize proteins on target cell surfaces using receptors.<br />
<br />
B. The target surface proteins trigger the killing or non-killing response from the immune cells.<br />
<br />
<br />
'''Cytoskeleton''' - by Derek Wong<br />
<br />
A. Cytoskeletal proteins are important in forming the connection between immune cells and target cells, the immune synapse.<br />
<br />
<br />
'''Cell Death''' - by Daniel Hostetter<br />
<br />
A. The activation of the killing process results in...<br />
<br />
1. Cytotoxic agents released into target cells.<br />
<br />
2. Death signal docking on target receptors triggering apoptosis.<br />
<br />
<br />
'''Intracellular signaling''' - by David Pincus<br />
<br />
A. The inside of the cell is like a Rube Goldberg device<br />
<br />
B. Writers, Erasers and Readers - kinases, phosphatases and effectors<br />
<br />
C. Second messengers<br />
<br />
D. Small G proteins, GTPases and ATPases<br />
<br />
<br />
<br />
<br />
===='''Week 2: June 21- 25'''==== <br />
<br />
<br />
<br />
'''Logic Gates''' - by Reid Williams<br />
<br />
A. The independent parts of proteins can be rearranged to produce a protein that acts differently. <br />
<br />
B. These new proteins can be used to screen for the many different types of cancer and make the immune cells respond in a certain way.<br />
<br />
C. Application: Cancer cells vary greatly in their surface protein expression (markers). We can use these markers as inputs that act as prerequisites for a certain immune cell action (i.e. killing and not killing).<br />
<br />
<br />
'''Wrap-up and Intro to Team challenge''' - by Raquel Gomes and James Onuffer<br />
<br />
A. Summary of everything learned during bootcamp<br />
<br />
B. Intro to challenges<br />
<br />
<br />
Boot camp continues onto week two. This week, our lectures focused on more of the creative aspect of synthetic biology. We learned about modularity and how parts can be put together into devices. We also studied Boolean logic and the various types of logic gates like AND or NOT gates. <br />
<br />
Then after a summary of all the material we’ve learned over the course of boot camp, we were broken up into two groups for a '''team challenge'''. <br />
Both teams had two challenges: One was to design synthetic logic gates for cancer cell recognition and killing by cytotoxic killer cells based upon differential tumor antigen expression. The other challenge was to engineer modulators that enhanced cell mediated cytotoxicity using synthetic biology. Each group then had about two days to brainstorm, select, and tailor ideas for a final presentation to our advisers and instructors. The ideas for modulators of cell cytotoxicity were then categorized into two groups, (a) improving the cargo aspect of cell killing, and (b) improving the signaling of killing.<br />
<br />
Each team was to come up with a few complete and practical projects under their topic. We had to look though the ideas we already had and pick out the ones we liked the best. We would then work exclusively on those ideas, finalizing and refining those ideas to present again to our instructors and advisers. After presenting, we were advised on the ideas based on feasibility within the period of time, interest, and practical application.<br />
<br />
With a general project in mind, we began our first steps towards actually creating our projects. Before we could actually start synthesizing our parts, we needed to make primers for our parts since we were using a unique form of combinatorial cloning, Aar1 cloning. This required our parts to have both the Aar1 site and special complimentary binding sites. As we had never had any primer creating experience, we were given a lesson in how to use computer programs like APE and gene designer that would not only help with primer creation but alignments and more. We were also taught the basics of primer creation such as melting temperature and GC% content. This would all help in the weeks ahead with the various parts we had to synthesize, alignments we had to make, and more.<br />
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<h3 style="font-weight:bold;">Bootcamp Gallery</h3><hr><br><br />
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{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/MedalTeam:UCSF/Medal2010-10-28T03:26:57Z<p>Ryanliang: </p>
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<h3 style="font-weight:bold;">The UCSF team has fulfilled the following criteria to meet the gold medal requirements:</h3><br />
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<p><b>1. Submitted new BioBrick parts (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K321003" target="_blank">BBa_K321003</a> & <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K321004" target="_blank">BBa_K321004</a>) and entered detailed information regarding these parts in the Registry of Parts. The information included a demonstration of the parts’ functions and characterization of their operation. [Silver Medal requirements]</b></p><br><br />
<br />
<p><b>2. Characterized an existing BioBrick Part and entered this information back on the Registry. [Gold Medal requirement]</b><br><br><br />
<br />
Our team has further characterized the BioBrick part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K209400" target="_blank">BBa_k209400</a> by showing a previously unknown cellular function conferred by this part. Developed by the UCSF 2009 iGEM team, the HM4D synthetic GPCR part was originally found to successfully mediate neutrophil chemotaxis towards the synthetic ligand CNO. (See project <a href="https://2009.igem.org/Team:UCSF" target="_blank">Cell-Bots</a>) As the process of chemotaxis involves activation of cellular kinases, we hypothesized that this synthetic GPCR might enhance the Killer cell killing signal, which is the focus of our project this year and also involves cellular kinases. When we incorporated this part into killers cells, we found that it significantly increased killer cell activation. See the results <br />
<a href="https://2010.igem.org/Team:UCSF/Project/Signaling" target="_blank">here</a>.</p><br><br />
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<p><b>3. Assisted other iGEM teams with their projects. [Gold Medal requirement]</b><br><br><br />
<br />
In collaboration with <a href="https://2010.igem.org/Team:Paris_Liliane_Bettencourt" target="_blank">Paris Liliane Bettencourt</a> and <a href="https://2010.igem.org/Team:Peking" target="_blank">Peking University</a>, the UCSF team has contributed to developing a variety of visual protocols for techniques widely used in synthetic biology. (See Paris Liliane Bettencourt's <a href="https://2010.igem.org/Team:Paris_Liliane_Bettencourt/Collaboration" target="_blank">collaboration</a> page) These protocol images are being used in <a href="http://synbioworld.org/openprotocol/" target="_blank">openProtocol</a> by Paris Liliane Bettencourt to help write consensus protocols for common laboratory techniques.</p><br><br />
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<br />
<p>PDF versions of some of the visual protocols:<br><br><br />
<a href="https://static.igem.org/mediawiki/2010/7/76/Pcr_purification.pdf" target="_blank">PCR Purification</a><br><br />
<a href="https://static.igem.org/mediawiki/2010/d/dd/Miniprep.pdf" target="_blank">Miniprep</a><br />
</p><br />
<br />
<br><br />
<br><br />
<p>THANK YOU PARIS AND PKU! IT WAS FUN!!!</p><br />
<br><br />
<br />
<p>In addition to the visual protocols, the UCSF team has helped teams <a href="https://2010.igem.org/Team:METU_Turkey" target="_blank">Metu Turkey</a>, <a href="https://2010.igem.org/Team:Penn_State" target="_blank">Penn State</a>, and <br />
<a href="https://2010.igem.org/Team:Warsaw" target="_blank">Warsaw</a> with their surveys. These surveys covered the topics of Categorizing Registry Parts, Genetically Modified Organisms, and Synthetic Biology.</p><br><br><br />
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__NOTOC__<br />
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{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-28T02:57:14Z<p>Ryanliang: </p>
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==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
[[Image:UCSFnewgranuleagents-06.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
===Brief Introduction===<br />
<br /><br />
[[Image:UCSFMailaddress-05.jpg|400px]]<br />
<br /><br /><br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br /><br />
[[Image:UCSFGranuleloadingaddress-04.jpg|400px]]<br />
<br /><br /><br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|'''#'''||'''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]'''||'''Plasmid Name'''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells and started to select for cells expressing our construct for our assay by selecting with the selection agent G418. The cells used for the assay were mixed populations as the selection was not complete.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
SUMMARY OF RESULTS<br />
(See below for more details)<br />
colocalization stats images ready for wiki<br />
{|cellspacing="0"<br />
! Construct<br />
! GFP looks granular<br />
! Granule stain worked<br />
! GFP and granule stain appear colocalized<br />
! Location of granules<br />
! Colocalization statistics<br />
|-<br />
|GZMB-eGFP-CDMPR (56)<br />
|yes<br />
|yes<br />
|yes<br />
|Center<br />
|Pearson's Correlation: 0.831 Mander's Overlap: 0.967<br />
|-<br />
|GZMB-eGFP-LIMP (59)<br />
|yes<br />
|Kind of<br />
|Not really<br />
|Edges<br />
|Pearson's Correlation: 0.577 Mander's Overlap: 0.993<br />
|-<br />
|GZMB-eGFP-Ymotif (60)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|throughout cell, mostly central<br />
|Pearson's Correlation: 0.829 Mander's Overlap: 0.936<br />
|-<br />
|GZMB-eGFP-STP (61)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|Edges<br />
|Pearson's Correlation: 0.774 Mander's Overlap: 0.976<br />
|-<br />
|pMAX-GFP<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|-<br />
|GZMM-eGFP-GILT (63)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|GZMM-eGFP-STP (67)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|NKL-untransfected<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|}<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
===Future application===<br />
<br /><br />
[[Image:UCSFnewgranuleagents-07.jpg|center|600px]]<br />
<br /><br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
http://www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSFTeam:UCSF2010-10-27T23:50:33Z<p>Ryanliang: /* Project Description */</p>
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===Project Description===<br />
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Natural killer (NK) cells of the immune system identify cancer and virally-infected cells and kill them. These potent killers travel throughout the body, recognizing proteins and other molecules on the surface of cells. In order to differentiate between healthy and diseased cells, NK cells use a variety of receptors, which bind to specific ligands at the target cells’ surface. The balance between activating and inhibitory signals will tell the NK cell if the target cell is diseased or healthy, respectively. If the target cell is deemed potentially dangerous, the NK cell grips the target cell tightly and creates an immunological synapse at the site of adhesion. Within this immunological synapse, the NK cell releases cytotoxic granules to kill the target cell without harming any nearby cells allowing for a direct, apoptotic death.<br />
<br />
Our team will focus on improving NK cells’ specificity and killing efficiency towards certain cancer types. By using synthetic biology tools and logic gates’ design, we hope to create powerful killing biomachines for the fight against cancer. Our newly engineered synthetic devices would have the potential to enhance current adoptive cell-based immunotherapy for cancer patients.<br />
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===Project Description===<br />
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Natural killer (NK) cells of the immune system identify cancer and virally-infected cells and kill them. These potent killers travel throughout the body, recognizing proteins and other molecules on the surface of cells. In order to differentiate between healthy and diseased cells, NK cells use a variety of receptors, which bind to specific ligands at the target cells’ surface. The balance between activating and inhibitory signals will tell the NK cell if the target cell is diseased or healthy, respectively. If the target cell is deemed potentially dangerous, the NK cell grips the target cell tightly and creates an immunological synapse at the site of adhesion. Within this immunological synapse, the NK cell releases cytotoxic granules to kill the target cell without harming any nearby cells allowing for a direct, apoptotic death.<br />
<br />
Our team will focus on improving NK cells’ specificity and killing efficiency towards certain cancer types. By using synthetic biology tools and logic gates’ design, we hope to create powerful killing biomachines for the fight against cancer. Our newly engineered synthetic devices would have the potential to enhance current adoptive cell-based immunotherapy for cancer patients.<br />
<br />
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</a><br />
<a href="https://2010.igem.org/Team:UCSF/Project/Signaling"><img src="https://static.igem.org/mediawiki/2010/9/9a/UCSF_signaling_home_icon.png" width="208" border="0" alt="Stronger Signaling" /><br />
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<a href="https://2010.igem.org/Team:UCSF/Project/Arsenal" ><img src="https://static.igem.org/mediawiki/2010/3/35/UCSF_arsenal_home_icon.png" border="0" width="208" alt="Better Arsenal" /><br />
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<h3 style="color:black;">TEAM</h3><br />
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<a href="https://2010.igem.org/Team:UCSF/Team"><img src="https://static.igem.org/mediawiki/2010/f/ff/Team_photo.png" /></a><br />
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<h3 style="color:black;">SPONSORS</h3><br />
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<a href="https://2010.igem.org/Team:UCSF/Sponsors"><img src="https://static.igem.org/mediawiki/2010/3/38/Sponsors_2010.png" width="200px" /></a><br />
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Follow us on <a href="http://twitter.com/#!/iGEM_UCSF">Twitter!</a><br />
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__NOTOC__</div>Ryanlianghttp://2010.igem.org/Team:UCSF/TeamTeam:UCSF/Team2010-10-27T23:42:22Z<p>Ryanliang: /* Students */</p>
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[[Image:Team_photo_large.png]]<br><br />
Team composed of 7 students from Lincoln High School, 3 first year students from SF City College and 1 undergraduate from Peking University.<br />
<br />
===Students===<br />
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<a href="http://www.dropbox.com/gallery/12435548/1/teamphotos?h=ac34b2" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9c/UCSF_GROUPPHOTOZ.jpg" width="550px" border="0" alt="UCSF 2010 Students Gallery" /></a><br />
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<center>Images Courtesy of June Park</center><br />
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<h4 style="color:black; font-weight:bold;">Carmen Zhou</h4><br />
My name is Carmen Zhou and I will soon be entering UCSD as a freshman molecular biology major. The seeding of my interest in biotechnology began when I first observed my transformed plate of fluorescing bacteria under a black light. A silly seed, I know, but having been able to see an actual result of one of my experiments first hand was something quite thrilling. This single seed grew as my knowledge of the causes and effects of diseases expanded, which made biology seem more dynamic, disgusting, and like it was begging to be changed. I guess that is where I stand today-on an open field full of possibilities to reverse such diseases.<br />
And that is where iGEM comes in. Although I initially joined iGEM as a means to get to learn more about the techniques bioengineers use and to just get an idea of how research is conducted, I was pleased to find out that my iGEM experience was going to be one of those possibilities in the open field. The combination of health, cancer killing, new techniques, and silly mentors just surpassed my expectations and made this summer unforgettable. I can now see myself zooming through lab work with confidence and even landing a research position as an undergraduate!<br />
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<h4 style="color:black; font-weight:bold;">Lianna Fung</h4><br />
I just recently graduated from Abraham Lincoln High School and am now attending UCSD. I have always been interested in science and the limitless possibilities it can yield. Biotechnology in particular caught my interest after taking a course on it in high school. It fascinated me that we had reached a point where we could modify and improve upon the biology of organisms for specific purposes. This fascination caused me to seek out more opportunities to learn and improve my experience in the field. This led me to my desire to participate in iGEM. iGEM seemed a perfect combination of what I wanted and more. It was a chance to work with others as a team in a welcoming environment. It also gave me the chance to learn new skills that I would normally not be exposed to until further down in my education.<br />
My iGEM experience was interesting. It could be tiring and frustrating at times, but it was also very fun and rewarding. iGEM also creates a sort of independence in people that isn’t as easy to find in the classroom. Overall, the iGEM experience was wonderful and well worth the long hours spent on it. As a bonus, I got to meet some very interesting people as well and make some good friends too.<br />
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<h4 style="color:black; font-weight:bold;">Connor Grant</h4><br />
My name is Connor Grant and I just graduated from Abraham Lincoln High School and will be a freshman at UCSD next year. I almost didn't join iGEM because it conflicted with soccer, but in the end I decided (with some encouragement from my biotech teacher) to spend the summer in the lab at UCSF. iGEM was a great way to learn lab techniques that are used very often in company and University research all over the world. It was good to get experience working in a lab and interacting with post docs and PHD students. It was a good experience over all and I'm glad I did it.<br />
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<h4 style="color:black; font-weight:bold;">Hannah Yan</h4><br />
I recently graduated from Abraham Lincoln High School of San Francisco and am attending Barnard College. For me, iGEM was a chance for me to see if I wanted get into the field of science and to do something awesome during the summer. It really has been a great experience, with its ups and downs. Despite all the failures I have encountered while doing my cloning and minipreps, the ecstatic feeling I get when something worked made everything worthwhile. During iGEM, I was able to use the skills I had learned in biotechnology class and I learned a few new ones as well. I think my experience at iGEM will help me a lot when I decide to pursue the field of science in the future. My summer has been extremely academic, but being able to work with friends and on such a great project has been a lot of fun.<br />
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<h4 style="color:black; font-weight:bold;">John Elam</h4><br />
Hey, I'm John Elam from this year's UCSF iGEM team. I was born and raised in San Francisco and am currently attending UC Davis, majoring in molecular biology and biochemistry. I joined iGEM because I knew it would be a great chance to actually do some real lab work before college, and also because I really liked the basic idea for the project that got pitched to us in May. As of right now a four year degree from Davis is all that I am certain about; as for medical school or graduate school, I'd certainly like to go but you never know what will happen in the future. I played football for three years in high school but currently have no plans to play in college. I'm looking forward to presenting at the Jamboree and hope to learn a lot while I'm there.<br />
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<h4 style="color:black; font-weight:bold;">Crystal Liu</h4><br />
Hello! I'm Crystal Liu, and I am currently an undergraduate at UC Davis majoring in Biochemistry and Molecular Biology. I first heard about the UCSF iGEM team as a freshman in high school, and decided that it was an experience I really wanted to be a part of at the end of my senior year. Four years later, I made it onto the team! I definitely had one of the best summers of my life. Working in a lab really opened my eyes to the world of research and helped me understand firsthand why progress and results aren't immediate. In addition to labwork, I am extremely grateful to have met all the amazing people from the Lim Lab & CPL, as well as everyone else who contributed their impressive skills and knowledge to the 2010 UCSF iGEM team. :) On a side note, I love expensive chocolate.<br />
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<h4 style="color:black; font-weight:bold;">Sam Zorn</h4><br />
Although am still a senior at Abraham Lincoln High School, I am an active participant on the iGEM team. Lab work has been my passion for the last few years of my life. When I was picked for the iGEM team, I was ecstatic. Since that day I have been committed to working as hard as I could to make our project successful. iGEM has given me a real world career experience that has helped me decide on my academic path. The field of synthetic biology has appealed to me greatly, and this summer that I spent at iGEM has more than fulfilled my expectations. I hope that my summer here will not only help me get into college, but also help kick start my career as a bench researcher. When i'm neither working in the labs nor at school, I enjoy getting out and being active. My favorite sports are soccer, and parkour, although I also like to run track, swim, and spar in various martial arts.<br />
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===International Student===<br />
<h4 style="color:black; font-weight:bold;">Min Lin</h4><br />
I'm Min Lin from China; I just got my Bachelor degree in Biological Science from Peking University. This is my second year in iGEM; I'm also in Peking University iGEM 2009 team. I think I've learned a lot in iGEM, and the knowledge will be really useful to me in the future. I like our project this year, because I feel that medicine is a very promising field for synthetic biology applications, although we are still at very first steps. I've been enjoying the nice environment and weather in San Francisco during the summer. And it is really an unforgettable experience to work in iGEM in the Cell Propulsion Lab.<br />
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===Super Buddies===<br />
<h4 style="color:black; font-weight:bold;">Ryan Liang</h4><br />
This is my second year of iGEM and I must say that I am excited to do this for a second round! I am a student in City College of San Francisco with an intent on transferring to UC Davis. Early on I had a passion for the arts and pursued it up until high school where I was introduced to biotechnology and science as an industry. This is when I realized that science is more than just reading and memorization - it is life and the most basic fundamentals of each and every one of us. iGEM allowed me to bring forth my passion for creativity in correlation with synthetic biology. iGEM has encapsulated synthetic biology into such a fun and innovative experience that getting the opportunity to participate once again is truly a blessing.<br />
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<h4 style="color:black; font-weight:bold;">Ethan Chan</h4><br />
IGEM has always been such a great opportunity for all the teams to come up with project ideas are that out of the ordinary. The idea of developing a project that has never been done before is the most exciting part for me. I believe that the work done by iGEM teams have impacted the field so much that it will only get better. I am currently working towards attaining a Biotechnology degree at City College of San Francisco. I am hoping to transfer to UC Davis afterwards. After finding out UCSF's project for the 2010 year, I knew I wanted to come back. Throughout this summer, I have learned to work with mammalian cells. Before the iGEM experience, I was only familiar with prokaryotic cells. iGEM has expanded my skills and knowledge in so many ways. All the skills that i have learned will definitely help me in the future. With all these great pharmaceuticals that are being produced, the field of synthetic biology have proven its effectiveness and its future applications. I am looking forward to being a part of this field in the near future.<br />
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<h4 style="color:black; font-weight:bold;">Eric Wong</h4><br />
Good Afternoon, my name is Eric Wong and i am a graduate of Abraham Lincoln High School class of 2009. I am current pursuing my education in CCSF hoping to transfer into the UC system with the intended major of Molecular Cell Development. I was apart of the iGem team last year and in the time span of 4 months i have learned so many various things from running Assays, Cloning, Troubleshooting and analyzing Experiments and Data. I enjoyed the experience so much so that i decided to come back this year as a mentor for this year's team. I view this and last years iGem experience as something more than just a competition, I has given me the necessary education experience to prepare me for this field of study and allow me to learn about different project from various teams.<br><br><br />
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===UCSF iGEM Program===<br />
<h4 style="color:black; font-weight:bold;">Raquel Gomes</h4><br />
I run the UCSF iGEM program for the last three years. I really enjoy designing all the educational components of the Program but especially the 2-week bootcamp. I love teaching and having the iGEM students around all summer. I will miss all their craziness.<br />
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===Advisors===<br />
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<h4 style="color:black; font-weight:bold;">James Onuffer</h4><br />
This is my second year of involvement and the second year the Cell Propulsion Lab has hosted the team. It was quite an experience to set up the program this year, especially since this is a subject area that we had not begun working on in the Cell Propulsion Lab. The immune response to cancer is a challenging topic to take on (especially coupled with synthetic biology) and required an intensive two week bootcamp to teach the students basic concepts and endogenous systems/parts that they should be aware of. We challenged the students to come up with designs for synthetic cytotoxicity logic gates and increasing the cytotoxic response. It was quite rewarding to see them propose various devices during their team challenge and to see them take charge of getting them prioritized, made , and tested. Things did not always go smoothly, after all this goes with the territory. They had to learn to be organized, think on their feet, and be problem solvers.......quite a growth opportunity that I’m sure will stay with them for the rest of their lives. <br />
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<h4 style="color:black; font-weight:bold;">Wendell Lim</h4><br />
I have had a lab at UCSF for 15 years. The most rewarding thing is working with bright, open-minded young scientists and seeing them develop. Its great to see that most of the iGEM kids that we have worked with have continued to be excited about science and synthetic biology.<br />
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===Buddies===<br />
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<h4 style="color:black; font-weight:bold;">Jason Park</h4><br />
I am a 5th year MD/PhD student in the UCSF School of Medicine and the UCSF / UC Berkeley Joint Graduate Group in Bioengineering. I'm originally from the Los Angeles area and went to college at MIT. I've been in the Cell Propulsion Lab for about two years and I am co-advised by Dr. Wendell Lim and Dr. Bruce Conklin. I became a buddy for iGEM because I thought it would be fun working with bright, motivated high school and undergraduate students for the summer and I knew I would get good experience learning to be a better mentor. (Also, I had been interested in participating in iGEM as an undergraduate at MIT but never ended up doing it!) The best thing about being part of iGEM this summer has been working with and teaching students - together going through the learning process of doing lab research with all of its ups and downs.<br />
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<h4 style="color:black; font-weight:bold;">Chia-Yung Wu</h4><br />
I am a transplant from MIT, where I participated in iGEM as a graduate advisor for the MIT team in 2008 and 2009. This summer, I joined the Lim/Cell Propulsion labs as a postdoc. It has been a great pleasure working with the UCSF team.<br />
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<h4 style="color:black; font-weight:bold;">Russell Gordley</h4><br />
I majored in Biochemistry at Swarthmore College, and performed my graduate studies with Carlos Barbas at the Scripps Research Institute in La Jolla, CA. I am interested in using synthetic biology to understand and enhance the evolvability of biological systems.<br />
<br />
After my freshman year in college, I took a summer internship with Jim Stivers at the National Institute of Standards and Technology. The practice of research turned out to be far more complex and interesting than I could have imagined--an intellectual marathon whose path is revealed with each new data set and disproven hypothesis. I am happy to participate in a program that immerses high school students in the research process. For anyone interested in a life of science, this is a great time to join the pursuit!<br />
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<h4 style="color:black; font-weight:bold;">Jesse Zalatan</h4><br />
I got started in science research with a summer internship after my junior year in high school and I was hooked! I majored in Biochemical Sciences at Harvard and went on to get my Ph.D. in Chemistry at Stanford, where I studied how enzymes speed up chemical reactions that are otherwise incredibly slow. My current research at UCSF is focused on cell signaling, where enzymes plays an important role. I am trying to understand how signaling enzymes maintain specificity for the correct targets and avoid signaling mixups. Being introduced to science research in high school inspired me to pursue science in college and beyond, and I got involved with the iGEM team to help share that experience with new students.<br />
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<h4 style="color:black; font-weight:bold;">Wilson Wong</h4><br />
My name is Wilson Wong and I am a postdoc at Wendell Lim's lab. I got my undergraduate degree in chemical engineering at UC Berkeley and graduate degree in chemical engineering at UCLA. I am currently working on applying synthetic biology to rewire human T-cell receptor signaling dynamics. I am interested in helping iGEM because I have always enjoy teaching and mentoring. My iGEM experience has been great. I am very happy with the progress they've made so far and I have a wonderful time working with them.<br />
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<h4 style="color:black; font-weight:bold;">Krista McNally</h4><br />
I am an Associate Specialist at UCSF and handle the Tissue Culture needs for the Cell Propulsion Laboratory. Before coming to UCSF, I was a Research Associate at a Medical Device company performing immunohistochemisty, histology, and thermal imaging. This is the first time I’ve encountered the iGEM program, and it was a great experience to see these young people work so hard as a team to accomplish a meaningful project. During the course of the summer, I gave a lecture to the Team on Tissue Culture and relevant Biosafety concerns, trained some of the students individually to do tissue culture work, and assisted with their use of the Flow Cytometer. I was really impressed with their commitment and diligence during the summer, and enjoyed having so many upbeat and energetic people in the lab. <br />
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<h4 style="color:black; font-weight:bold;">Silinda Neou</h4><br />
I majored in Biochemistry from Cal State East Bay and now work at UCSF in the Cell Propulsion Lab. I love scientific research and have been chasing a career in research ever since my first internship at a microbiology lab. This summer I got the pleasure of interacting with the iGEM students and watching their overall scientific development. They are a great bunch of kids.<br />
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<h4 style="color:black; font-weight:bold;">Jared Toettcher</h4><br />
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<h4 style="color:black; font-weight:bold;">Samantha Liang</h4><br />
I’m currently a second-year graduate student at UCSF in the Biochemistry and Molecular Biology Program, where I am a member of Zev Gartner’s lab. My research involves using chemical techniques to make protein therapeutics more specific and effective. During my undergrad, I majored in Bioengineering at UC Berkeley and worked in Chris Anderson’s lab for 3 years. At Berkeley, I was on the iGEM team in 2006 and loved it so much that I joined again in 2007. I think that iGEM is a unique opportunity for students to conduct research in a fun team context, and also gives them to skills to perform research independently in the future. Because of this, I wanted to stay involved in iGEM during grad school, and that’s why I’m a buddy for the awesome UCSF team this year!<br />
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===Instructors===<br />
'''Special thanks to the following instructors for the seminars you provided during our bootcamp!'''<br><br />
<h4 style="color:black; font-weight:bold;">Derek Wong</h4><br />
Taunton Lab<br><br />
http://cmp.ucsf.edu/faculty/pdb_show.html?id=taunton<br><br />
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<h4 style="color:black; font-weight:bold;">Dan Hostetter</h4><br />
Craik Lab<br><br />
http://www.craiklab.ucsf.edu<br><br />
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<h4 style="color:black; font-weight:bold;">David Pincus</h4><br />
El-Samad Lab<br><br />
http://biochemistry.ucsf.edu/labs/elsamad/people/people.html<br><br />
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<h4 style="color:black; font-weight:bold;">Reid Williams</h4><br />
Lim Lab<br><br />
http://limlab.ucsf.edu/<br><br />
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{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/FunStuffTeam:UCSF/FunStuff2010-10-27T23:33:08Z<p>Ryanliang: </p>
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<h3 style="font-weight:bold;">Killer Cell Lovin' Flyer (suggested retail price: $19.95)</h3><br />
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<a href="https://static.igem.org/mediawiki/2010/c/c2/UCSF_HAHAHAH.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/c2/UCSF_HAHAHAH.jpg" border="0" alt="Get Some Killer Cell Loving'" width="400"/></a><br />
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Not sure what this flyer means? Check out <a href="http://www.youtube.com/watch?v=qHQ9CtCv778&feature=related" target="_blank">this video</a>.<br />
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<h3 style="font-weight:bold;">Team Photo Gallery</h3><hr><br><br />
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<h3 style="font-weight:bold;">The Irresistible Frisbee & its pouch! (requested donation: $9.95)</h3><hr><br><br />
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<a href="https://static.igem.org/mediawiki/2010/a/a3/UCSF_fling_bag.png" target="_blank"><br />
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<h3 style="font-weight:bold;">Killer Animations</h3><hr><br><br />
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onmouseover="mouseOver()" <br />
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Click on image to watch the ninja move!<br />
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<object width="100%" height="400"><param name="allowFullScreen" value="true" /><param name="movie" value="http://www.vuvox.com/collage_express/collage.swf?collageID=029aea37e3"/><embed src="http://www.vuvox.com/collage_express/collage.swf?collageID=029aea37e3" allowFullScreen="true" type="application/x-shockwave-flash" width="100%" height="400"></embed></object><br />
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<p>Press play and enjoy!</p><br />
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__NOTOC__</div>Ryanlianghttp://2010.igem.org/Team:UCSF/FunStuffTeam:UCSF/FunStuff2010-10-27T23:22:31Z<p>Ryanliang: </p>
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<h3 style="font-weight:bold;">Killer Cell Lovin' Flyer (suggested retail price: $19.95)</h3><br />
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Not sure what this flyer means? Check out <a href="http://www.youtube.com/watch?v=qHQ9CtCv778&feature=related" target="_blank">this video</a>.<br />
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<h3 style="font-weight:bold;">Team Photo Gallery</h3><hr><br><br />
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<h3 style="font-weight:bold;">The Irresistible Frisbee & its pouch! (requested donation: $9.95)</h3><hr><br><br />
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<h3 style="font-weight:bold;">Killer Animations</h3><hr><br><br />
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Click on image to watch the ninja move!<br />
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{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/FunStuffTeam:UCSF/FunStuff2010-10-27T23:21:02Z<p>Ryanliang: </p>
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<h3 style="font-weight:bold;">Killer Cell Lovin' Flyer (suggested retail price: $19.95)</h3><br />
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<a href="https://static.igem.org/mediawiki/2010/c/c2/UCSF_HAHAHAH.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/c2/UCSF_HAHAHAH.jpg" border="0" alt="Get Some Killer Cell Loving'" width="400"/></a><br />
<br />
<br><br />
Not sure what this flyer means? Check out <a href="http://www.youtube.com/watch?v=qHQ9CtCv778&feature=related" target="_blank">this video</a>.<br />
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<h3 style="font-weight:bold;">Team Photo Gallery</h3><hr><br><br />
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<h3 style="font-weight:bold;">The Irresistible Frisbee & its pouch! (requested donation: $9.95)</h3><hr><br><br />
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<h3 style="font-weight:bold;">Killer Animations</h3><hr><br><br />
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Click on image to watch the ninja move!<br />
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{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/TeamTeam:UCSF/Team2010-10-27T23:04:37Z<p>Ryanliang: /* Super Buddies */</p>
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[[Image:Team_photo_large.png]]<br />
Team composed of 7 students from Lincoln High School, 3 first year students from SF City College and 1 undergraduate from Peking University.<br />
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<br />
===Students===<br />
<h4 style="color:black; font-weight:bold;">Carmen Zhou</h4><br />
My name is Carmen Zhou and I will soon be entering UCSD as a freshman molecular biology major. The seeding of my interest in biotechnology began when I first observed my transformed plate of fluorescing bacteria under a black light. A silly seed, I know, but having been able to see an actual result of one of my experiments first hand was something quite thrilling. This single seed grew as my knowledge of the causes and effects of diseases expanded, which made biology seem more dynamic, disgusting, and like it was begging to be changed. I guess that is where I stand today-on an open field full of possibilities to reverse such diseases.<br />
And that is where iGEM comes in. Although I initially joined iGEM as a means to get to learn more about the techniques bioengineers use and to just get an idea of how research is conducted, I was pleased to find out that my iGEM experience was going to be one of those possibilities in the open field. The combination of health, cancer killing, new techniques, and silly mentors just surpassed my expectations and made this summer unforgettable. I can now see myself zooming through lab work with confidence and even landing a research position as an undergraduate!<br />
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<h4 style="color:black; font-weight:bold;">Lianna Fung</h4><br />
I just recently graduated from Abraham Lincoln High School and am now attending UCSD. I have always been interested in science and the limitless possibilities it can yield. Biotechnology in particular caught my interest after taking a course on it in high school. It fascinated me that we had reached a point where we could modify and improve upon the biology of organisms for specific purposes. This fascination caused me to seek out more opportunities to learn and improve my experience in the field. This led me to my desire to participate in iGEM. iGEM seemed a perfect combination of what I wanted and more. It was a chance to work with others as a team in a welcoming environment. It also gave me the chance to learn new skills that I would normally not be exposed to until further down in my education.<br />
My iGEM experience was interesting. It could be tiring and frustrating at times, but it was also very fun and rewarding. iGEM also creates a sort of independence in people that isn’t as easy to find in the classroom. Overall, the iGEM experience was wonderful and well worth the long hours spent on it. As a bonus, I got to meet some very interesting people as well and make some good friends too.<br />
<br />
<h4 style="color:black; font-weight:bold;">Connor Grant</h4><br />
My name is Connor Grant and I just graduated from Abraham Lincoln High School and will be a freshman at UCSD next year. I almost didn't join iGEM because it conflicted with soccer, but in the end I decided (with some encouragement from my biotech teacher) to spend the summer in the lab at UCSF. iGEM was a great way to learn lab techniques that are used very often in company and University research all over the world. It was good to get experience working in a lab and interacting with post docs and PHD students. It was a good experience over all and I'm glad I did it.<br />
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<h4 style="color:black; font-weight:bold;">Hannah Yan</h4><br />
I recently graduated from Abraham Lincoln High School of San Francisco and am attending Barnard College. For me, iGEM was a chance for me to see if I wanted get into the field of science and to do something awesome during the summer. It really has been a great experience, with its ups and downs. Despite all the failures I have encountered while doing my cloning and minipreps, the ecstatic feeling I get when something worked made everything worthwhile. During iGEM, I was able to use the skills I had learned in biotechnology class and I learned a few new ones as well. I think my experience at iGEM will help me a lot when I decide to pursue the field of science in the future. My summer has been extremely academic, but being able to work with friends and on such a great project has been a lot of fun.<br />
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<h4 style="color:black; font-weight:bold;">John Elam</h4><br />
Hey, I'm John Elam from this year's UCSF iGEM team. I was born and raised in San Francisco and am currently attending UC Davis, majoring in molecular biology and biochemistry. I joined iGEM because I knew it would be a great chance to actually do some real lab work before college, and also because I really liked the basic idea for the project that got pitched to us in May. As of right now a four year degree from Davis is all that I am certain about; as for medical school or graduate school, I'd certainly like to go but you never know what will happen in the future. I played football for three years in high school but currently have no plans to play in college. I'm looking forward to presenting at the Jamboree and hope to learn a lot while I'm there.<br />
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<h4 style="color:black; font-weight:bold;">Crystal Liu</h4><br />
Hello! I'm Crystal Liu, and I am currently an undergraduate at UC Davis majoring in Biochemistry and Molecular Biology. I first heard about the UCSF iGEM team as a freshman in high school, and decided that it was an experience I really wanted to be a part of at the end of my senior year. Four years later, I made it onto the team! I definitely had one of the best summers of my life. Working in a lab really opened my eyes to the world of research and helped me understand firsthand why progress and results aren't immediate. In addition to labwork, I am extremely grateful to have met all the amazing people from the Lim Lab & CPL, as well as everyone else who contributed their impressive skills and knowledge to the 2010 UCSF iGEM team. :) On a side note, I love expensive chocolate.<br />
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<h4 style="color:black; font-weight:bold;">Sam Zorn</h4><br />
Although am still a senior at Abraham Lincoln High School, I am an active participant on the iGEM team. Lab work has been my passion for the last few years of my life. When I was picked for the iGEM team, I was ecstatic. Since that day I have been committed to working as hard as I could to make our project successful. iGEM has given me a real world career experience that has helped me decide on my academic path. The field of synthetic biology has appealed to me greatly, and this summer that I spent at iGEM has more than fulfilled my expectations. I hope that my summer here will not only help me get into college, but also help kick start my career as a bench researcher. When i'm neither working in the labs nor at school, I enjoy getting out and being active. My favorite sports are soccer, and parkour, although I also like to run track, swim, and spar in various martial arts.<br />
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<br />
===International Student===<br />
<h4 style="color:black; font-weight:bold;">Min Lin</h4><br />
I'm Min Lin from China; I just got my Bachelor degree in Biological Science from Peking University. This is my second year in iGEM; I'm also in Peking University iGEM 2009 team. I think I've learned a lot in iGEM, and the knowledge will be really useful to me in the future. I like our project this year, because I feel that medicine is a very promising field for synthetic biology applications, although we are still at very first steps. I've been enjoying the nice environment and weather in San Francisco during the summer. And it is really an unforgettable experience to work in iGEM in the Cell Propulsion Lab.<br />
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<br />
===Super Buddies===<br />
<h4 style="color:black; font-weight:bold;">Ryan Liang</h4><br />
This is my second year of iGEM and I must say that I am excited to do this for a second round! I am a student in City College of San Francisco with an intent on transferring to UC Davis. Early on I had a passion for the arts and pursued it up until high school where I was introduced to biotechnology and science as an industry. This is when I realized that science is more than just reading and memorization - it is life and the most basic fundamentals of each and every one of us. iGEM allowed me to bring forth my passion for creativity in correlation with synthetic biology. iGEM has encapsulated synthetic biology into such a fun and innovative experience that getting the opportunity to participate once again is truly a blessing.<br />
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<h4 style="color:black; font-weight:bold;">Ethan Chan</h4><br />
IGEM has always been such a great opportunity for all the teams to come up with project ideas are that out of the ordinary. The idea of developing a project that has never been done before is the most exciting part for me. I believe that the work done by iGEM teams have impacted the field so much that it will only get better. I am currently working towards attaining a Biotechnology degree at City College of San Francisco. I am hoping to transfer to UC Davis afterwards. After finding out UCSF's project for the 2010 year, I knew I wanted to come back. Throughout this summer, I have learned to work with mammalian cells. Before the iGEM experience, I was only familiar with prokaryotic cells. iGEM has expanded my skills and knowledge in so many ways. All the skills that i have learned will definitely help me in the future. With all these great pharmaceuticals that are being produced, the field of synthetic biology have proven its effectiveness and its future applications. I am looking forward to being a part of this field in the near future.<br />
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<h4 style="color:black; font-weight:bold;">Eric Wong</h4><br />
Good Afternoon, my name is Eric Wong and i am a graduate of Abraham Lincoln High School class of 2009. I am current pursuing my education in CCSF hoping to transfer into the UC system with the intended major of Molecular Cell Development. I was apart of the iGem team last year and in the time span of 4 months i have learned so many various things from running Assays, Cloning, Troubleshooting and analyzing Experiments and Data. I enjoyed the experience so much so that i decided to come back this year as a mentor for this year's team. I view this and last years iGem experience as something more than just a competition, I has given me the necessary education experience to prepare me for this field of study and allow me to learn about different project from various teams.<br><br><br />
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<h3 style="font-weight:bold;">Student Gallery</h3><hr><br><br />
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<br />
===UCSF iGEM Program===<br />
<h4 style="color:black; font-weight:bold;">Raquel Gomes</h4><br />
I run the UCSF iGEM program for the last three years. I really enjoy designing all the educational components of the Program but especially the 2-week bootcamp. I love teaching and having the iGEM students around all summer. I will miss all their craziness.<br />
<br />
<br />
===Advisors===<br />
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<h4 style="color:black; font-weight:bold;">James Onuffer</h4><br />
This is my second year of involvement and the second year the Cell Propulsion Lab has hosted the team. It was quite an experience to set up the program this year, especially since this is a subject area that we had not begun working on in the Cell Propulsion Lab. The immune response to cancer is a challenging topic to take on (especially coupled with synthetic biology) and required an intensive two week bootcamp to teach the students basic concepts and endogenous systems/parts that they should be aware of. We challenged the students to come up with designs for synthetic cytotoxicity logic gates and increasing the cytotoxic response. It was quite rewarding to see them propose various devices during their team challenge and to see them take charge of getting them prioritized, made , and tested. Things did not always go smoothly, after all this goes with the territory. They had to learn to be organized, think on their feet, and be problem solvers.......quite a growth opportunity that I’m sure will stay with them for the rest of their lives. <br />
<br />
<h4 style="color:black; font-weight:bold;">Wendell Lim</h4><br />
I have had a lab at UCSF for 15 years. The most rewarding thing is working with bright, open-minded young scientists and seeing them develop. Its great to see that most of the iGEM kids that we have worked with have continued to be excited about science and synthetic biology.<br />
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<br />
===Buddies===<br />
<br />
<h4 style="color:black; font-weight:bold;">Jason Park</h4><br />
I am a 5th year MD/PhD student in the UCSF School of Medicine and the UCSF / UC Berkeley Joint Graduate Group in Bioengineering. I'm originally from the Los Angeles area and went to college at MIT. I've been in the Cell Propulsion Lab for about two years and I am co-advised by Dr. Wendell Lim and Dr. Bruce Conklin. I became a buddy for iGEM because I thought it would be fun working with bright, motivated high school and undergraduate students for the summer and I knew I would get good experience learning to be a better mentor. (Also, I had been interested in participating in iGEM as an undergraduate at MIT but never ended up doing it!) The best thing about being part of iGEM this summer has been working with and teaching students - together going through the learning process of doing lab research with all of its ups and downs.<br />
<br />
<h4 style="color:black; font-weight:bold;">Chia-Yung Wu</h4><br />
I am a transplant from MIT, where I participated in iGEM as a graduate advisor for the MIT team in 2008 and 2009. This summer, I joined the Lim/Cell Propulsion labs as a postdoc. It has been a great pleasure working with the UCSF team.<br />
<br />
<h4 style="color:black; font-weight:bold;">Russell Gordley</h4><br />
I majored in Biochemistry at Swarthmore College, and performed my graduate studies with Carlos Barbas at the Scripps Research Institute in La Jolla, CA. I am interested in using synthetic biology to understand and enhance the evolvability of biological systems.<br />
<br />
After my freshman year in college, I took a summer internship with Jim Stivers at the National Institute of Standards and Technology. The practice of research turned out to be far more complex and interesting than I could have imagined--an intellectual marathon whose path is revealed with each new data set and disproven hypothesis. I am happy to participate in a program that immerses high school students in the research process. For anyone interested in a life of science, this is a great time to join the pursuit!<br />
<br />
<h4 style="color:black; font-weight:bold;">Jesse Zalatan</h4><br />
I got started in science research with a summer internship after my junior year in high school and I was hooked! I majored in Biochemical Sciences at Harvard and went on to get my Ph.D. in Chemistry at Stanford, where I studied how enzymes speed up chemical reactions that are otherwise incredibly slow. My current research at UCSF is focused on cell signaling, where enzymes plays an important role. I am trying to understand how signaling enzymes maintain specificity for the correct targets and avoid signaling mixups. Being introduced to science research in high school inspired me to pursue science in college and beyond, and I got involved with the iGEM team to help share that experience with new students.<br />
<br />
<h4 style="color:black; font-weight:bold;">Wilson Wong</h4><br />
My name is Wilson Wong and I am a postdoc at Wendell Lim's lab. I got my undergraduate degree in chemical engineering at UC Berkeley and graduate degree in chemical engineering at UCLA. I am currently working on applying synthetic biology to rewire human T-cell receptor signaling dynamics. I am interested in helping iGEM because I have always enjoy teaching and mentoring. My iGEM experience has been great. I am very happy with the progress they've made so far and I have a wonderful time working with them.<br />
<br />
<h4 style="color:black; font-weight:bold;">Krista McNally</h4><br />
I am an Associate Specialist at UCSF and handle the Tissue Culture needs for the Cell Propulsion Laboratory. Before coming to UCSF, I was a Research Associate at a Medical Device company performing immunohistochemisty, histology, and thermal imaging. This is the first time I’ve encountered the iGEM program, and it was a great experience to see these young people work so hard as a team to accomplish a meaningful project. During the course of the summer, I gave a lecture to the Team on Tissue Culture and relevant Biosafety concerns, trained some of the students individually to do tissue culture work, and assisted with their use of the Flow Cytometer. I was really impressed with their commitment and diligence during the summer, and enjoyed having so many upbeat and energetic people in the lab. <br />
<br />
<h4 style="color:black; font-weight:bold;">Silinda Neou</h4><br />
I majored in Biochemistry from Cal State East Bay and now work at UCSF in the Cell Propulsion Lab. I love scientific research and have been chasing a career in research ever since my first internship at a microbiology lab. This summer I got the pleasure of interacting with the iGEM students and watching their overall scientific development. They are a great bunch of kids.<br />
<br />
<h4 style="color:black; font-weight:bold;">Jared Toettcher</h4><br />
<br />
<h4 style="color:black; font-weight:bold;">Samantha Liang</h4><br />
I’m currently a second-year graduate student at UCSF in the Biochemistry and Molecular Biology Program, where I am a member of Zev Gartner’s lab. My research involves using chemical techniques to make protein therapeutics more specific and effective. During my undergrad, I majored in Bioengineering at UC Berkeley and worked in Chris Anderson’s lab for 3 years. At Berkeley, I was on the iGEM team in 2006 and loved it so much that I joined again in 2007. I think that iGEM is a unique opportunity for students to conduct research in a fun team context, and also gives them to skills to perform research independently in the future. Because of this, I wanted to stay involved in iGEM during grad school, and that’s why I’m a buddy for the awesome UCSF team this year!<br />
<br><br><br />
<br />
===Instructors===<br />
'''Special thanks to the following instructors for the seminars you provided during our bootcamp!'''<br><br />
<h4 style="color:black; font-weight:bold;">Derek Wong</h4><br />
Taunton Lab<br><br />
http://cmp.ucsf.edu/faculty/pdb_show.html?id=taunton<br><br />
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<h4 style="color:black; font-weight:bold;">Dan Hostetter</h4><br />
Craik Lab<br><br />
http://www.craiklab.ucsf.edu<br><br />
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<h4 style="color:black; font-weight:bold;">David Pincus</h4><br />
El-Samad Lab<br><br />
http://biochemistry.ucsf.edu/labs/elsamad/people/people.html<br><br />
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<h4 style="color:black; font-weight:bold;">Reid Williams</h4><br />
Lim Lab<br><br />
http://limlab.ucsf.edu/<br><br />
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[[Image:Team_photo_large.png]]<br />
Team composed of 7 students from Lincoln High School, 3 first year students from SF City College and 1 undergraduate from Peking University.<br />
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===Students===<br />
<h4 style="color:black; font-weight:bold;">Carmen Zhou</h4><br />
My name is Carmen Zhou and I will soon be entering UCSD as a freshman molecular biology major. The seeding of my interest in biotechnology began when I first observed my transformed plate of fluorescing bacteria under a black light. A silly seed, I know, but having been able to see an actual result of one of my experiments first hand was something quite thrilling. This single seed grew as my knowledge of the causes and effects of diseases expanded, which made biology seem more dynamic, disgusting, and like it was begging to be changed. I guess that is where I stand today-on an open field full of possibilities to reverse such diseases.<br />
And that is where iGEM comes in. Although I initially joined iGEM as a means to get to learn more about the techniques bioengineers use and to just get an idea of how research is conducted, I was pleased to find out that my iGEM experience was going to be one of those possibilities in the open field. The combination of health, cancer killing, new techniques, and silly mentors just surpassed my expectations and made this summer unforgettable. I can now see myself zooming through lab work with confidence and even landing a research position as an undergraduate!<br />
<br />
<h4 style="color:black; font-weight:bold;">Lianna Fung</h4><br />
I just recently graduated from Abraham Lincoln High School and am now attending UCSD. I have always been interested in science and the limitless possibilities it can yield. Biotechnology in particular caught my interest after taking a course on it in high school. It fascinated me that we had reached a point where we could modify and improve upon the biology of organisms for specific purposes. This fascination caused me to seek out more opportunities to learn and improve my experience in the field. This led me to my desire to participate in iGEM. iGEM seemed a perfect combination of what I wanted and more. It was a chance to work with others as a team in a welcoming environment. It also gave me the chance to learn new skills that I would normally not be exposed to until further down in my education.<br />
My iGEM experience was interesting. It could be tiring and frustrating at times, but it was also very fun and rewarding. iGEM also creates a sort of independence in people that isn’t as easy to find in the classroom. Overall, the iGEM experience was wonderful and well worth the long hours spent on it. As a bonus, I got to meet some very interesting people as well and make some good friends too.<br />
<br />
<h4 style="color:black; font-weight:bold;">Connor Grant</h4><br />
My name is Connor Grant and I just graduated from Abraham Lincoln High School and will be a freshman at UCSD next year. I almost didn't join iGEM because it conflicted with soccer, but in the end I decided (with some encouragement from my biotech teacher) to spend the summer in the lab at UCSF. iGEM was a great way to learn lab techniques that are used very often in company and University research all over the world. It was good to get experience working in a lab and interacting with post docs and PHD students. It was a good experience over all and I'm glad I did it.<br />
<br />
<h4 style="color:black; font-weight:bold;">Hannah Yan</h4><br />
I recently graduated from Abraham Lincoln High School of San Francisco and am attending Barnard College. For me, iGEM was a chance for me to see if I wanted get into the field of science and to do something awesome during the summer. It really has been a great experience, with its ups and downs. Despite all the failures I have encountered while doing my cloning and minipreps, the ecstatic feeling I get when something worked made everything worthwhile. During iGEM, I was able to use the skills I had learned in biotechnology class and I learned a few new ones as well. I think my experience at iGEM will help me a lot when I decide to pursue the field of science in the future. My summer has been extremely academic, but being able to work with friends and on such a great project has been a lot of fun.<br />
<br />
<h4 style="color:black; font-weight:bold;">John Elam</h4><br />
Hey, I'm John Elam from this year's UCSF iGEM team. I was born and raised in San Francisco and am currently attending UC Davis, majoring in molecular biology and biochemistry. I joined iGEM because I knew it would be a great chance to actually do some real lab work before college, and also because I really liked the basic idea for the project that got pitched to us in May. As of right now a four year degree from Davis is all that I am certain about; as for medical school or graduate school, I'd certainly like to go but you never know what will happen in the future. I played football for three years in high school but currently have no plans to play in college. I'm looking forward to presenting at the Jamboree and hope to learn a lot while I'm there.<br />
<br />
<h4 style="color:black; font-weight:bold;">Crystal Liu</h4><br />
Hello! I'm Crystal Liu, and I am currently an undergraduate at UC Davis majoring in Biochemistry and Molecular Biology. I first heard about the UCSF iGEM team as a freshman in high school, and decided that it was an experience I really wanted to be a part of at the end of my senior year. Four years later, I made it onto the team! I definitely had one of the best summers of my life. Working in a lab really opened my eyes to the world of research and helped me understand firsthand why progress and results aren't immediate. In addition to labwork, I am extremely grateful to have met all the amazing people from the Lim Lab & CPL, as well as everyone else who contributed their impressive skills and knowledge to the 2010 UCSF iGEM team. :) On a side note, I love expensive chocolate.<br />
<br />
<h4 style="color:black; font-weight:bold;">Sam Zorn</h4><br />
Although am still a senior at Abraham Lincoln High School, I am an active participant on the iGEM team. Lab work has been my passion for the last few years of my life. When I was picked for the iGEM team, I was ecstatic. Since that day I have been committed to working as hard as I could to make our project successful. iGEM has given me a real world career experience that has helped me decide on my academic path. The field of synthetic biology has appealed to me greatly, and this summer that I spent at iGEM has more than fulfilled my expectations. I hope that my summer here will not only help me get into college, but also help kick start my career as a bench researcher. When i'm neither working in the labs nor at school, I enjoy getting out and being active. My favorite sports are soccer, and parkour, although I also like to run track, swim, and spar in various martial arts.<br />
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<br />
===International Student===<br />
<h4 style="color:black; font-weight:bold;">Min Lin</h4><br />
I'm Min Lin from China; I just got my Bachelor degree in Biological Science from Peking University. This is my second year in iGEM; I'm also in Peking University iGEM 2009 team. I think I've learned a lot in iGEM, and the knowledge will be really useful to me in the future. I like our project this year, because I feel that medicine is a very promising field for synthetic biology applications, although we are still at very first steps. I've been enjoying the nice environment and weather in San Francisco during the summer. And it is really an unforgettable experience to work in iGEM in the Cell Propulsion Lab.<br />
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===Super Buddies===<br />
<h4 style="color:black; font-weight:bold;">Ryan Liang</h4><br />
This is my second year of iGEM and I must say that I am excited to do this for a second round! I am a student in City College of San Francisco with an intent on transferring to UC Davis. Early on I had a passion for the arts and pursued it up until high school where I was introduced to biotechnology and science as an industry. This is when I realized that science is more than just reading and memorization - it is life and the most basic fundamentals of each and every one of us. iGEM allowed me to bring forth my passion for creativity in correlation with synthetic biology. iGEM has encapsulated synthetic biology into such a fun and innovative experience that getting the opportunity to participate once again is truly a blessing.<br />
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<h4 style="color:black; font-weight:bold;">Ethan Chan</h4><br />
IGEM has always been such a great opportunity for all the teams to come up with project ideas are that out of the ordinary. The idea of developing a project that has never been done before is the most exciting part for me. I believe that the work done by iGEM teams have impacted the field so much that it will only get better. I am currently working towards attaining a Biotechnology degree at City College of San Francisco. I am hoping to transfer to UC Davis afterwards. After finding out UCSF's project for the 2010 year, I knew I wanted to come back. Throughout this summer, I have learned to work with mammalian cells. Before the iGEM experience, I was only familiar with prokaryotic cells. iGEM has expanded my skills and knowledge in so many ways. All the skills that i have learned will definitely help me in the future. With all these great pharmaceuticals that are being produced, the field of synthetic biology have proven its effectiveness and its future applications. I am looking forward to being a part of this field in the near future.<br />
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<h4 style="color:black; font-weight:bold;">Eric Wong</h4><br />
Good Afternoon, my name is Eric Wong and i am a graduate of Abraham Lincoln High School class of 2009. I am current pursuing my education in CCSF hoping to transfer into the UC system with the intended major of Molecular Cell Development. I was apart of the iGem team last year and in the time span of 4 months i have learned so many various things from running Assays, Cloning, Troubleshooting and analyzing Experiments and Data. I enjoyed the experience so much so that i decided to come back this year as a mentor for this year's team. I view this and last years iGem experience as something more than just a competition, I has given me the necessary education experience to prepare me for this field of study and allow me to learn about different project from various teams.<br><br><br />
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===UCSF iGEM Program===<br />
<h4 style="color:black; font-weight:bold;">Raquel Gomes</h4><br />
I run the UCSF iGEM program for the last three years. I really enjoy designing all the educational components of the Program but especially the 2-week bootcamp. I love teaching and having the iGEM students around all summer. I will miss all their craziness.<br />
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<br />
===Advisors===<br />
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<h4 style="color:black; font-weight:bold;">James Onuffer</h4><br />
This is my second year of involvement and the second year the Cell Propulsion Lab has hosted the team. It was quite an experience to set up the program this year, especially since this is a subject area that we had not begun working on in the Cell Propulsion Lab. The immune response to cancer is a challenging topic to take on (especially coupled with synthetic biology) and required an intensive two week bootcamp to teach the students basic concepts and endogenous systems/parts that they should be aware of. We challenged the students to come up with designs for synthetic cytotoxicity logic gates and increasing the cytotoxic response. It was quite rewarding to see them propose various devices during their team challenge and to see them take charge of getting them prioritized, made , and tested. Things did not always go smoothly, after all this goes with the territory. They had to learn to be organized, think on their feet, and be problem solvers.......quite a growth opportunity that I’m sure will stay with them for the rest of their lives. <br />
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<h4 style="color:black; font-weight:bold;">Wendell Lim</h4><br />
I have had a lab at UCSF for 15 years. The most rewarding thing is working with bright, open-minded young scientists and seeing them develop. Its great to see that most of the iGEM kids that we have worked with have continued to be excited about science and synthetic biology.<br />
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===Buddies===<br />
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<h4 style="color:black; font-weight:bold;">Jason Park</h4><br />
I am a 5th year MD/PhD student in the UCSF School of Medicine and the UCSF / UC Berkeley Joint Graduate Group in Bioengineering. I'm originally from the Los Angeles area and went to college at MIT. I've been in the Cell Propulsion Lab for about two years and I am co-advised by Dr. Wendell Lim and Dr. Bruce Conklin. I became a buddy for iGEM because I thought it would be fun working with bright, motivated high school and undergraduate students for the summer and I knew I would get good experience learning to be a better mentor. (Also, I had been interested in participating in iGEM as an undergraduate at MIT but never ended up doing it!) The best thing about being part of iGEM this summer has been working with and teaching students - together going through the learning process of doing lab research with all of its ups and downs.<br />
<br />
<h4 style="color:black; font-weight:bold;">Chia-Yung Wu</h4><br />
I am a transplant from MIT, where I participated in iGEM as a graduate advisor for the MIT team in 2008 and 2009. This summer, I joined the Lim/Cell Propulsion labs as a postdoc. It has been a great pleasure working with the UCSF team.<br />
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<h4 style="color:black; font-weight:bold;">Russell Gordley</h4><br />
I majored in Biochemistry at Swarthmore College, and performed my graduate studies with Carlos Barbas at the Scripps Research Institute in La Jolla, CA. I am interested in using synthetic biology to understand and enhance the evolvability of biological systems.<br />
<br />
After my freshman year in college, I took a summer internship with Jim Stivers at the National Institute of Standards and Technology. The practice of research turned out to be far more complex and interesting than I could have imagined--an intellectual marathon whose path is revealed with each new data set and disproven hypothesis. I am happy to participate in a program that immerses high school students in the research process. For anyone interested in a life of science, this is a great time to join the pursuit!<br />
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<h4 style="color:black; font-weight:bold;">Jesse Zalatan</h4><br />
I got started in science research with a summer internship after my junior year in high school and I was hooked! I majored in Biochemical Sciences at Harvard and went on to get my Ph.D. in Chemistry at Stanford, where I studied how enzymes speed up chemical reactions that are otherwise incredibly slow. My current research at UCSF is focused on cell signaling, where enzymes plays an important role. I am trying to understand how signaling enzymes maintain specificity for the correct targets and avoid signaling mixups. Being introduced to science research in high school inspired me to pursue science in college and beyond, and I got involved with the iGEM team to help share that experience with new students.<br />
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<h4 style="color:black; font-weight:bold;">Wilson Wong</h4><br />
My name is Wilson Wong and I am a postdoc at Wendell Lim's lab. I got my undergraduate degree in chemical engineering at UC Berkeley and graduate degree in chemical engineering at UCLA. I am currently working on applying synthetic biology to rewire human T-cell receptor signaling dynamics. I am interested in helping iGEM because I have always enjoy teaching and mentoring. My iGEM experience has been great. I am very happy with the progress they've made so far and I have a wonderful time working with them.<br />
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<h4 style="color:black; font-weight:bold;">Krista McNally</h4><br />
I am an Associate Specialist at UCSF and handle the Tissue Culture needs for the Cell Propulsion Laboratory. Before coming to UCSF, I was a Research Associate at a Medical Device company performing immunohistochemisty, histology, and thermal imaging. This is the first time I’ve encountered the iGEM program, and it was a great experience to see these young people work so hard as a team to accomplish a meaningful project. During the course of the summer, I gave a lecture to the Team on Tissue Culture and relevant Biosafety concerns, trained some of the students individually to do tissue culture work, and assisted with their use of the Flow Cytometer. I was really impressed with their commitment and diligence during the summer, and enjoyed having so many upbeat and energetic people in the lab. <br />
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<h4 style="color:black; font-weight:bold;">Silinda Neou</h4><br />
I majored in Biochemistry from Cal State East Bay and now work at UCSF in the Cell Propulsion Lab. I love scientific research and have been chasing a career in research ever since my first internship at a microbiology lab. This summer I got the pleasure of interacting with the iGEM students and watching their overall scientific development. They are a great bunch of kids.<br />
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<h4 style="color:black; font-weight:bold;">Jared Toettcher</h4><br />
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<h4 style="color:black; font-weight:bold;">Samantha Liang</h4><br />
I’m currently a second-year graduate student at UCSF in the Biochemistry and Molecular Biology Program, where I am a member of Zev Gartner’s lab. My research involves using chemical techniques to make protein therapeutics more specific and effective. During my undergrad, I majored in Bioengineering at UC Berkeley and worked in Chris Anderson’s lab for 3 years. At Berkeley, I was on the iGEM team in 2006 and loved it so much that I joined again in 2007. I think that iGEM is a unique opportunity for students to conduct research in a fun team context, and also gives them to skills to perform research independently in the future. Because of this, I wanted to stay involved in iGEM during grad school, and that’s why I’m a buddy for the awesome UCSF team this year!<br />
<br><br><br />
<br />
===Instructors===<br />
'''Special thanks to the following instructors for the seminars you provided during our bootcamp!'''<br><br />
<h4 style="color:black; font-weight:bold;">Derek Wong</h4><br />
Taunton Lab<br><br />
http://cmp.ucsf.edu/faculty/pdb_show.html?id=taunton<br><br />
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<h4 style="color:black; font-weight:bold;">Dan Hostetter</h4><br />
Craik Lab<br><br />
http://www.craiklab.ucsf.edu<br><br />
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<h4 style="color:black; font-weight:bold;">David Pincus</h4><br />
El-Samad Lab<br><br />
http://biochemistry.ucsf.edu/labs/elsamad/people/people.html<br><br />
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<h4 style="color:black; font-weight:bold;">Reid Williams</h4><br />
Lim Lab<br><br />
http://limlab.ucsf.edu/<br><br />
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<h3 style="font-weight:bold;">Killer Cell Lovin' Flyer (suggested retail price: $19.95)</h3><br />
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Not sure what this flyer means? Check out <a href="http://www.youtube.com/watch?v=qHQ9CtCv778&feature=related" target="_blank">this video</a>.<br />
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<h3 style="font-weight:bold;">Team Photo Gallery</h3><hr><br><br />
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<h3 style="font-weight:bold;">The Irresistible Frisbee & its pouch! (requested donation: $9.95)</h3><hr><br><br />
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Click on image to watch the ninja move!<br />
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{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Notes/BootcampTeam:UCSF/Notes/Bootcamp2010-10-27T22:19:43Z<p>Ryanliang: </p>
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=== '''SUMMER BOOTCAMP''' ===<br />
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Before the summer Bootcamp Raquel Gomes and the super buddies (students from UCSF iGEM 2009) came to Lincoln High School for three afterschool sessions where they taught us about synthetic biology, immunology, UCSF iGEM 2009 project and techniques the super buddies were learning in the lab. We also were given papers to read and discuss. As soon as our school year was over a two week bootcamp started at UCSF.<br />
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===='''Week 1: June 14-18'''====<br />
<br />
We kicked off our summer with a brief introduction of the iGEM team and Lim Lab members after a tour of the UCSF campus. It didn’t take long for us to plunge right into lectures though. Each morning we would learn about a new topic that falls under the broader category of immunity and end with an assignments that test our understanding of that topic. The highlights of our lectures are organized with the help of the following handy table:<br />
<br />
<br />
'''''Seminar Topic and Highlights'''''<br />
<br />
<br />
'''Intro to Cancer and Synthetic Biology''' - By Raquel Gomes<br />
<br />
A. How cancer develops<br />
<br />
B. Synthetic Biology design<br />
<br />
C. Introduction to topics covered during bootcamp and instructors <br />
<br />
D. Main rules to work in lab<br />
<br />
<br />
'''Immune Response''' - by Raquel Gomes<br />
<br />
A. Immune cells recognize proteins on target cell surfaces using receptors.<br />
<br />
B. The target surface proteins trigger the killing or non-killing response from the immune cells.<br />
<br />
<br />
'''Cytoskeleton''' - by Derek Wong<br />
<br />
A. Cytoskeletal proteins are important in forming the connection between immune cells and target cells, the immune synapse.<br />
<br />
<br />
'''Cell Death''' - by Daniel Hostetter<br />
<br />
A. The activation of the killing process results in...<br />
<br />
1. Cytotoxic agents released into target cells.<br />
<br />
2. Death signal docking on target receptors triggering apoptosis.<br />
<br />
<br />
'''Intracellular signaling''' - by David Pincus<br />
<br />
A. The inside of the cell is like a Rube Goldberg device<br />
<br />
B. Writers, Erasers and Readers - kinases, phosphatases and effectors<br />
<br />
C. Second messengers<br />
<br />
D. Small G proteins, GTPases and ATPases<br />
<br />
<br />
<br />
<br />
===='''Week 2: June 21- 25'''==== <br />
<br />
<br />
<br />
'''Logic Gates''' - by Reid Williams<br />
<br />
A. The independent parts of proteins can be rearranged to produce a protein that acts differently. <br />
<br />
B. These new proteins can be used to screen for the many different types of cancer and make the immune cells respond in a certain way.<br />
<br />
C. Application: Cancer cells vary greatly in their surface protein expression (markers). We can use these markers as inputs that act as prerequisites for a certain immune cell action (i.e. killing and not killing).<br />
<br />
<br />
'''Wrap-up and Intro to Team challenge''' - by Raquel Gomes and James Onuffer<br />
<br />
A. Summary of everything learned during bootcamp<br />
<br />
B. Intro to challenges<br />
<br />
<br />
Boot camp continues onto week two. This week, our lectures focused on more of the creative aspect of synthetic biology. We learned about modularity and how parts can be put together into devices. We also studied Boolean logic and the various types of logic gates like AND or NOT gates. <br />
<br />
Then after a summary of all the material we’ve learned over the course of boot camp, we were broken up into two groups for a '''team challenge'''. <br />
Both teams had two challenges: One was to design synthetic logic gates for cancer cell recognition and killing by cytotoxic killer cells based upon differential tumor antigen expression. The other challenge was to engineer modulators that enhanced cell mediated cytotoxicity using synthetic biology. Each group then had about two days to brainstorm, select, and tailor ideas for a final presentation to our advisers and instructors. The ideas for modulators of cell cytotoxicity were then categorized into two groups, (a) improving the cargo aspect of cell killing, and (b) improving the signaling of killing.<br />
<br />
Each team was to come up with a few complete and practical projects under their topic. We had to look though the ideas we already had and pick out the ones we liked the best. We would then work exclusively on those ideas, finalizing and refining those ideas to present again to our instructors and advisers. After presenting, we were advised on the ideas based on feasibility within the period of time, interest, and practical application.<br />
<br />
With a general project in mind, we began our first steps towards actually creating our projects. Before we could actually start synthesizing our parts, we needed to make primers for our parts since we were using a unique form of combinatorial cloning, Aar1 cloning. This required our parts to have both the Aar1 site and special complimentary binding sites. As we had never had any primer creating experience, we were given a lesson in how to use computer programs like APE and gene designer that would not only help with primer creation but alignments and more. We were also taught the basics of primer creation such as melting temperature and GC% content. This would all help in the weeks ahead with the various parts we had to synthesize, alignments we had to make, and more.<br />
<br />
<br />
<html><br />
<br><br><br />
<h3 style="font-weight:bold;">Bootcamp Gallery</h3><hr><br><br />
<br />
<div align="center"><br />
<a href="http://www.dropbox.com/gallery/12435548/1/PublicBootcamp?h=2ffc6c" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/ce/UcsfGallerybc.png" border="0" alt="UCSF 2010 Bootcamp Gallery" width="675px" /></a><br />
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__TOC__<br />
{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Notes/BootcampTeam:UCSF/Notes/Bootcamp2010-10-27T22:15:00Z<p>Ryanliang: /* Week 2: June 21- 25 */</p>
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=== '''SUMMER BOOTCAMP''' ===<br />
<br />
Before the summer Bootcamp Raquel Gomes and the super buddies (students from UCSF iGEM 2009) came to Lincoln High School for three afterschool sessions where they taught us about synthetic biology, immunology, UCSF iGEM 2009 project and techniques the super buddies were learning in the lab. We also were given papers to read and discuss. As soon as our school year was over a two week bootcamp started at UCSF.<br />
<br />
<br />
<br />
===='''Week 1: June 14-18'''====<br />
<br />
We kicked off our summer with a brief introduction of the iGEM team and Lim Lab members after a tour of the UCSF campus. It didn’t take long for us to plunge right into lectures though. Each morning we would learn about a new topic that falls under the broader category of immunity and end with an assignments that test our understanding of that topic. The highlights of our lectures are organized with the help of the following handy table:<br />
<br />
<br />
'''''Seminar Topic and Highlights'''''<br />
<br />
<br />
'''Intro to Cancer and Synthetic Biology''' - By Raquel Gomes<br />
<br />
A. How cancer develops<br />
<br />
B. Synthetic Biology design<br />
<br />
C. Introduction to topics covered during bootcamp and instructors <br />
<br />
D. Main rules to work in lab<br />
<br />
<br />
'''Immune Response''' - by Raquel Gomes<br />
<br />
A. Immune cells recognize proteins on target cell surfaces using receptors.<br />
<br />
B. The target surface proteins trigger the killing or non-killing response from the immune cells.<br />
<br />
<br />
'''Cytoskeleton''' - by Derek Wong<br />
<br />
A. Cytoskeletal proteins are important in forming the connection between immune cells and target cells, the immune synapse.<br />
<br />
<br />
'''Cell Death''' - by Daniel Hostetter<br />
<br />
A. The activation of the killing process results in...<br />
<br />
1. Cytotoxic agents released into target cells.<br />
<br />
2. Death signal docking on target receptors triggering apoptosis.<br />
<br />
<br />
'''Intracellular signaling''' - by David Pincus<br />
<br />
A. The inside of the cell is like a Rube Goldberg device<br />
<br />
B. Writers, Erasers and Readers - kinases, phosphatases and effectors<br />
<br />
C. Second messengers<br />
<br />
D. Small G proteins, GTPases and ATPases<br />
<br />
<br />
<br />
<br />
===='''Week 2: June 21- 25'''==== <br />
<br />
<br />
<br />
'''Logic Gates''' - by Reid Williams<br />
<br />
A. The independent parts of proteins can be rearranged to produce a protein that acts differently. <br />
<br />
B. These new proteins can be used to screen for the many different types of cancer and make the immune cells respond in a certain way.<br />
<br />
C. Application: Cancer cells vary greatly in their surface protein expression (markers). We can use these markers as inputs that act as prerequisites for a certain immune cell action (i.e. killing and not killing).<br />
<br />
<br />
'''Wrap-up and Intro to Team challenge''' - by Raquel Gomes and James Onuffer<br />
<br />
A. Summary of everything learned during bootcamp<br />
<br />
B. Intro to challenges<br />
<br />
<br />
Boot camp continues onto week two. This week, our lectures focused on more of the creative aspect of synthetic biology. We learned about modularity and how parts can be put together into devices. We also studied Boolean logic and the various types of logic gates like AND or NOT gates. <br />
<br />
Then after a summary of all the material we’ve learned over the course of boot camp, we were broken up into two groups for a '''team challenge'''. <br />
Both teams had two challenges: One was to design synthetic logic gates for cancer cell recognition and killing by cytotoxic killer cells based upon differential tumor antigen expression. The other challenge was to engineer modulators that enhanced cell mediated cytotoxicity using synthetic biology. Each group then had about two days to brainstorm, select, and tailor ideas for a final presentation to our advisers and instructors. The ideas for modulators of cell cytotoxicity were then categorized into two groups, (a) improving the cargo aspect of cell killing, and (b) improving the signaling of killing.<br />
<br />
Each team was to come up with a few complete and practical projects under their topic. We had to look though the ideas we already had and pick out the ones we liked the best. We would then work exclusively on those ideas, finalizing and refining those ideas to present again to our instructors and advisers. After presenting, we were advised on the ideas based on feasibility within the period of time, interest, and practical application.<br />
<br />
With a general project in mind, we began our first steps towards actually creating our projects. Before we could actually start synthesizing our parts, we needed to make primers for our parts since we were using a unique form of combinatorial cloning, Aar1 cloning. This required our parts to have both the Aar1 site and special complimentary binding sites. As we had never had any primer creating experience, we were given a lesson in how to use computer programs like APE and gene designer that would not only help with primer creation but alignments and more. We were also taught the basics of primer creation such as melting temperature and GC% content. This would all help in the weeks ahead with the various parts we had to synthesize, alignments we had to make, and more.<br />
<br />
<br />
Ryan - add photos of bootcamp and team challenge!<br />
<br><br><br />
<h3 style="font-weight:bold;">Bootcamp Gallery</h3><hr><br><br />
<br />
<div align="center"><br />
<a href="http://www.dropbox.com/gallery/12435548/1/PublicBootcamp?h=2ffc6c" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/ce/UcsfGallerybc.png" border="0"/ alt="UCSF Team Gallery"></a><br />
</div><br />
<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
__TOC__<br />
{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Notes/BootcampTeam:UCSF/Notes/Bootcamp2010-10-27T22:08:08Z<p>Ryanliang: /* Week 2: June 21- 25 */</p>
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{{Template:UCSF/BannerAndNav}}<br />
{{Template:UCSF/LeftStart}}<br />
<br />
<br />
<br />
=== '''SUMMER BOOTCAMP''' ===<br />
<br />
Before the summer Bootcamp Raquel Gomes and the super buddies (students from UCSF iGEM 2009) came to Lincoln High School for three afterschool sessions where they taught us about synthetic biology, immunology, UCSF iGEM 2009 project and techniques the super buddies were learning in the lab. We also were given papers to read and discuss. As soon as our school year was over a two week bootcamp started at UCSF.<br />
<br />
<br />
<br />
===='''Week 1: June 14-18'''====<br />
<br />
We kicked off our summer with a brief introduction of the iGEM team and Lim Lab members after a tour of the UCSF campus. It didn’t take long for us to plunge right into lectures though. Each morning we would learn about a new topic that falls under the broader category of immunity and end with an assignments that test our understanding of that topic. The highlights of our lectures are organized with the help of the following handy table:<br />
<br />
<br />
'''''Seminar Topic and Highlights'''''<br />
<br />
<br />
'''Intro to Cancer and Synthetic Biology''' - By Raquel Gomes<br />
<br />
A. How cancer develops<br />
<br />
B. Synthetic Biology design<br />
<br />
C. Introduction to topics covered during bootcamp and instructors <br />
<br />
D. Main rules to work in lab<br />
<br />
<br />
'''Immune Response''' - by Raquel Gomes<br />
<br />
A. Immune cells recognize proteins on target cell surfaces using receptors.<br />
<br />
B. The target surface proteins trigger the killing or non-killing response from the immune cells.<br />
<br />
<br />
'''Cytoskeleton''' - by Derek Wong<br />
<br />
A. Cytoskeletal proteins are important in forming the connection between immune cells and target cells, the immune synapse.<br />
<br />
<br />
'''Cell Death''' - by Daniel Hostetter<br />
<br />
A. The activation of the killing process results in...<br />
<br />
1. Cytotoxic agents released into target cells.<br />
<br />
2. Death signal docking on target receptors triggering apoptosis.<br />
<br />
<br />
'''Intracellular signaling''' - by David Pincus<br />
<br />
A. The inside of the cell is like a Rube Goldberg device<br />
<br />
B. Writers, Erasers and Readers - kinases, phosphatases and effectors<br />
<br />
C. Second messengers<br />
<br />
D. Small G proteins, GTPases and ATPases<br />
<br />
<br />
<br />
<br />
===='''Week 2: June 21- 25'''==== <br />
<br />
<br />
<br />
'''Logic Gates''' - by Reid Williams<br />
<br />
A. The independent parts of proteins can be rearranged to produce a protein that acts differently. <br />
<br />
B. These new proteins can be used to screen for the many different types of cancer and make the immune cells respond in a certain way.<br />
<br />
C. Application: Cancer cells vary greatly in their surface protein expression (markers). We can use these markers as inputs that act as prerequisites for a certain immune cell action (i.e. killing and not killing).<br />
<br />
<br />
'''Wrap-up and Intro to Team challenge''' - by Raquel Gomes and James Onuffer<br />
<br />
A. Summary of everything learned during bootcamp<br />
<br />
B. Intro to challenges<br />
<br />
<br />
Boot camp continues onto week two. This week, our lectures focused on more of the creative aspect of synthetic biology. We learned about modularity and how parts can be put together into devices. We also studied Boolean logic and the various types of logic gates like AND or NOT gates. <br />
<br />
Then after a summary of all the material we’ve learned over the course of boot camp, we were broken up into two groups for a '''team challenge'''. <br />
Both teams had two challenges: One was to design synthetic logic gates for cancer cell recognition and killing by cytotoxic killer cells based upon differential tumor antigen expression. The other challenge was to engineer modulators that enhanced cell mediated cytotoxicity using synthetic biology. Each group then had about two days to brainstorm, select, and tailor ideas for a final presentation to our advisers and instructors. The ideas for modulators of cell cytotoxicity were then categorized into two groups, (a) improving the cargo aspect of cell killing, and (b) improving the signaling of killing.<br />
<br />
Each team was to come up with a few complete and practical projects under their topic. We had to look though the ideas we already had and pick out the ones we liked the best. We would then work exclusively on those ideas, finalizing and refining those ideas to present again to our instructors and advisers. After presenting, we were advised on the ideas based on feasibility within the period of time, interest, and practical application.<br />
<br />
With a general project in mind, we began our first steps towards actually creating our projects. Before we could actually start synthesizing our parts, we needed to make primers for our parts since we were using a unique form of combinatorial cloning, Aar1 cloning. This required our parts to have both the Aar1 site and special complimentary binding sites. As we had never had any primer creating experience, we were given a lesson in how to use computer programs like APE and gene designer that would not only help with primer creation but alignments and more. We were also taught the basics of primer creation such as melting temperature and GC% content. This would all help in the weeks ahead with the various parts we had to synthesize, alignments we had to make, and more.<br />
<br />
<br />
Ryan - add photos of bootcamp and team challenge!<br />
<br><br><br />
<h3 style="font-weight:bold;">Bootcamp Gallery</h3><hr><br><br />
<br />
<div align="center"><br />
<a href="http://www.dropbox.com/gallery/12435548/1/PublicBootcamp?h=2ffc6c" target="_blank"><img src="https://static.igem.org/mediawiki/2010/4/44/UCSF_team_gallery_framed.png" border="0"/ alt="UCSF Team Gallery"></a><br />
</div><br />
<br />
<br />
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<br />
{{Template:UCSF/RightStart}}<br />
__TOC__<br />
{{Template:UCSF/RightEnd}}</div>Ryanlianghttp://2010.igem.org/File:UcsfGallerybc.pngFile:UcsfGallerybc.png2010-10-27T21:57:54Z<p>Ryanliang: </p>
<hr />
<div></div>Ryanlianghttp://2010.igem.org/Team:UCSF/Notes/AttributionsTeam:UCSF/Notes/Attributions2010-10-27T21:51:11Z<p>Ryanliang: /* Roles of people involved in making the IGEM experience the funnest summer ever!!! .... well almost */</p>
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==='''Roles of people involved in making the IGEM experience the funnest summer ever!!! .... well almost'''===<br />
<br />
<br />
'''Advisors''' - Advised us on project feasibility and best ideas worth pursuing.<br />
<br />
'''Buddies''' - Postdocs and graduate students that took some time during the summer to teach us techniques and to help us trouble shoot. <br />
<br />
'''Instructors''' - Postdocs and graduate students that taught us seminars during bootcamp.<br />
<br />
'''Supper Buddies''' - Veteran students that were part of the UCSF iGEM 2009 team. We came back to UCSF in February to start setting up everything for the new students. We go to college at San Francisco City College.<br />
<br />
'''Students''' - Cloning Gods. At the end of the summer we discovered the Qiagen robot - if we only knew about it earlier we could have cloned everything there is to be cloned.<br />
<br />
'''International Student''' - Min Lin is from Peking University and he pretty much knows how to do almost everything, like a true ninja master.<br />
<br />
'''Raquel Gomes''' - Directs the UCSF iGEM program. We first met her when she came to taught us about basic principles of immunology and synthetic biology in the after school sessions at Lincoln High school during the Spring. She is in charge of our iGEM education. She loves giving us assignments. <br />
<br />
'''James Onuffer''' - He knows everything and also runs the CPL lab! Any question we might ask he will give us an answer way bigger than we expected. :) When things would not work out he would help us find a solution or send us the right way - when the killing assays did not work he helped us figure out what other assays we could possibly use.<br />
<br />
==='''Project design and Labs that contributed to our project'''===<br />
<br />
This summer our project was organized around engineering immune cells with enhanced anti-cancer activity: engineered cancer killer cells (natural killer (NK) cells and cytotoxic T-cells (CTL)). Our host lab was the Cell Propulsion Lab (CPL) which is an NIH-funded nanomedicine development center at UCSF /UC Berkeley. The CPL is using synthetic biology to engineer mammalian cell signaling. It has primarily been interested in synthetic scaffolds and feedback loops to enhance and control cell signaling and studying the modularity of chemotaxis mechanisms.<br />
For this year’s iGEM team sponsorship, the CPL was interested in having us explore and work on cell mediated cytotoxicity, which is a subject area that they had not begun working on. We needed to obtain new cell lines and gene constructs from collaborators for our project as they were not already available in the lab. Additionally we needed to establish gene transfection techniques and killing assays. We obtained several natural killer cell lines from Lewis Lanier’s lab at UCSF. We also obtained plasmids containing single-chain antibody (scFv) constructs against the cancer antigens mesothelin and CD19 from the laboratory of Michael Milone at the University of Pennsylvania. Our host lab advised us on this early system work and also set up the Material Transfer Agreements (MTAs) for the scFv constructs as they are proprietary.<br />
How was this years project put together and accomplished? Earlier in the year, Ryan Liang and Ethan Chan from last year’s iGEM team started working in the lab to start growing the cell lines, work out plasmid transient transfections, and killing assays. This early work was planned with feedback and advice from members of the CPL lab. This way Ryan and Ethan could get a jump start on some of the basic systems of the project before the rest of us arrived in June. The summer goes fast, so the project area needs to be defined and planning is necessary so we can accomplish something in 3 months.<br />
<br />
Once all of us arrived for the summer, the advisors and instructors arranged a two-week Bootcamp where we learned about cytotoxic cells, the immune response to cancer, principles of protein signaling modules and circuits, and synthetic biology. They also introduced to us the previously described use of scFv modules with signaling domains to direct the cytotoxic response towards cancer cells (Chimeric Antigen Receptors (CARs) as well as other receptors and signaling domains important in the cytotoxicity response. At the end of the Bootcamp we were challenged by our advisors with two topics for a team challenge: 1.) how could we create synthetic logic gates to address different cancer vs normal cell antigen expression scenarios, and 2.) how could we create enhanced killing through the use of synthetic biology. We split into two teams and brainstormed and then we presented our designs to the entire group. From the first challenge we proposed to use combinations of activating and inhibitor modules on chimeric antigen receptors to create gates such as an ANDN (AND NOT) gate; these devices would mimic the self vs. cancer responses of natural receptors on NK cells. However our devices could be engineered to detect any cancer antigen for which an antibody could be raised. Although individual activating CARs had been reported by others, no one had used them in combination to create logic gates. For the second challenge we presented several ways to enhance cell killing by adding multiple signaling domains to one CAR receptor and also by engineering new granule components to overcome cancer resistance.<br />
<br />
After the team challenge, the real work started. We were challenged to propose, list, and design the actual components of our devices. We generated lists and then in discussion and brainstorming with our advisors, we set a priority list for construction. We followed the same modular cloning to create our devices using the restriction enzyme AarI used by last year’s team. Advisors in the CPL ordered the clones we decided upon primarily from Open Biosystems. Once they arrived we went to work. We sequenced the clones to be sure they were correct and then we designed PCR primers for cloning and were in charge of all aspects of device construction and analysis. During the construction of the initial CAR devices, we came up with the idea of sending GFP to the granules of cytotoxic cells using motifs from granule proteins and started the work on this as well.<br />
<br />
We made many devices for assay (we had a lot of ideas :) ), but unfortunately we ran into a problem with our idea to use cytotoxicity assays to measure the killing response. The problem was that the transient transfection/expression of our devices by electroporation was too low to see responses (<20% efficiency). The large amount of untransfected cells dominated the response. In brainstorming with our advisors we decided to use/substitute T cell activation assays as a reporter of potential killing response as they should be correlated. The granule loading devices were looked at by microscopy with advice and supervision of buddies and advisors. Both of these techniques would allow us to look at transient transfected cell responses without a lot of background response from untransfected cells.<br />
<br />
With all of this, we were only able to run a few experiments before the summer was over. We were only able to validate two ANDN gates and the loading of GFP into granules. It’s too bad that the killing assays didn’t work out with our system. Our advisors mentioned that either viral methods of gene delivery or stable cell line creation would probably be necessary to run the cytotoxicity assays so that high expression levels could be obtained. It was a great experience to come up with devices using information from the literature and natural systems. Using combinations of CARs to construct logic gates had not been reported before and it was also very cool to see that we can use cellular zipcodes to localize proteins to granules. The CPL is interested in some of our devices and will hopefully follow up after iGEM is over.<br />
<br><br />
<br><br />
<br><br />
See more on who did what in our [https://2010.igem.org/Team:UCSF/Notes/Timeline project timeline page].<br />
<br><br />
<br><br />
<br><br />
<br />
{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Notes/AttributionsTeam:UCSF/Notes/Attributions2010-10-27T21:50:46Z<p>Ryanliang: /* Roles of people involved in making the IGEM experience the funnest summer ever!!! .... well almost */</p>
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==='''Roles of people involved in making the IGEM experience the funnest summer ever!!! .... well almost'''===<br />
<br />
<br />
'''Advisors''' - Advised us on project feasibility and best ideas worth pursuing.<br />
<br />
'''Buddies''' - Postdocs and graduate students that took some time during the summer to teach us techniques and to help us trouble shoot. <br />
<br />
'''Instructors''' - Postdocs and graduate students that taught us seminars during bootcamp.<br />
<br />
'''Supper Buddies''' - veteran students that were part of the UCSF iGEM 2009 team. We came back to UCSF in February to start setting up everything for the new students. We go to college at San Francisco City College.<br />
<br />
'''Students''' - cloning Gods. At the end of the summer we discovered the Qiagen robot - if we only knew about it earlier we could have cloned everything there is to be cloned.<br />
<br />
'''International Student''' - Min Lin is from Peking University and he pretty much knows how to do almost everything, like a true ninja master.<br />
<br />
'''Raquel Gomes''' - directs the UCSF iGEM program. We first met her when she came to taught us about basic principles of immunology and synthetic biology in the after school sessions at Lincoln High school during the Spring. She is in charge of our iGEM education. She loves giving us assignments. <br />
<br />
'''James Onuffer''' - He knows everything and also runs the CPL lab! Any question we might ask he will give us an answer way bigger than we expected. :) When things would not work out he would help us find a solution or send us the right way - when the killing assays did not work he helped us figure out what other assays we could possibly use.<br />
<br />
==='''Project design and Labs that contributed to our project'''===<br />
<br />
This summer our project was organized around engineering immune cells with enhanced anti-cancer activity: engineered cancer killer cells (natural killer (NK) cells and cytotoxic T-cells (CTL)). Our host lab was the Cell Propulsion Lab (CPL) which is an NIH-funded nanomedicine development center at UCSF /UC Berkeley. The CPL is using synthetic biology to engineer mammalian cell signaling. It has primarily been interested in synthetic scaffolds and feedback loops to enhance and control cell signaling and studying the modularity of chemotaxis mechanisms.<br />
For this year’s iGEM team sponsorship, the CPL was interested in having us explore and work on cell mediated cytotoxicity, which is a subject area that they had not begun working on. We needed to obtain new cell lines and gene constructs from collaborators for our project as they were not already available in the lab. Additionally we needed to establish gene transfection techniques and killing assays. We obtained several natural killer cell lines from Lewis Lanier’s lab at UCSF. We also obtained plasmids containing single-chain antibody (scFv) constructs against the cancer antigens mesothelin and CD19 from the laboratory of Michael Milone at the University of Pennsylvania. Our host lab advised us on this early system work and also set up the Material Transfer Agreements (MTAs) for the scFv constructs as they are proprietary.<br />
How was this years project put together and accomplished? Earlier in the year, Ryan Liang and Ethan Chan from last year’s iGEM team started working in the lab to start growing the cell lines, work out plasmid transient transfections, and killing assays. This early work was planned with feedback and advice from members of the CPL lab. This way Ryan and Ethan could get a jump start on some of the basic systems of the project before the rest of us arrived in June. The summer goes fast, so the project area needs to be defined and planning is necessary so we can accomplish something in 3 months.<br />
<br />
Once all of us arrived for the summer, the advisors and instructors arranged a two-week Bootcamp where we learned about cytotoxic cells, the immune response to cancer, principles of protein signaling modules and circuits, and synthetic biology. They also introduced to us the previously described use of scFv modules with signaling domains to direct the cytotoxic response towards cancer cells (Chimeric Antigen Receptors (CARs) as well as other receptors and signaling domains important in the cytotoxicity response. At the end of the Bootcamp we were challenged by our advisors with two topics for a team challenge: 1.) how could we create synthetic logic gates to address different cancer vs normal cell antigen expression scenarios, and 2.) how could we create enhanced killing through the use of synthetic biology. We split into two teams and brainstormed and then we presented our designs to the entire group. From the first challenge we proposed to use combinations of activating and inhibitor modules on chimeric antigen receptors to create gates such as an ANDN (AND NOT) gate; these devices would mimic the self vs. cancer responses of natural receptors on NK cells. However our devices could be engineered to detect any cancer antigen for which an antibody could be raised. Although individual activating CARs had been reported by others, no one had used them in combination to create logic gates. For the second challenge we presented several ways to enhance cell killing by adding multiple signaling domains to one CAR receptor and also by engineering new granule components to overcome cancer resistance.<br />
<br />
After the team challenge, the real work started. We were challenged to propose, list, and design the actual components of our devices. We generated lists and then in discussion and brainstorming with our advisors, we set a priority list for construction. We followed the same modular cloning to create our devices using the restriction enzyme AarI used by last year’s team. Advisors in the CPL ordered the clones we decided upon primarily from Open Biosystems. Once they arrived we went to work. We sequenced the clones to be sure they were correct and then we designed PCR primers for cloning and were in charge of all aspects of device construction and analysis. During the construction of the initial CAR devices, we came up with the idea of sending GFP to the granules of cytotoxic cells using motifs from granule proteins and started the work on this as well.<br />
<br />
We made many devices for assay (we had a lot of ideas :) ), but unfortunately we ran into a problem with our idea to use cytotoxicity assays to measure the killing response. The problem was that the transient transfection/expression of our devices by electroporation was too low to see responses (<20% efficiency). The large amount of untransfected cells dominated the response. In brainstorming with our advisors we decided to use/substitute T cell activation assays as a reporter of potential killing response as they should be correlated. The granule loading devices were looked at by microscopy with advice and supervision of buddies and advisors. Both of these techniques would allow us to look at transient transfected cell responses without a lot of background response from untransfected cells.<br />
<br />
With all of this, we were only able to run a few experiments before the summer was over. We were only able to validate two ANDN gates and the loading of GFP into granules. It’s too bad that the killing assays didn’t work out with our system. Our advisors mentioned that either viral methods of gene delivery or stable cell line creation would probably be necessary to run the cytotoxicity assays so that high expression levels could be obtained. It was a great experience to come up with devices using information from the literature and natural systems. Using combinations of CARs to construct logic gates had not been reported before and it was also very cool to see that we can use cellular zipcodes to localize proteins to granules. The CPL is interested in some of our devices and will hopefully follow up after iGEM is over.<br />
<br><br />
<br><br />
<br><br />
See more on who did what in our [https://2010.igem.org/Team:UCSF/Notes/Timeline project timeline page].<br />
<br><br />
<br><br />
<br><br />
<br />
{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/FunStuffTeam:UCSF/FunStuff2010-10-27T21:42:24Z<p>Ryanliang: </p>
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<h3 style="font-weight:bold;">Killer Cell Lovin' Flyer (Suggested Retail Price)</h3><br />
<hr><br><br />
<br />
<div align="center"><br />
<a href="https://static.igem.org/mediawiki/2010/c/c2/UCSF_HAHAHAH.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/c2/UCSF_HAHAHAH.jpg" border="0" alt="Get Some Killer Cell Loving'" width="400"/></a><br />
<br />
<br><br />
Not sure what this flyer means? Check out <a href="http://www.youtube.com/watch?v=qHQ9CtCv778&feature=related" target="_blank">this video</a>.<br />
</div><br />
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<br><br><br />
<h3 style="font-weight:bold;">Team Photo Gallery</h3><hr><br><br />
<br />
<div align="center"><br />
<a href="http://www.dropbox.com/gallery/12793331/1/WikiGalleryPicks?h=8fb6ea" target="_blank"><img src="https://static.igem.org/mediawiki/2010/4/44/UCSF_team_gallery_framed.png" border="0"/ alt="UCSF Team Gallery"></a><br />
</div><br />
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<br><br><br />
<h3 style="font-weight:bold;">The Irresistible Frisbee & its pouch! (Requested Donation $4.95 + SH) </h3><hr><br><br />
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<div align="center"><br />
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<a href="https://static.igem.org/mediawiki/2010/e/e4/UCSF_fling_disk_lg.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2010/e/e4/UCSF_fling_disk_lg.png" alt="UCSF frisbee design" width="400" border="0" /></a><br />
<br />
<a href="https://static.igem.org/mediawiki/2010/a/a3/UCSF_fling_bag.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2010/a/a3/UCSF_fling_bag.png" alt="UCSF frisbee pouch" width="230" border="0" /></a><br />
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<h3 style="font-weight:bold;">Killer Animations</h3><hr><br><br />
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<img src="https://static.igem.org/mediawiki/2010/8/8f/UCSF_Igem_Ninjas_animated_web_transparent.gif"/><br />
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onmouseover="mouseOver()" <br />
onmouseout="mouseOut()" <br />
onclick="onClick()" <br />
height="250"><br />
<br><br />
Click on image to watch the ninja move!<br />
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{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Notes/AttributionsTeam:UCSF/Notes/Attributions2010-10-27T19:38:08Z<p>Ryanliang: </p>
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==='''Roles of people involved in making the IGEM experience the funnest summer ever!!! .... well almost'''===<br />
<br />
<br />
'''Advisors''' - Advised us on project feasibility and best ideas worth pursuing.<br />
<br />
'''Buddies''' - Postdocs and graduate students that took some time during the summer to teach us techniques and to help us trouble shoot. <br />
<br />
'''Instrutors''' - Postdocs and graduate students that taught us seminars during bootcamp.<br />
<br />
'''Supper Buddies''' - veteran students that were part of the UCSF iGEM 2009 team. We came back to UCSF in February to start setting up everything for the new students. We go to college at San Francisco City College.<br />
<br />
'''Students''' - cloning Gods. At the end of the summer we discovered the Qiagen robot - if we only knew about it earlier we could have cloned everything there is to be cloned.<br />
<br />
'''International Student''' - Min Lin is from Peking University and he pretty much knows how to do almost everything, like a true ninja master.<br />
<br />
'''Raquel Gomes''' - directs the UCSF iGEM program. We first met her when she came to taught us about basic principles of immunology and synthetic biology in the after school sessions at Lincoln High school during the Spring. She is in charge of our iGEM education. She loves giving us assignments. <br />
<br />
'''James Onuffer''' - He knows everything and also runs the CPL lab! Any question we might ask he will give us an answer way bigger than we expected. :) When things would not work out he would help us find a solution or send us the right way - when the killing assays did not work he helped us figure out what other assays we could possibly use.<br />
<br />
<br />
<br />
==='''Project design and Labs that contributed to our project'''===<br />
<br />
This summer our project was organized around engineering immune cells with enhanced anti-cancer activity: engineered cancer killer cells (natural killer (NK) cells and cytotoxic T-cells (CTL)). Our host lab was the Cell Propulsion Lab (CPL) which is an NIH-funded nanomedicine development center at UCSF /UC Berkeley. The CPL is using synthetic biology to engineer mammalian cell signaling. It has primarily been interested in synthetic scaffolds and feedback loops to enhance and control cell signaling and studying the modularity of chemotaxis mechanisms.<br />
For this year’s iGEM team sponsorship, the CPL was interested in having us explore and work on cell mediated cytotoxicity, which is a subject area that they had not begun working on. We needed to obtain new cell lines and gene constructs from collaborators for our project as they were not already available in the lab. Additionally we needed to establish gene transfection techniques and killing assays. We obtained several natural killer cell lines from Lewis Lanier’s lab at UCSF. We also obtained plasmids containing single-chain antibody (scFv) constructs against the cancer antigens mesothelin and CD19 from the laboratory of Michael Milone at the University of Pennsylvania. Our host lab advised us on this early system work and also set up the Material Transfer Agreements (MTAs) for the scFv constructs as they are proprietary.<br />
How was this years project put together and accomplished? Earlier in the year, Ryan Liang and Ethan Chan from last year’s iGEM team started working in the lab to start growing the cell lines, work out plasmid transient transfections, and killing assays. This early work was planned with feedback and advice from members of the CPL lab. This way Ryan and Ethan could get a jump start on some of the basic systems of the project before the rest of us arrived in June. The summer goes fast, so the project area needs to be defined and planning is necessary so we can accomplish something in 3 months.<br />
<br />
Once all of us arrived for the summer, the advisors and instructors arranged a two-week Bootcamp where we learned about cytotoxic cells, the immune response to cancer, principles of protein signaling modules and circuits, and synthetic biology. They also introduced to us the previously described use of scFv modules with signaling domains to direct the cytotoxic response towards cancer cells (Chimeric Antigen Receptors (CARs) as well as other receptors and signaling domains important in the cytotoxicity response. At the end of the Bootcamp we were challenged by our advisors with two topics for a team challenge: 1.) how could we create synthetic logic gates to address different cancer vs normal cell antigen expression scenarios, and 2.) how could we create enhanced killing through the use of synthetic biology. We split into two teams and brainstormed and then we presented our designs to the entire group. From the first challenge we proposed to use combinations of activating and inhibitor modules on chimeric antigen receptors to create gates such as an ANDN (AND NOT) gate; these devices would mimic the self vs. cancer responses of natural receptors on NK cells. However our devices could be engineered to detect any cancer antigen for which an antibody could be raised. Although individual activating CARs had been reported by others, no one had used them in combination to create logic gates. For the second challenge we presented several ways to enhance cell killing by adding multiple signaling domains to one CAR receptor and also by engineering new granule components to overcome cancer resistance.<br />
<br />
After the team challenge, the real work started. We were challenged to propose, list, and design the actual components of our devices. We generated lists and then in discussion and brainstorming with our advisors, we set a priority list for construction. We followed the same modular cloning to create our devices using the restriction enzyme AarI used by last year’s team. Advisors in the CPL ordered the clones we decided upon primarily from Open Biosystems. Once they arrived we went to work. We sequenced the clones to be sure they were correct and then we designed PCR primers for cloning and were in charge of all aspects of device construction and analysis. During the construction of the initial CAR devices, we came up with the idea of sending GFP to the granules of cytotoxic cells using motifs from granule proteins and started the work on this as well.<br />
<br />
We made many devices for assay (we had a lot of ideas :) ), but unfortunately we ran into a problem with our idea to use cytotoxicity assays to measure the killing response. The problem was that the transient transfection/expression of our devices by electroporation was too low to see responses (<20% efficiency). The large amount of untransfected cells dominated the response. In brainstorming with our advisors we decided to use/substitute T cell activation assays as a reporter of potential killing response as they should be correlated. The granule loading devices were looked at by microscopy with advice and supervision of buddies and advisors. Both of these techniques would allow us to look at transient transfected cell responses without a lot of background response from untransfected cells.<br />
<br />
With all of this, we were only able to run a few experiments before the summer was over. We were only able to validate two ANDN gates and the loading of GFP into granules. It’s too bad that the killing assays didn’t work out with our system. Our advisors mentioned that either viral methods of gene delivery or stable cell line creation would probably be necessary to run the cytotoxicity assays so that high expression levels could be obtained. It was a great experience to come up with devices using information from the literature and natural systems. Using combinations of CARs to construct logic gates had not been reported before and it was also very cool to see that we can use cellular zipcodes to localize proteins to granules. The CPL is interested in some of our devices and will hopefully follow up after iGEM is over.<br />
<br><br />
<br><br />
<br><br />
See more on who did what in our [https://2010.igem.org/Team:UCSF/Notes/Timeline project timeline page].<br />
<br><br />
<br><br />
<br><br />
<br />
{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Notes/AttributionsTeam:UCSF/Notes/Attributions2010-10-27T19:37:20Z<p>Ryanliang: /* Roles of people involved in making the IGEM experience the funnest summer ever!!! .... well almost */</p>
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==='''Roles of people involved in making the IGEM experience the funnest summer ever!!! .... well almost'''===<br />
<br />
<br />
'''Advisors''' - Advised us on project feasibility and best ideas worth pursuing.<br />
<br />
'''Buddies''' - Postdocs and graduate students that took some time during the summer to teach us techniques and to help us trouble shoot. <br />
<br />
'''Instrutors''' - Postdocs and graduate students that taught us seminars during bootcamp.<br />
<br />
'''Supper Buddies''' - veteran students that were part of the UCSF iGEM 2009 team. We came back to UCSF in February to start setting up everything for the new students. We go to college at San Francisco City College.<br />
<br />
'''Students''' - cloning Gods. At the end of the summer we discovered the Qiagen robot - if we only knew about it earlier we could have cloned everything there is to be cloned.<br />
<br />
'''International Student''' - Min Lin is from Peking University and he pretty much knows how to do almost everything, like a true ninja master.<br />
<br />
'''Raquel Gomes''' - directs the UCSF iGEM program. We first met her when she came to taught us about basic principles of immunology and synthetic biology in the after school sessions at Lincoln High school during the Spring. She is in charge of our iGEM education. She loves giving us assignments. <br />
<br />
'''James Onuffer''' - He knows everything and also runs the CPL lab! Any question we might ask he will give us an answer way bigger than we expected. :) When things would not work out he would help us find a solution or send us the right way - when the killing assays did not work he helped us figure out what other assays we could possibly use.<br />
<br />
==='''Project design and Labs that contributed to our project'''===<br />
<br />
This summer our project was organized around engineering immune cells with enhanced anti-cancer activity: engineered cancer killer cells (natural killer (NK) cells and cytotoxic T-cells (CTL)). Our host lab was the Cell Propulsion Lab (CPL) which is an NIH-funded nanomedicine development center at UCSF /UC Berkeley. The CPL is using synthetic biology to engineer mammalian cell signaling. It has primarily been interested in synthetic scaffolds and feedback loops to enhance and control cell signaling and studying the modularity of chemotaxis mechanisms.<br />
For this year’s iGEM team sponsorship, the CPL was interested in having us explore and work on cell mediated cytotoxicity, which is a subject area that they had not begun working on. We needed to obtain new cell lines and gene constructs from collaborators for our project as they were not already available in the lab. Additionally we needed to establish gene transfection techniques and killing assays. We obtained several natural killer cell lines from Lewis Lanier’s lab at UCSF. We also obtained plasmids containing single-chain antibody (scFv) constructs against the cancer antigens mesothelin and CD19 from the laboratory of Michael Milone at the University of Pennsylvania. Our host lab advised us on this early system work and also set up the Material Transfer Agreements (MTAs) for the scFv constructs as they are proprietary.<br />
How was this years project put together and accomplished? Earlier in the year, Ryan Liang and Ethan Chan from last year’s iGEM team started working in the lab to start growing the cell lines, work out plasmid transient transfections, and killing assays. This early work was planned with feedback and advice from members of the CPL lab. This way Ryan and Ethan could get a jump start on some of the basic systems of the project before the rest of us arrived in June. The summer goes fast, so the project area needs to be defined and planning is necessary so we can accomplish something in 3 months.<br />
<br />
Once all of us arrived for the summer, the advisors and instructors arranged a two-week Bootcamp where we learned about cytotoxic cells, the immune response to cancer, principles of protein signaling modules and circuits, and synthetic biology. They also introduced to us the previously described use of scFv modules with signaling domains to direct the cytotoxic response towards cancer cells (Chimeric Antigen Receptors (CARs) as well as other receptors and signaling domains important in the cytotoxicity response. At the end of the Bootcamp we were challenged by our advisors with two topics for a team challenge: 1.) how could we create synthetic logic gates to address different cancer vs normal cell antigen expression scenarios, and 2.) how could we create enhanced killing through the use of synthetic biology. We split into two teams and brainstormed and then we presented our designs to the entire group. From the first challenge we proposed to use combinations of activating and inhibitor modules on chimeric antigen receptors to create gates such as an ANDN (AND NOT) gate; these devices would mimic the self vs. cancer responses of natural receptors on NK cells. However our devices could be engineered to detect any cancer antigen for which an antibody could be raised. Although individual activating CARs had been reported by others, no one had used them in combination to create logic gates. For the second challenge we presented several ways to enhance cell killing by adding multiple signaling domains to one CAR receptor and also by engineering new granule components to overcome cancer resistance.<br />
<br />
After the team challenge, the real work started. We were challenged to propose, list, and design the actual components of our devices. We generated lists and then in discussion and brainstorming with our advisors, we set a priority list for construction. We followed the same modular cloning to create our devices using the restriction enzyme AarI used by last year’s team. Advisors in the CPL ordered the clones we decided upon primarily from Open Biosystems. Once they arrived we went to work. We sequenced the clones to be sure they were correct and then we designed PCR primers for cloning and were in charge of all aspects of device construction and analysis. During the construction of the initial CAR devices, we came up with the idea of sending GFP to the granules of cytotoxic cells using motifs from granule proteins and started the work on this as well.<br />
<br />
We made many devices for assay (we had a lot of ideas :) ), but unfortunately we ran into a problem with our idea to use cytotoxicity assays to measure the killing response. The problem was that the transient transfection/expression of our devices by electroporation was too low to see responses (<20% efficiency). The large amount of untransfected cells dominated the response. In brainstorming with our advisors we decided to use/substitute T cell activation assays as a reporter of potential killing response as they should be correlated. The granule loading devices were looked at by microscopy with advice and supervision of buddies and advisors. Both of these techniques would allow us to look at transient transfected cell responses without a lot of background response from untransfected cells.<br />
<br />
With all of this, we were only able to run a few experiments before the summer was over. We were only able to validate two ANDN gates and the loading of GFP into granules. It’s too bad that the killing assays didn’t work out with our system. Our advisors mentioned that either viral methods of gene delivery or stable cell line creation would probably be necessary to run the cytotoxicity assays so that high expression levels could be obtained. It was a great experience to come up with devices using information from the literature and natural systems. Using combinations of CARs to construct logic gates had not been reported before and it was also very cool to see that we can use cellular zipcodes to localize proteins to granules. The CPL is interested in some of our devices and will hopefully follow up after iGEM is over.<br />
<br><br />
<br><br />
<br><br />
See more on who did what in our [https://2010.igem.org/Team:UCSF/Notes/Timeline project timeline page].<br />
<br><br />
<br><br />
<br><br />
<br />
{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/SignalingTeam:UCSF/Project/Signaling2010-10-27T19:35:45Z<p>Ryanliang: </p>
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<h3 style="font-weight:bold;">Stronger Signaling</h3><br />
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'''Goal''': Engineer a stronger signaling response on killer cells that will lead to a more effective/faster elimination of the cancer cell.<br />
<br />
<br />
'''Approach''': After brainstorming during our team challenge we came up with three different approaches to achieve stronger signaling:<br />
<br />
1. Co-stimulation by transfection of killer cells with a synthetic GPCR<br />
<br />
2. Reengineer killer cell’s receptors to have multiple activation domains<br />
<br />
3. Bypassing steps in the signaling cascade by engineering direct connections between the killer cell’s receptor and downstream effectors.<br />
<br />
<br />
'''Devices''':<br />
<br />
1. '''Co-stimulation''' – we successfully used a device from last year’s UCSF team (registry part# '''BBA_K209400''') and showed enhanced signaling activation<br />
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2. '''Multiple activation domains''' – we designed and constructed new devices for this approach but did not get to test them<br />
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3. '''Bypassing steps in activation cascade''' – we designed a few devices for this approach but did not had enough time to assemble them from parts to devices.<br />
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'''Brief Introduction'''<br />
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In this part of our project we wanted to create devices that made our synthetic cancer killers more effective than their natural counterparts at eliminating cancer cells by enhancing the activation mechanisms that lead to the killing response. Once our synthetic cancer killers confirmed they were in the presence of a cancer cell (using the devices developed in the GREATER PRECISION part of our project) they would take no time to eliminate it and could rapidly move on to the next target.<br />
Natural immune killers use receptor-associated intracellular activation domains to tell the inside of the cell that it needs to get ready to kill the cancer cell. This means that these '''intracellular activation domains''', such as ITAMs (Immunoreceptor Tyrosine-based Activation Motifs), once activated recruit and activate '''kinases''' and a '''cascade of signaling events''' is initiated culminating in the formation of the immune synapse between the killer cell and the cancer cell. The killer cell then releases killing agents into the immune synapse that tell the cancer cell to apoptose (die!). We thought of three ways to improve the activation of the killer cell (described in more detail below): (1) Co-stimulation using a synthetic GPCR to enhance strength of killing response; (2) Engineer receptors with multiple activation domains to increase the level of activation; and (3) Bypass steps on the activation cascade to possibly improve speed of response.<br />
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'''1. Stronger killing by Co-stimulation with a synthetic GPCR'''<br />
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''Concept, Experimental design and Results''<br />
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One way we thought of enhancing the killing response was to increase the amount of kinases activated, as these seem to have a central role in the induction of killing on the killer cells. For that we looked into other receptors that once activated would also lead to the activation of kinases. Last year, the UCSF iGEM team used synthetic G protein-coupled receptors (GPCRs) to engineer immune cells to move (chemotax) towards new targets. In the future these synthetic cellbots would be able to detect specific chemicals released by tumors and move towards them more efficiently (for more info on this project please see UCSF iGEM 2009). GPCRs are activated by small molecule ligands instead of cell surface proteins. Because intracellular kinases are involved in chemotaxis, we hypothesized that the synthetic GPCRs used by the 2009 team that showed to mediate chemotaxis could possibly enhance the killing response by increasing the overall amount of kinases activated (Figure 1).<br />
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[[Image:GPCR_Stronger_Signaling.png]]<br />
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'''''Figure 1'''''<br />
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To determine whether a synthetic GPCR can enhance activation of killing response, we transfected T cells with a synthetic GPCR from last year’s team (part# BBA_K209400), that recognizes the small synthetic molecule called CNO. Initially we wanted to use a killing assay to measure the level of immune killer cell activation but unfortunately this assay did not work out (please click here for a detailed explanation). So to measure the level of activation of our T cells we used an assay that measures T cell activation through the production of interleukin 2 (IL-2). IL-2 is a signaling molecule that is expressed by activated T cells. In our assay non-transfected and transfected T cells were incubated with and without CNO (synthetic GPCR’s ligand). After incubation the levels of IL-2 produced (presence of IL-2 in supernatant) were measured by ELISA assay. You can see our results in the '''graph below'''. The first and second bars (from the left to the right) on the graph below represent non-transfected T cells that were in the absence or presence of CNO, respectively. The third and fourth bars represent the synthetic GPCR-tranfected T cells that were in the absence or presence of CNO, respectively. The data shows that T-cells transfected with synthetic GPCR and in the presence of CNO (ligand) show a higher level of activation. The results indicate that the use of a synthetic GPCR can increase killer cell activation, therefore achieving the goal of enhanced signaling.<br />
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[[Image:GPCR_graph.png|620px]]<br />
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'''''Results Figure''''': Enhancing killer cell activation with a synthetic GPCR<br />
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A synthetic GPCR shown to mediate immune cell migration towards the ligand CNO (results of UCSF iGEM team 2009 project) was introduced to T cells as a potential way to increase the level of kinase activation. IL-2 production was used as a measure of T-cell activation. The results show that when in the presence of CNO, T cells transfected with the synthetic GPCR , have a higher level of activation than non-transfected T-cells.<br />
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'''''Devices:''''' We used a device from last year [http://partsregistry.org/wiki/index.php?title=Part:BBa_K209400'''(part# BBA_K209400)'''] that codes for the synthetic GPCR.<br />
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'''''Future Application'''''<br />
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Our experimental model suggests a synthetic biology approach to eliminate cancer cells more effectively. If we could engineer synthetic GPCRs that recognize small molecules secreted by cancer cells, we could make killer cells move faster to a cancer site (based on UCSF 2009 project results) AND once at the cancer site our synthetic killers would also be equipped to eliminate the cancer more efficiently. The additional kinase activation caused by the synthetic GPCRs would augment the normal activation signal to enhance the killing response of the killer cell.<br />
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'''2. Stronger Killing by Engineering receptors with multiple activation domains'''<br />
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''Concept and Experimental design''<br />
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As mentioned in the introduction above when killer cell receptors bind to cancer antigens their intracellular activation domains (such as ITAMs) get activated and lead to the activation of kinases. So another approach to increase the amount of kinases activated and ultimately enhancement of the killing response would be to activate more activation domains. A small input would lead to a amplified output. With this in mind our team came up with the idea of constructing receptors with multiple activation domains so when one receptor would become activated it would lead to the activation of a higher number of kinases (Figure 2). Examples of immune killer cell’s receptors activation domains are CD3zeta, FCRgamma, DAP10.<br />
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[[Image:Stronger_signaling_approach2.jpg|620px]]<br />
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'''''Figure 2'''''<br />
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Using chimeric antigen receptors (CARs) as external sensors for the presence of specific antigens we built various receptors with possibly different degrees of amplification. Attached to the antigen receptor would be multiple intracellular activation domains such as CD3z,FCRgamma and DAP10. This increased number of activation domains allows for a stronger signal to be transmitted further into the cell from the binding of one ligand to the antigen receptor. The increased amplification of the activation signal should lead to a stronger response by killer cells to induce cytotoxicity.<br />
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'''''Devices:''''' We were able to build, but not test, the following devices:<br />
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CAR-'''CD3z-CD3z'''<br />
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CAR-'''DAP10-CD3z'''<br />
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CAR-'''DAP10-DAP10'''<br />
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CAR-'''FCRg-CD3z'''<br />
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CAR-'''FCRg-DAP10'''<br />
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CAR = chimeric antigen receptor scFv (single-chain antibody protein domain) from Milone lab at University of Pennsylvania. They gave us mouse anti-humanCD19 and anti-humanMesothelin plasmids for us to use as a PCR source for cloning. These antibodies were developed by Ira Pastan at NCI and Dario Campana at Saint Jude's.<br />
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'''NOTE''': Although we have these devices ready to be tested we did not submit them to the registry of parts as they are not in this year’s required biobrick format. All these devices were built under biobrick standard RFC28 and not moved to pSB1C3 due to time constraints.<br />
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'''3. Stronger killing by bypassing steps in the activation signaling cascade'''<br />
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''Concept and Design''<br />
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As discussed in the introduction, the activation signaling cascade (or pathway) connect the input (activation by binding cancer antigen) with the ultimate output (cancer cell killing). These signaling cascades while effective at regulating the ultimate response by the killer cell, can be slow because once the ligand binds to the receptor, the signal has to pass through the long chain of molecules that make up the signaling pathway. We would like to engineer a possible faster pathway by bypassing certain intermediate steps of the pathway (for example regulatory steps). As a result, we would cut down on the number of steps needed to be taken before reaching the desired output (Figure 3).<br />
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[[Image:stronger_signaling_approach3.png]]<br />
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'''''Figure 3'''''<br />
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One way to achieve bypassing steps of the signaling pathway would be to make devices with the receptor bound directly to a downstream effector of the activation pathway. In this way we could skip steps in the signaling process and reduce the amount of time it takes a killer cell to get ready to kill a cancer cell. We can visualize this process through the use of a simple, hypothetical signaling pathway consisting of the receptor and three activation pathway proteins, A, B and C. If the activated receptor signlas to A which in turn signals to B which signals to C which causes cell killing, the process could be made much quicker if we could engineer the receptor to “talk” directly to C, skipping A and B entirely. The new signaling pathway would then have the receptor signal to C which causes cell killing. Obviously there are some drawbacks of this design such as loss of amplification by the signaling cascade. Still it would be interesting to see if the by “shortening” the signaling pathway we would loose some regulatory steps and lead to a “faster’ killing response by the killer cell.<br />
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'''''Devices:''''' We did not have the time to build any of the devices we designed during the team challenge for this approach.<br />
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__NOTOC__</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/Future_ImplicationsTeam:UCSF/Project/Future Implications2010-10-27T19:31:00Z<p>Ryanliang: </p>
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==='''Future Implications'''===<br />
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[[Image:The_future.png]]<br />
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We expect that in the Future our '''''Synthetic Cancer Killers''''' would be equipped with '''GREATER PRECISION''' in the detection of cancer, '''STRONGER SIGNALING''' that would lead to a faster and stronger killing response, and '''BETTER ARSENAL''' to kill any kind of cancer they encounter (resistant or not)! <br />
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We would achieve this by modifying parts in the natural system (see other sections for more info).<br />
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[[Image:Calling_the_troops.png]]<br />
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Clinically, we envision to engineer Killer cells from cancer patients with our devices to make them powerful synthetic cancer killers. Upon transfusion of these engineered cells into the patient the cancer would be more efficiently eliminated. [http://www.ncbi.nlm.nih.gov/pubmed/20467460 review on adoptive immunotherapy]<br />
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{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/Future_ImplicationsTeam:UCSF/Project/Future Implications2010-10-27T19:30:45Z<p>Ryanliang: </p>
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==='''Future Implications'''===<br />
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[[Image:The_future.png]]<br />
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We expect that in the Future our '''''Synthetic Cancer Killers''''' would be equipped with '''GREATER PRECISION''' in the detection of cancer, '''STRONGER SIGNALING''' that would lead to a faster and stronger killing response, and '''BETTER ARSENAL''' to kill any kind of cancer they encounter (resistant or not)! <br />
<br />
We would achieve this by modifying parts in the natural system (see other sections for more info).<br />
<br />
<br />
[[Image:Calling_the_troops.png]]<br />
<br />
Clinically, we envision to engineer Killer cells from cancer patients with our devices to make them powerful synthetic cancer killers. Upon transfusion of these engineered cells into the patient the cancer would be more efficiently eliminated.[http://www.ncbi.nlm.nih.gov/pubmed/20467460 review on adoptive immunotherapy]<br />
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{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/File:Calling_the_troops.pngFile:Calling the troops.png2010-10-27T19:22:21Z<p>Ryanliang: </p>
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<div></div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/Future_ImplicationsTeam:UCSF/Project/Future Implications2010-10-27T19:20:18Z<p>Ryanliang: /* Future Implications */</p>
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==='''Future Implications'''===<br />
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[[Image:The_future.png]]<br />
<br />
We expect that in the Future our '''''Synthetic Cancer Killers''''' would be equipped with '''GREATER PRECISION''' in the detection of cancer, '''STRONGER SIGNALING''' that would lead to a faster and stronger killing response, and '''BETTER ARSENAL''' to kill any kind of cancer they encounter (resistant or not)! <br />
<br />
We would achieve this by modifying parts in the natural system (see other sections for more info).<br />
<br />
<br />
[[Image:Calling_the_troops.png]]<br />
<br />
Clinically, we envision to engineer Killer cells from cancer patients with our devices to make them powerful synthetic cancer killers. Upon transfusion of these engineered cells into the patient the cancer would be more efficiently eliminated.<br />
<br />
<br />
{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/Future_ImplicationsTeam:UCSF/Project/Future Implications2010-10-27T19:12:08Z<p>Ryanliang: /* Future Implications */</p>
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==='''Future Implications'''===<br />
<br />
<br />
[[Image:The_future.png]]<br />
<br />
We expect that in the Future our '''''Synthetic Cancer Killers''''' would be equipped with '''GREATER PRECISION''' in the detection of cancer, '''STRONGER SIGNALING''' that would lead to a faster and stronger killing response, and '''BETTER ARSENAL''' to kill any kind of cancer they encounter (resistant or not)! <br />
<br />
We would achieve this by modifying parts in the natural system (see other sections for more info).<br />
<br />
<br />
<br />
<br />
<br />
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{{Template:UCSF/WholeBlockEnd}}</div>Ryanlianghttp://2010.igem.org/Team:UCSF/Project/Future_ImplicationsTeam:UCSF/Project/Future Implications2010-10-27T19:06:36Z<p>Ryanliang: /* Future Implications */</p>
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==='''Future Implications'''===<br />
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===Future Implications===<br />
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===Graphic showing the main assays used to show T cell Activation===<br />
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<p><b>MANY THANKS to <a href="https://2010.igem.org/Team:Peking/Team/WYWang">Weiye Wang</a> from Peking University Team for drawing us such a beautiful banner</b></p><br />
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