From 2009.igem.org

Latest revision as of 22:17, 21 October 2009

Engineering SPEED: Creating Synthetic Brakes and Accelerators

Motivation: Why is this useful?

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 PhosphatidylInositol(4,5)bisphosphate (PIP2) to PhosphatidylInositol(3,4,5)trisphosphate (PIP3) (two signaling lipids in the plasma membrane) at the front of our cells. In a positive feedback loop PIP3 triggers the formation of more PIP3 at the front while similarly PIP2 leads to more PIP2 production at the sides and rear of the cell. This system sets the axis of polarity of the cell. The PIP3 patch at the front aligns the actin network and accordingly functions as a ‘turbo boost’ pushing the cell forward.

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.

Approach

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.

Similarly a PIP3 binding domain fused to a PIP2 producing catalytic domain could put PIP2 in place of PIP3 and thereby act as a negative feedback loop.

Synthetic protein based feedback loops: On the left: a positive feedback loop made of a PIP3 binding domain fused to a PIP3 generating enzyme. On the right: a negative feedback loop made of a PIP3 binding domain fused to a PIP2 generating enzyme.

Results

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!

Here is an overview of all feedback loops we screened and the effect they had on the speed of cells. We used automated cell tracking on more than 196 hours worth of movies (note: one movie is 10 minutes!) and identified strains that moved faster or slower at a very stringent statistical cutoff (p<0.0001).

This way we were able to identify 7 brakes and 1 accelerator!

Check out the movie of one of our strong brakes BBa_K209066: PTEN-RasC dominant active (da) compared to wildtype.

PTEN fused to RasC da (PIP2 binding - PIP3 generating); speed: 3.5 um/min Cells were plated in buffer and basic motile behavior was recorded for 10 minutes taking 1 picture every 15 seconds.

wildtype; speed: 5.9 um/min Cells were plated in buffer and basic motile behavior was recorded for 10 minutes taking 1 picture every 15 seconds.

This is indeed an effect of fusing the particular localization to the catalytic domain as neither of them alone has such a strong effect. Access further data and details here.

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:

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!

NEXT - PAYLOAD

Methods

Engineering Speed:

• Cloning: Aar1 Shuffle - besides BBFRFC28, here is an actual step by step scheme we used for some of our combinatorial assembly of parts
• Dicty: Transformation Protocol - here is how we get our constructs into Dicty
• Dicty: Motility Assay Protocol - here is how we prepare and film Dicty cells

Selected Reading

G protein signaling events are activated at the leading edge of chemotactic cells. Parent CA, Blacklock BJ, Froehlich WM, Murphy DB, Devreotes PN. Cell. 1998 Oct 2;95(1):81-91.

PI3-kinase signaling contributes to orientation in shallow gradients and enhances speed in steep chemoattractant gradients. Bosgraaf L, Keizer-Gunnink I, Van Haastert PJ. J Cell Sci. 2008 Nov 1;121(Pt 21):3589-97.

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