Team:Chiba/Project

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Introduction

Fig.1 Project desgin
"Team : Chiba - E.coli time manager"

　　We control the timing of gene expression by using multiple signaling devices.To this end,we utilize molecules associated with Quorum sensing, a phenomenon that allows bacteria to communicate with each other.Our project uses two classes of bacteria: senders and receivers. Senders produce signaling molecules, and Receivers are activated only after a particular concentration of this molecule is reached.Although different quorum sensing species have slightly different signaling molecules, these molecules are not completely specific to their hosts and cross-species reactivity is observed [http://www3.interscience.wiley.com/journal/119124142/abstract (M.K Winson et al.FEMS Microbiology Letters,1998)]. Communication using non-endogenous molecules is less sensitive, and requires a higher signal concentration to take effect.This results in slower activation of receivers.


Motivation

Fig.2 Team logo










Project Design

Our project uses two classes of bacteria: senders and receivers.Senders produce　signaling molecules, and　receivers are activated only after a particular concentration of this　molecule is reached.The communication using non-endogenous molecules is less sensitive,and it requires higher signal concentration to take effect.This results in slower activation of receivers.

About Quorum Sensing

Fig.3 Constructed circuit of Quorum sensing.

　　Quorum sensing is a cell-to-cell signaling action of bacteria. They detect the cell density of the same species and coordinate the expression behavior of their cells. Species of Gram-Negative signaling transfer molecules (so-called autoinducer) is a series of acyl homoserine lactone (AHL). The signals are synthesized from S-adenosylmethionine(SAM) by a synthase protein and once they have reached a threshold　concentration,they bound to a transcriptional regulatory　protein to induce expression of target genes.

More about Quorum Sensing

  • [http://parts.mit.edu/registry/index.php/Featured_Parts:Cell-Cell-Signaling Cell-Cell-Signaling]
  • [http://www.che.caltech.edu/groups/fha/quorum.html About Quorum sensing]

Controlling the time of a cell-to-cell signaling action

Communication using non-endogenous molecules is less sensitive, and requires a higher signal concentration to take effect.This results in slower activation of receivers.

  • Sender Phase
    • Quorum-Sensing Cross-talk
Fig.4 AHL variety

　　 AHLs produced by different bacteria differ only in the length of the acyl-chain moiety and substitution at position C-3.([http://partsregistry.org/Part:BBa_F2620:Specificity BBa_F2620:Specificity])


  • Receiver Phase
    • LuxR/Plux mutants show
  1. a greater response to 3OC6HSL ([http://authors.library.caltech.edu/5553/ C. H. Collins.et al.Mol.Microbiol.2005.])
  2. a increase in sensitivity to 3OC12HSL ([http://mic.sgmjournals.org/cgi/content/abstract/151/11/3589 B. Koch.et al.Microbiology (2005)]).
    • AHL reporter with aiiA
Express LuxR and aiiA constantly. AiiA degrades AHL as signaling molecule. Express GFP when the AHL concentration exceed the capacity of aiiA.
This enables the delay of the activation time of receiver.

About Quorum Sensing

　　Quorum sensing is a cell-to-cell signaling action of bacteria. They detect the cell density of the same species and coordinate the expression behavior of their cells. Species of Gram-Negative signaling transfer molecules (so-called autoinducer) is a series of acyl homoserine lactone (AHL). The signals are synthesized from S-adenosylmethionine(SAM) by a synthase protein and once they have reached a threshold　concentration,they bound to a transcriptional regulatory　protein to induce expression of target genes.

More about Quorum Sensing

  • [http://parts.mit.edu/registry/index.php/Featured_Parts:Cell-Cell-Signaling Cell-Cell-Signaling]
  • [http://www.che.caltech.edu/groups/fha/quorum.html About Quorum sensing]

　　

What our system requires

  • Communication module
  • Quick output

How Our System Works

Experiments

Quorum-Sensing Cross-talk

Fig.5 Senders crosstalk
  • Senders
    • [http://partsregistry.org/Part:BBa_K084007 plac+rbs+LasI]

Tet-lasi chiba.gif

    • [http://partsregistry.org/Part:BBa_K084008 plac+rbs+RhlI]

Tet-rhli chiba.gif

    • [http://partsregistry.org/Part:BBa_K084012 plac+rbs+LuxI]

Tet-luxi chiba.gif

  • Receiver
    • [http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (Express GFP in response to AHL)]

Reporter 9002 chiba.gif


LuxR/Plux mutants

AiiA Receiver

Method

To characterize quorum sensing crosstalk, constitutive AHL senders were mixed with constitutive receivers and mesure fluorescence intensity.

  1. Transform Senders into E.coli strains(JW1908/XL10GOLD) and Receiver into E.coli strain(JW1908).
  2. Inoculated them independently in liquid media. Incubated at 37℃ 12h.
  3. Washed Senders and receiver.
  4. Mix them.
  5. Incubated at 37℃ or 30℃.
  6. Measured intensity of green fluorescence at regular time intervals.

Result

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