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Team:UCSF/Notebook
From 2009.igem.org
In the beginning of the summer our team was divided into two groups - one group focused on Engineering NAVIGATION and the other group focused on Engineering SPEED. The group that engineered NAVIGATION worked with HL60 cells - HL60 Team. The group that engineered SPEED worked with Dictyostelium - Dicty Team.
HL60 Team
GOAL: Our goals for this project are to screen 30 various different GPCR's (G couple protein receptors) to determine which receptors mediate chemotaxis using the high through put transwell assay. After the first phase screening using 5-6 day differentiated HL-60 cell transfected with known receptors is completed, we move on to the secondary phase of screening with viable chemotatic receptors. During the second phase of screening we fused "Actin Modulators", "PIP3 Modulators", and "GEF Activators" in hopes of an accelerated chemotatic response.
ENGINEERING NAVIGATION:
- Can we send our cells to new targets? Inserting new Sensors to HL60 (GPCR Screening)
- Can we make our new engineered cells more or less sensitive to the new signals? Tuning receptor sensitivity by attaching recycling modules to the new GPCRs
Milestones:
Step 1. Clone receptors, N-terminal tags, C-terminal domains. Generate fusions. (everyone)
Step 2. Transwell screen to identify constructs that affect migration (Ryan, Eric)
Step 3. Time lapse microscopy to determine directional migration (Hansi, Jackie, Katja)
Step 4. Establish method for attaching beads to cells (Katja)
Team members
Eric Wong's notebook: I worked on cloning, PCR, Gel purification and extraction, and was apart of the GPCR screening team.
Jackie Tam's notebook: I was responsible for cloning, microscopy, and video analysis.
Ryan Liang's notebook: I cloned parts to be used in the GPCR screens, transfected HL-60 cells with various different GPCRs, ran transwell assays, and analyzed/compared transwell assay results.
Hansi Liu's notebook: I established a protocol for time-lapse microscopy of HL-60 cells crawling in gradients, and collected that data.
MAKING CELLS CARRY PAYLOAD:
- Can we make our cells carry stuff? - proof-of-concept: attach beads to cells
Team members
Katja Kolar's notebook: I did microscopy with wt and hM4D-transfected HL-60 cells, experiments for the "Payload" part of the project, and cloned HL-60 team constructs.
Dicty Team
GOAL: Our goal was to build brakes and accelerators for motile cell. We challenged ourselves to engineer feedback loops that would affect the localization of PIP3 within the cell.
ENGINEERING SPEED:
- Can we send our cells move faster or slower? Creating brakes and accelerators by modulating PIP3 polarity - building synthetic feedback loops by fusing catalytic and localization domains.
Milestones:
Step 1. PCR parts: catalytic and localization domains Ryan
Step 2. Create combinations of localization domains and catalytic domains (feedback elements) everyone
Step 3. Generate Dictyostelium strains expressing feedback elements everyone
Step 4. Measure motility parameters (speed, directionality) everyone
Step 5. Analyze data. Create histograms and compare if our new feedback elements had any effect on Dicty. Do they cause them to move faster or slower? everyoneTeam members
Allen Cai's notebook: I worked mostly on the cloning part of the project. I ligated a lot of the localization and catalytic domains together. I also took care of some dicty strains and made movies of them.
Alex Smith's notebook: I transformed newly engineered constructs into wild type Dictyostelium discoideum, took movies of cells, analyzed data, and briefly experimented with rapamycin mediated recruitment.
Edna Miao's notebook: I transformed new constructs into Dicty, analyzed data, and worked with various different Dicty strains.
Ethan Chan's notebook: I constructed Dicty cells with new feedback elements, analyzed data, and worked with various different Dicty strains.
Ryan Quan's notebook: I worked on team dicty and taught the students some basic cloning techniques but mainly learned many new things along with them.
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