Team:ETH Zurich/Overview/CircuitDesign

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Can you feel the smoke tonight?

Circuit Design

To realize the smoke detecting optosensor (see Project Idea), we implemented two distinct network designs: one SmoColi sensing acethaldehyde (see Figure 1) and one sensing xylene (see Figure 2). Both designs share the same signal transduction and processing units, and a similar design in the signal detection unit.

Signal Pre-Processing

The first step for both designs is to map an absolute acetaldehyde (xylene) concentration into a spatial signal. This first step is done in a channel by diffusion and degradation of acetaldehyde (xylene) a spatial gradient is produced.

Signal Detection

Figure 1: Molecular mechanism of SmoColi with acetaldehyde sensor

Figure 2: Molecular mechanism of SmoColi with xylene sensor.

For Design 1 (Design 2), the small signaling molecule acetaldehyde (xylene) is supplied in the extra-cellular medium. Acetaldehyde (xylene) can diffuse through cellular membranes and associates with its constitutively expressed receptor AlcR (XylR). Upon binding, the AlcR-acetaldehyde (XylR-xylene) complex can bind to its cognate operator, and inhibits (activates) the transcription of the genes under the control of the P_AlcR (P_U) promoter.

Since for the further signal processing it was necessary to have a POPS signal proportional to the signaling molecule concentration, and since the acetaldehyde sensor inhibits transcription, we decided to include an additional unit to invert the transduced signal downstream of acetaldehyde (this element is not necessary for the xylene sensor): we put TetR under the transcriptional control of AlcR, and since TetR acts as a transcriptional inhibitor for downstream proteins, the transduced acetaldehyde signal has a similar qualitative input/output relation as the transduced xylene sensor without inverter.

Signal Processing I: the Band-Pass Filter

Figure 3: 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 sensor molecule (violett), receptively. Non active interactions and non expressed Proteins are indicated light colors.

To realize the spatial green fluorescence band indicating the acetaldehyde (xylene) concentration, we implemented a signal processing unit, which transduces the incoming signal if and only if the signal is neither too strong nor too weak (band-pass filter [1]):

TetR represses (XylR enhances) the expression of the LacI_M1 repressor (codon-modified LacI) and the lambda repressor CI. Thus, high acetaldehyde (xylene) concentrations result in high cytoplasmic levels of CI and of LacI_M1. Since LacI_M1 represses the expression of the green fluorescent protein (GFP), no green fluorescence signal can be detected for high concentrations.

Low acetaldehyde (xylene) concentrations result in low LacI_M1 and CI concentrations. Although in this case, LacI_M1 does not repress GFP production, GFP is transcriptional repressed by the non-codon modified LacI, which is expressed at low CI concentrations. Only intermediate signals results in a moderate level of CI and LacI_M1. The repression of CI is significantly higher than the one of LacI_M1. While moderate levels of CI effectively shuts off LacI expression, the LacI_M1 concentration is to low to repress GFP

Signal Processing II: the Alarm Signal

To realize the alarm signal for toxic acetaldehyde (xylene) concentrations, we put the N-Acyl homoserine lactone (AHL) Synthase LuxI under the transcriptional control of CI, which is thus expressed only at low acetaldehyde (xylene) concentrations. LuxI syntesizes the small signaling molecule AHL which can quickly diffuse through the cell membrane in the extracellular space. Due to its relatively high diffusion constant, AHL diffuses through the whole tube and, thus, is present even in cells where no AHL is produced. Inside a cell, AHL can bind to its constitutively expressed receptor protein LuxR, and the thus activated LuxR represses the transcription of a red fluorescent protein (RFP). Thus, if the acetaldehyde concentration is below a threshold at least for a large enough minority of the cells in the channel, RFP is repressed in all cells. Only if the acetaldehyde concentration is above the threshold for all cells, no AHL is present in the channel and RFP is produced. Thus, the alarm signal RFP can be used to signal critical and potentially dangerous acetaldehyde (xylene) concentrations.

References

[1] [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html Subhayu Basu, Yoram Gerchman1, Cynthia H. Collins, Frances H. Arnold & Ron Weiss: A synthetic multicellular system for programmed pattern formation, Nature 2005, 434: 1130-11342]

@@ Line 22: / Line 22: @@
 [[File:ETH_states_of_bandpass.png|300px|right|thumb|'''Figure 3: 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 sensor molecule (violett), receptively. Non active interactions and non expressed Proteins are indicated light colors.]]
-To realize the spatial green fluorescence band indicating the acetaldehyde (xylene) concentration, we implemented a signal processing unit, which transduces the incoming signal if and only if the signal is neither too strong nor too weak (band-pass filter [[#Ref2|[2]]]):
+To realize the spatial green fluorescence band indicating the acetaldehyde (xylene) concentration, we implemented a signal processing unit, which transduces the incoming signal if and only if the signal is neither too strong nor too weak (band-pass filter [[#Ref1|[1]]]):
 TetR represses (XylR enhances) the expression of the LacI<sub>M1</sub> repressor (codon-modified LacI) and the lambda repressor CI. Thus, high acetaldehyde (xylene) concentrations result in high cytoplasmic levels of CI and of LacI<sub>M1</sub>. Since LacI<sub>M1</sub> represses the expression of the green fluorescent protein (GFP), no green fluorescence signal can be detected for high concentrations.
@@ Line 30: / Line 30: @@
 === Signal Processing II: the Alarm Signal ===
 To realize the alarm signal for toxic acetaldehyde (xylene) concentrations, we put the N-Acyl homoserine lactone (AHL) Synthase LuxI under the transcriptional control of CI, which is thus expressed only at low acetaldehyde (xylene) concentrations. LuxI syntesizes the small signaling molecule AHL which can quickly diffuse through the cell membrane in the extracellular space. Due to its relatively high diffusion constant, AHL diffuses through the whole tube and, thus, is present even in cells where no AHL is produced. Inside a cell, AHL can bind to its constitutively expressed receptor protein LuxR, and the thus activated LuxR represses the transcription of a red fluorescent protein (RFP). Thus, if the acetaldehyde concentration is below a threshold at least for a large enough minority of the cells in the channel, RFP is repressed in all cells. Only if the acetaldehyde concentration is above the threshold for all cells, no AHL is present in the channel and RFP is produced. Thus, the alarm signal RFP can be used to signal critical and potentially dangerous acetaldehyde (xylene) concentrations.
+|}
+{|class="roundContainer"
+|
+== References ==
+<span id="Ref1">[1] [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html Subhayu Basu, Yoram Gerchman1, Cynthia H. Collins, Frances H. Arnold & Ron Weiss: '''A synthetic multicellular system for programmed pattern formation''', Nature 2005, 434: 1130-11342]</span>
 |}