Circuit design
 Circuit operations for SmoColi exposed to medium, low and zero xylene concentration resulting in a GFP band. Triangles indicating the gradient of AHL (blue) and xylene(violett), receptively. Non active interactions and non expressed Proteins are indicated light colors. SmoColi is a bacterio quantifier which can be activated with different signal, we implemented two of them acetaldehyde and xylene.
The concentration gradient which is needed for SmoColi is either naturally or achieved by synthetic cellular degradation. For example in case of Xylene we included the upper Tol pathway of Pseudomonas putida in SmoColi [1]. In contrast acetaldeydhde is naturally degraded.
In our system we can use different small molceules as an activating input for the bandpass-filter [2]. The repressor which is activated by the small molecule is constantly expressed and enhances the expression of the LacIM1 repressor (codon-modified LacI) and the lambda repressor CI, which are under control of the Xyl-Promotor, respectively. High xylene concentration results in high cytoplasmic levels of CI and of LacIM1 and repression of the green fluorescent protein (GFP). Cells that are far from the input have low xylene concentrations, because of xylene degradation. Accordingly, LacIM1 and CI are only expressed at basal level. Without repression of the lamda promoter wild-type LacI is produced and represses the production of GFP. At the same point the N-Acyl homoserine lactone (AHL) Synthase LuxI is produced, AHL binds to LuxR which is constantly expressed and represses the red fluorescent protein (RFP). AHL is a quorum-sensing molecule with a high diffusion rate, it diffuses through the whole tube and represses RFP production in the whole tube, even in cells where no AHL is produced.
 Circuit operations for SmoColi exposed to very high xylene concentration resulting in an alarmsystem which turns the whole channel red. The triangle is indicating the xylene concentration. Non active interactions and non expressed Proteins are indicated light colors. If the xylene concentration is too high and it can not be degraded within the tube, no AHL is produced. Without AHL LuxR does not repress RFP and the whole tube turns red. To obtain better dynamics we tagged GFP and TetR with LVA tags.
Finally we can also use a negative input for our system, in this case we have to introduce an additional inverter to invert the negative input signal into a positive one. Therefore the tetracycline repressor protein (TetR) was used in case of AlcR. If acetalydeyhde is present AlcR binds to the promotor of TetR and inhibit its represssion, resulting in no TetR.
|
"
