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.

The idea of this module is du to the iGEM London Imperial College 2010 Team work on Parasight [1].

Staphylococcus aureus secretes an enzyme, exfoliative toxin B [2] 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 [3] [4] receptor:

the extracellular part is the extracellular part of Tap [5], a dipeptide receptor involved in the chemotaxism
the intracellular part is the intracellular part of EnvZ [6], a histidine kinase involved in the osmoregulation

Once the dipeptide is bound to the Tap part [7], the EnvZ part allows the phosphorylation of OmpR [8] [9], a transcriptional activator which is constitutively produced.

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 [10].
Adenyl cyclase [11] 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] https://2010.igem.org/Team:Imperial_College_London/Modules/Detection

[2] 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.

[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] 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.

[5] Polypeptide: Tap

[6] Protein: EnvZ sensory histidine kinase

[7] Michael D. Manson, Volker Blank, Gabriele Brade. (1986). Peptide chemotaxis in E. coli involves the Tap signal transducer and the dipeptide permease. Nature. Vol. 321.

[8] 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.

[9] ompR expression

[10] 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.

[11] Enzyme: adenyl cyclase

[12]

Team:Grenoble/Biology/Network

From 2012.igem.org