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Biology - TNT-sensing Pathway
Personal note

One of the reasons for studying biology for me has always been that scientific knowledge is power. The exhilarating and empowering IGEM experience has finally helped me understand that I really am one of the key holders to progress.

It has been said many times that science is not truly science unless it allows us to prove its concepts by creating things. Certainly for biology, the culmination and the peak of our knowledge will result in this ability, more precisely in the ability to apply engineering concepts to biological knowledge, to be able to demonstrate the facts through re-recreating them. I believe that to say that we are successfully doing this already would be an overstatement; nonetheless, I do believe that the steady path to our “masterhood” of biology has begun, and it is the taking part in this exciting, intriguing and perhaps mysterious new beginning that is the most rewarding part of being involved in IGEM.

The moment a landmine encounters soils, it leaks TNT (Jenkins et al., 2001). TNT will be detected by a computationally designed synthetic receptor, TNT.R1 (Looger et al., 2003). Since TNT.R1 is derived from ribose-binding protein and wild-type E. coli swims towards ribose, we hypothesize that E. coli cells expressing TNT.R1 will chemotaxi towards TNT. To verify this we modeled a chemotaxis experiment. To see the experimental results in detail please click here.
Upon interaction with TNT, the receptor (BBa_K216002) will undergo a conformational change, allowing it to interact with Trg-EnvZ (a.k.a Trz) fusion protein (BBa_K216004). Previously, Trg-EnvZ was entered into the registry, unfortunately, the sequencing results were inconsistent. Furthermore, we failed to revive stabs received from the registry. As such, we contacted Dr Hazelbauer and colleges who kindly provided us with the plasmid carrying the fusion protein (Baumgartner et al., 1994).
Subsequently, Trg-EnvZ also undergoes conformational change and autophosphorylates. Eventually, it phosphorylates the secondary messenger ompR, and phosphorylated omp-R activates the ompC promoter (BBa_R0082).
Activation of PompC will result in upregulation of enhanced yellow fluorescent protein (eyfp) and onr. Onr encodes PETN reductase, a nitroreductase from Enterobactor cloacae (Accession #: U68759, BBa_K216006). PETN reductase will reduce TNT to nitrites (French et al., 1998) that would feed into the nitrite/nitrate detection system.

Figure 1 TNT binds to TNT.R1 in the periplasm. The TNT-TNT.R1 complex induces a conformational change in the Trg-EnvZ (Trz) fusion protein. Trg-EnvZ autophosphorylates and subsequently phosphorylates ompR. Phosphorylated ompR activates transcription.
You will also notice that EYFP is not excited by visible light, therefore no signal will be detected in the presence of TNT only. However, in the presence of nitrite, a blue-green light that will subsequently excite EYFP will be produced (Figure 2).

TNT.R1 and the Trg-EnvZ are preiplasmic and transmembrane proteins respectively. Therefore, high levels of expression can potentially disrupt the plasma membrane and result in cell death. Hence, we decided to express both proteins under the control of a weak consitutive promoter (BBa_J23105). This is the same promoter used to control expression of the genes coding for aldehyde-producing enzymes in the nitrite/nitrate sensing system.
The secondary messenger ompR and promoter PompC occur naturally in E. coli. Their function is to activate gene transcription in response to osmotic stress (Maeda et al., 1990). Additionally, it has been found that the PompC activity varies in response to procaine concentration. This finding led us to characterize PompC activity with varying procaine concentration. The characterization was done using Jason Kelly’s promoter characterization kit. Click here for the results.
Edinburgh University iGEM Team 2009