Tag! / BioWire (Signal Transfer)

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Contents

Tag Project

2 States:

Not It State

  1. Bioluminesce a color different from the "it" cells
  2. Emit attractor for "it" cells
  3. Chemotax away from the repellent emitted by the "it" cells
  4. Emit repellent for all other "not-it" cells (optional)

It State

  1. Bioluminesce a color different from the "not it" cells
  2. Emit repellent for the "not it" cells
  3. Chemotax toward the attractant emitted by the "not it" cells

Toggle between the states

Steps/Checkpoints:

Begin with the "not it" states.

  1. Get cells to bioluminesce.
  2. Get cells to chemotax away from the repellent.
  3. Get cells to emit an attractant.
  4. Get cells to toggle to the "it" state.
    1. Change bioluminescence.
    2. Stop emitting attractant.
    3. Start emitting repellent.
    4. Switch chemotaxis toward attractant.
  5. Get cells to toggle back.

Biowire

Cell to cell "action potential" with chemical signaling.

  1. Wave of luminescence.
  2. Refractory period.

Signal transduction pathway is the same as for the Tag system. Incoherent feedforward loop to generate pulse signal.

Open Questions

How easy is it to modify the chemotaxis circuit to make bacteria chemotax to/from a molecule of choice? Has this been done before?

What problems did the MIT team run into last year?

What are the time scales on all of these parts? How long will it take a cell to switch state? Is there any way of speeding things up?

Speed

Crawl Speed 20 µm/speed
Speed of Expression 20min-6hour(Positive Feedback)
Calcium Event 200 µSec - 500milliSec
Size of cover slip 22 mm^2
Size of e. coli 1 µm
twice-life of e. coli 20 mins

Journal References

  1. Chemotaxis
    1. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=1093163&query_hl=3
    2. www.fas.harvard.edu/~jgao/igem
    3. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=2556636
    4. Here's a pretty nice overview of chemotaxis from a systems biology perspective (courtesy of MCB195). Chemotaxis book chapter and accompanying figures.
  2. Cell-Cell Signaling
    1. Biobricks catalog pages on cell-cell signaling and protein generators and the Biobricks overview of receivers. It seems like the only receivers we have are for 3OC6HSL, although there are generators for AI-1, 3OC6HSL, N-butyryl-HSL, and 3OC14HSL. We need to have two cell-cell signaling pathways. They say that "homoserine lactone [HSL] molecules with varying length acyl side chains can be used to ... permit different signaling channels to be used in the one system" but it's unclear if this has been done before.
    2. Design of artificial cell-cell communication using gene and metabolic networks. Bulter T, Lee SG, Wong WW, Fung E, Connor MR, Liao JC. These people used acetate as the intercellular signalling molecule and the nitrogen starvation pathway to regulate the response. A nice figure of their design. They chose acetate because it is constantly produced and secreted by all cells (they are interested in quorom sensing); we'd need to be able to regulate acetate production to make it a useful signal in our application.
    3. Computational design of receptor and sensor proteins with novel functions. Looger LL, Dwyer MA, Smith JJ, Hellinga HW. They design and test receivers of TNT, serotonin, and lactate. The response curves don't look to be quite as ultrasensitive as we might need them (see figure 3). Like Yin said, it is probably worth looking and seeing what other receivers the Hellinga group has constrcuted.
  3. Calcium Signalling
    1. Calcium Signaling in BacteriaDC Dominguez. Turn out that calcium signalling is involved in chemotaxis, and that bacteria that are being repelled show high calcium transients while bateria that are attracted show a decrease in intracellular calcium. So my original suggestion to use a calcium channel as part of the "tag" event might mess up chemotaxis... BUT it might be interesting to include a calcium-sensitive protein such as the Cameleon (see 2) in our design anyway because then we could visualize the switch immediately. We might have to think about putting in a more permanent signal though (ie turn on synthesis of GFP etc), to take place in parallel, if the time-scale of the transient is too fast.
    2. Protein-based Calcium Dyes Review. Several other protein-based (ie genetically encodable) calcium dyes are also described here. I know that aequorin has been used in bacteria, but not sure about the other ones. I thought the Cameleon was kind of cool - its supposed to fluoresce cyan and yellow in low calcium and yellow only in high calcium.--Paula 00:58, 6 Jun 2005 (EDT)
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