Roles of people involved in making the IGEM experience the funnest summer ever!!! .... well almost
Advisors - Advised us on project feasibility and best ideas worth pursuing.
Buddies - Postdocs and graduate students that took some time during the summer to teach us techniques and to help us trouble shoot.
Instructors - Postdocs and graduate students that taught us seminars during bootcamp.
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.
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.
International Student - Min Lin is from Peking University and he pretty much knows how to do almost everything, like a true ninja master.
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.
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.
Project design and Labs that contributed to our project
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. 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. 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.
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.
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.
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.
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.
See more on who did what in our project timeline page.