Network details

Our system is divided in two modules:

signaling module
amplification module

Signaling module

The signaling module allows our bacterial strain to integrate the input signal = the pathogene presence.

This is also one of our module of modeling.

Staphylococcus aureus secretes an enzyme, exfoliative toxin B which cut a specific amino-acids sequence (Desmoglein 1). This specific sequence can be used as a linker between a membrane protein and a dipeptide.
Once S. aureus is present, the linker is cut by the protease and the dipeptide is released.

The dipeptide binds to his receptor which is an engineered [1]receptor:

the extracellular part is the extracellular part of Tap, a dipeptide receptor involved in the chemotaxism
the intracellular part is the intracellular part of EnvZ, a kinase involved in the osmoregulation

Once the dipeptide is bound, the EnvZ part allows the phosphorylation of OmpR, a transcriptional activator.

Amplification module

The amplification module allows our bacterial strain to amplify the input signal and to produce an output signal = fluorescence.

This is also one of our module of modeling.

Internal amplification

Once OmpR is phosphorylated, it allows the production of adenyl cyclase by activating the OmpC promoter.
Adenyl cyclase is an enzyme which catalyse the conversion of ATP (Adenosine Tri-Phosphate) to cAMP (cyclic Adenosine Mono-Phosphate).

cAMP binds to CRP (C-reactive protein) and then this complex allows the production of AraC by activating the pMalT promoter.
In the presence of arabinose, AraC, with cAMP-CRP, activates the pAraBAD promoter, forming thus an "AND" gate, which allow the production of:

adenyl cyclase which reproduce cAMP, forming thus an amplification loop
GFP (Green Fluorescent Protein) = our output signal

External amplification

When one bacterium detecte S. aureus, it produces a lot of GFP and cAMP. cAMP can diffuse through the membrane and activates the amplification loop in all the neighbourings bacteria which can thus produce a lot of GFP and cAMP.
The result is an entire population which produce GFP whereas only one bacterium has detected the pathogen in the first place:

References

[1] Masayuki Amagi, Takayuki Yamaguchi, Yasushi Hanakawa, Koji Nishifuji, Motoyuki Sugai, John R. Stanley. Staphylococcal Exfoliative Toxin B Specifically Cleaves Desmoglein 1. (2002). The Journal of Investigative Dermatology. Vol. 118, No. 5.

[2]Siromi Weerasuriya, Brian M. Schneider, Michael D. Manson. (1998). Chimeric Chemoreceptors in Escherichia coli: Signaling properties of Tar-Tap and Tap-Tar Hybrids. Journal of Bacteriology. Vol. 180, No. 4, p. 914-920.

[3]J W Baumgartner, C Kim, R E Brissette, M Inouye, C Park, G L Hazelbauer. (1994). Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensors EnvZ. Journal of Bacteriology. Vol. 176, No. 4.

[4]Sheng Jian Cai, Masayori Inouye. (2002). EnvZ-OmpR Interaction and Osmoregulation in Escherichia coli. The Journal of Biological Chemistry. Vol. 277, No. 27, p.24155-24161.

[5]Sumio Maeda, Katsuhiko Takayanagi, Yoshifumi Nishimura, Takemi Maruyama, Kou Sato, and Takeshi Mizuno. (1991). Activation of the Osmoregulated ompC Gene by the OmpR Protein in Escherichia coli: A Study Involving Synthetic OmpR-Binding Sequences. Journal of Biochemistry. 110, 324-327.

Team:Grenoble/Biology/Network

From 2012.igem.org