Team:UCSF/Project

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Latest revision as of 01:00, 22 October 2009

Our team was inspired by the use of robots which perform tasks that would otherwise be difficult to accomplish. For example, the Mars rovers allow us to make observations and conduct physical experiments in harsh, remote locations otherwise inaccessible to humans. In many ways, the human body represents unexplored territory on a different scale. For example, we may understand many aspects of human disease. However, certain markers of disease are extremely difficult to detect (e.g., primary tumors) and treatment of disease can be hampered by the impracticality of performing invasive surgeries.

The "holy grail" of nanomedicine would be to develop microscopic robots that could travel anywhere in the body and perform complex, user-defined tasks. Such devices would have several key advantages over traditional, small-molecule therapies:

They could home to specific locations in the body (minimize off-target effects)
They could make decisions based on their external environment
They could perform more complicated functions

While the idea of microscopic, therapeutic robots may seem far-fetched, there are examples of such machines in nature. For example, neutrophils (a type of white blood cell) are capable of:

Detecting and homing to a wide range of chemical signals, at times localizing to very specific sites of inflammation
Triggering a variety of different pathways (extravasation, phagocytosis, apoptosis) in response to external signals
Navigating through different types of barriers (endothelial tissue, blood-brain barrier, etc)

Taking cues from nature, we were interested in harnessing or hijacking the function of complicated, natural cellular robots such as neutrophils to perform therapeutically useful tasks. In other words, we wanted to use neutrophils (or similarly motile cells) as a chassis for engineering. Minimally, we wanted to control how and when these cells move. Over the course of the summer, were were able to:

Control the NAVIGATION system of our cells (alter what the cells pursue, how strong a signal they require)
Control the SPEED of our cells (engineer accelerators and brakes)
Deliver a PAYLOAD with our cells

Our efforts toward these goals are described in the remainder of this site. Throughout our project, we worked with two model organisms: HL-60 (neutrophil-like) cells and Dictyostelium discoideum, a soil-dwelling amoeba that feeds on bacteria. Both cell types are common models for the study of chemotaxis (directed migration toward chemical signals), and each has its distinct advantages with respect to studying specific questions.

PROJECT SUMMARY

Part 1 - Engineering NAVIGATION

Part 2 - Engineering SPEED

Part 3 - Carrying a PAYLOAD

Part 4 - Future Application

Home	The Team	The Project	Parts Submitted to the Registry	Our summer experience	Notebook	Human Practices

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+Our team was inspired by the use of robots which perform tasks that would otherwise be difficult to accomplish.  For example, the Mars rovers allow us to make observations and conduct physical experiments in harsh, remote locations otherwise inaccessible to humans.  In many ways, the human body represents unexplored territory on a different scale.  For example, we may understand many aspects of human disease.  However, certain markers of disease are extremely difficult to detect (e.g., primary tumors) and treatment of disease can be hampered by the impracticality of performing invasive surgeries.
-<!--- The Mission, Experiments --->
-{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
+The "holy grail" of nanomedicine would be to develop microscopic robots that could travel anywhere in the body and perform complex, user-defined tasks.  Such devices would have several key advantages over traditional, small-molecule therapies:
-!align="center"|[[Team:UCSF|Home]]
-!align="center"|[[Team:UCSF/Team|The Team]]
-!align="center"|[[Team:UCSF/Project|The Project]]
-!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]
-!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]
-!align="center"|[[Team:UCSF/Notebook|Notebook]]
-!align="center"|[[Team:UCSF/Human Practices|Human Practices]]
-|}
-(''Or you can choose different headings.  But you must have a team page, a project page, and a notebook page.'')
+* They could home to specific locations in the body (minimize off-target effects)
+* They could make decisions based on their external environment
+* They could perform more complicated functions
-== '''Overall project''' ==
+While the idea of microscopic, therapeutic robots may seem far-fetched, there are examples of such machines in nature.  For example, neutrophils (a type of white blood cell) are capable of:
-Your abstract
+* Detecting and homing to a wide range of chemical signals, at times localizing to very specific sites of inflammation
+* Triggering a variety of different pathways (extravasation, phagocytosis, apoptosis) in response to external signals
+* Navigating through different types of barriers (endothelial tissue, blood-brain barrier, etc)
-== '''Part 1: Navigation: rewiring the cell to move toward new chemical signals'''==
-=== Motivation: ''why is this useful?'' ===
+Taking cues from nature, we were interested in harnessing or hijacking the function of complicated, natural cellular robots such as neutrophils to perform therapeutically useful tasks.  In other words, we wanted to use neutrophils (or similarly motile cells) as a chassis for engineering.  Minimally, we wanted to control how and when these cells move.  Over the course of the summer, were were able to:
-We envision a cellular robot that could travel to practically any site in the human body.  This would provide a flexible platform that could be used for a variety of therapeutic tasks.  The first step toward achieving this goal is to broaden the range of possible chemotactic targets for our cells.  Ideally, we could connect virtually any input to chemotaxis in a generalized way.
-=== Approach: ===
+* Control the '''NAVIGATION''' system of our cells (alter what the cells pursue, how strong a signal they require)
-Neutrophils sense most of their chemotactic signals through [http://en.wikipedia.org/wiki/G_protein_coupled_receptor G protein-coupled receptors] (GPCRs).  The spectrum of chemical signals to which these cells respond is therefore determined, at least in part, by the set of GPCRs they express.  Can this spectrum be broadened arbitrarily by the introduction of new GPCRs?  We tested this idea by expressing 23 exogenous GPCRs in HL-60 (neutrophil-like) cells.
+* Control the '''SPEED''' of our cells (engineer accelerators and brakes)
+* Deliver a '''PAYLOAD''' with our cells
-== '''Part 2: Speed: engineering accelerators and brakes: A cellular cruise control by modulating cell polarity with feedback loops''' ==
-=== Motivation: ''why is this useful?'' ===
+Our efforts toward these goals are described in the remainder of this site.  Throughout our project, we worked with two model organisms: HL-60 (neutrophil-like) cells and ''Dictyostelium discoideum'', a soil-dwelling amoeba that feeds on bacteria.  Both cell types are common models for the study of chemotaxis (directed migration toward chemical signals), and each has its distinct advantages with respect to studying specific questions.
-Just like we have control over speed in a car – we can brake or accelerate – it would be useful to engineer such behavior into our cellular nanorobots.
-Just think about it: We could '''speed cells up''' so that they reach their targets faster and '''stop them''' once they have arrived or do not behave properly.
-=== Background: ===
-For these experiments we chose ''Dictyostelium discoideum'' cells to test our prototypical brakes and accelerators quickly. We expect that our brakes and accelerators can be used in a plug and play fashion because Dicty’s way of movement is very similar to a neutrophil’s:
-When a receptor binds chemoattractant, it induces the conversion of PIP2 to PIP3 (two signaling lipids in the plasma membrane) at the front of our cells. In a '''positive feedback loop''' PIP3 triggers the formation of more PIP3 at the front while similarly PIP2 leads to more PIP2 production at the sides and rear of the cell. This system sets the axis of polarity of the cell. The PIP3 patch at the front functions as a ‘turbo boost’ pushing the cell forward.
+'''PROJECT SUMMARY'''
+Part 1 - [[Team:UCSF/Navigation|Engineering NAVIGATION]]
-[[Image:BACKGROUND1.jpg|500px|thumb|center|'''Polarized distribution of PIP3 and PIP2:''' A patch of PIP3 is localized at the front of a cell while PIP2 is at the back. Feedback loops are used to establish and maintain this polarized distribution. ]]
+Part 2 - [[Team:UCSF/SPEED|Engineering SPEED]]
-=== Approach: ===
+Part 3 - [[Team:UCSF/PAYLOAD|Carrying a PAYLOAD]]
-Inspired by nature we tried to build accelerators and brakes by introducing our own synthetic protein based feedback loops. We designed feedback elements by fusing localization and catalytic domains involved in PIP3 production and degradation to modulate localization and level of PIP3 and PIP2 in the cell.
-Here is an example of a positive feedback loop: a PIP3 binding localization domain fused to a PIP3 producing catalytic domain could produce more PIP3 where there is already PIP3- at the front. This might strengthen polarity and accelerate a cell.
+Part 4 - [[Team:UCSF/Future Applications|Future Application]]
-[[Image:positive_feedback_loop2.jpg|500px|thumb|center|'''Synthetic protein based feedback loops:''' One example: a positive feedback loop comprised of a PIP3 binding domain fused to a PIP3 generating enzyme.]]
-=== Results:  ===
+{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"
-Over the summer we assembled more than 100 fusions of localization and catalytic domains and screened whether they work. How? We measured the effect our constructs have on motility of ''Dictyostelium'' cells: stronger polarity should make cells faster while weaker polarity ought to slow them down!
+!align="center"|[[Team:UCSF|Home]]
+!align="center"|[[Team:UCSF/Team|The Team]]
-[[Here]] is an overview of all feedback loops we screened and the effect they had on the speed of cells. If you are interested what all the elements are check out our biobricks.
+!align="center"|[[Team:UCSF/Project|The Project]]
-We used automated cell tracking on more than 196 hours worth of movies (note: one movie is 10 minutes!) and identified strains that moved faster or slower at a very stringent statistical cutoff (p<0.0001).
+!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]
+!align="center"|[[Team:UCSF/Our summer experience|Our summer experience]]
+!align="center"|[[Team:UCSF/Notebook|Notebook]]
-This way we were able to identify '''7 brakes and 1 accelerator'''!
+!align="center"|[[Team:UCSF/Human Practices|Human Practices]]
+|}
-Check out the movie of '''one of our strong brakes (PTEN-RasC dominant active (da)) compared to wildtype'''. We hypothesize that this construct acts as a negative feedback loop on PIP2- (generating PIP3 where PIP2 should be) thereby confusing the cell with multiple fronts:
-[[Image:HO230.jpg|450px|thumb|right|PTEN (PIP2 binding) fused to RasC da (PIP3 generating); speed: 3.5 um/min]] [[Image:wt.jpg|450px|thumb|left|wildtype; speed: 5.9 um/min]]
-This is indeed an effect of fusing the particular localization to the catalytic domain as neither of them alone has such a strong effect ([[see details]]).
-=== Summary and outlook: ===
-We have screened more than 100 synthetic feedback elements for their ability to accelerate or slow down speed of cell motility. We have isolated a hand full of functional elements. Now we need to confirm the mechanism of action of these elements. In the future we would like to make them inducible by a signal from outside – like a stoplight!
-===Methods===
-*[[Media:Aar1_shuffle.pdf|Cloning: Aar1 Shuffle]] - Connecting all our fragments together.
-*[[Media:DICTY TRANSFORMATION.pdf|Dicty: transformation protocol]] - Used to transform into dicty
-*[[Media:DICTY-analyzing_motility.pdf|Dicty: Analyzing Motility protocol]] - Used to analyze the basic moitily of all cells