Using a network of E. coli to form a smart material
for low concentration detection!

'smart, self assembling material'

Neetd lots of editing

Beta System

According to the model "Why Should it Fail" of the Alpha System, we can see that it has some problems. We decided to test new methods to reduce the noise in our system. One idea was a new design – the Beta System, inspired by the noise reduction mechanism described by Goni-Moreno and Amos. (Goni-Moreno & Amos, 2012). We used a double repression Toggle Switch similar to that described by Gardner et al. (Gardner, Cantor, & Collins, 2000)), to filter the inputs of our system. This makes the cell-to-cell communication more accurate, while affording them an internal memory capacity.
This system consists of three main circuits:
(1) The Computation Circuit (which acts as the CPU and determines whether or not to activate the Toggle Switch).
(2) A Toggle Switch (which acts as the internal memory bank, by keeping the system active or inactive over long periods of time).
(3) A Signal Circuit (which acts as the "antenna" used for broadcasting and receiving cell to cell signals).

Computation Circuit

The Computation Circuit has been programmed to function as an OR gate:
Its first part consists of the gene LacI, which activates the Toggle Switch, and whose synthesis is regulated by the changeable promoter Pchangeable. This is the promoter activated by the substance we want to detect.
Its second part consists of the gene LacI, whose synthesis is regulated by the promoter Plux, which is activated by LuxR-AHL dimers, which result from the cell-to-cell communication.
This circuit determines whether or not to activate the toggle switch, according to the external signal (the substance being detected by the Pchangeable promoter), and the signals coming from the surrounding cell population (which activate the Plux promoter).

Toggle Switch

The Toggle Switch is how the cells "remember" data. As a default, the switch stays "off", until it receives a signal (in the form of LacI) from the Computation Circuit telling it to switch “on”. Once the Toggle Switch is "on" it remains activated indefinitely, unless it receives an external signal (IPTG) to switch “off”.
We based our system on the Toggle Switch from Gardner et. al. (Gardner, Cantor, & Collins, 2000):
"The toggle switch is composed of two repressors and two constitutive promoters [LacI and CI]. Each promoter is inhibited by the repressor that is transcribed by the opposing promoter. We selected this design for the toggle switch because it requires the fewest genes and cis-regulatory elements to achieve robust bistable behavior".
The system is switched "on" when the computation circuit produces the repressor LacI, which binds to Plac, thus inhibiting the expression of CI.


Without the presence of CI, the promoter PCI is uninhibited, and LacI and T7 RNA polymerase are synthesized. LacI inhibits the expression of CI, keeping our system in an "on" state, while the T7 RNA polymerase activates the Signal Circuit.
The system can be switched “off” by using IPTG, which induces Plac, causing CI expression and therefore repression of PCI, LacI and T7 RNA polymerase.

Signal circuit

The Signal Circuit is designed to facilitate cell-to-cell communication, which is based on the diffusion of a small quorum sensing molecule called AHL (N-Acyl Homoserine Lactose). AHL can diffuse through cell walls. For effective cell-to-cell communication, the cells must have two things:
(1) LuxR, a protein which binds to AHL (a "receiver").
(2) A genetic gate which can produce large amounts of AHL (an "antenna").

This circuit contains two parts:
The first part consists of the promoter Pcat, a constitutive promoter, which regulates LuxR expression in excess at all times. The LuxR protein can bind to AHL produced by neighboring cells, activating the Computational Circuit.
The second part consists of the promoter, PT7 RNA polymerase, which is controlled by the T7 polymerase synthesized by the Toggle Switch, and regulates the expression of LuxI – an enzyme that produces AHL. When the PT7 promoter is activated, it produces large amounts of AHL. This amplifies the signal produced by the toggle switch, before it is diffuses out through the lossy channel.

We believe that using the toggle switch is an improvement compared to the Alpha System, because Goni-Moreno and Amos’s analysis of the use of toggle switches for noise reduction showed positive results (Goni-Moreno & Amos, 2012).
We believe that amplified AHL production (using the T7 polymerase) would reduce the noise in our system because the error term in the Fokker-Planck equation (the equation commonly used for modeling noisy system) for this system, is reduced by a factor inversely proportional to this amplification (link to Ittai’s reference [13]).

Introduction

Some substances that we want to detect cannot diffuse into the cell or they do not activate promoters. To test for these substances we want utilize the E.coli’s EnvZ/ompR two-component signaling system (Forst & Roberts, 1994) by creating chimera proteins that detect the desired substance.

Figure 1: How a chimaera protein would use the EnvZ/ompR two-component signalling system to trigger our system

Taz is a chimaera protein of the cytoplasmic domain of EnvZ fused with the sensory domain of the transmemebrane aspartate receptor (TAR) (Tabor, Groban, & Voigt, 2009)

TaZ Construct

Completed and Biobricked

We found the receptor, tar-envZ biobrick (Bba_C0082) which contains the coding sequence for Taz. In order to use the Taz we added the promoter Pcat (Bba_I14033), an RBS (Bba_B0034) and double terminator (Bba_B0015). Thus we created the Taz construct biobrick BBa_K1343016. Click on the link to continue reading about our TaZ experimentation.

Gene Deletion

Failed to delete ackA-pta genes

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