Team:Technion-Israel/Project

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Revision as of 07:00, 16 October 2014

Safie by Technion-Israel

The Idea

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


Bio-detectors have been a big part of the iGEM projects ever since the competition first started, it's easy to see why: One of the simplest systems to build using our current tools for synthetic biology is a simple Input→Output "linear" (Promoter→Signaling Gene) bio-detector but this method has a major flaw:

In order to get a detection signal that's visible to the naked eye, we must have a LOT of bacteria change color (or any other signal). With the linear approach we find ourselves needing high concentration of the detected material for our system to be effective!

Now, while this issue is far from new and various teams have already tried to tackle this exact problem before, our team worked for a year on a new approach utilizing things like quorum sensing for inter-bacteria communication and signal amplification which is possible thanks to our creation of a synthetic bio-film using a revolutionary organic molecule called Azobenzene, resulting in what we refer to as a

'smart, self assembling material'


How It works

Neetd lots of editing

Beta System

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

*Figure of the Toggle Switch OFF mode – Avi*
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.
*Figure of the Toggle Switch ON mode – Avi*
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").
*Figure of the entire SC – Avi*
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]).

Azobenzene

(1) Our bacterium has two main features - one is a sensor and the second is azobenzene attached to the LPS. When the bacterium detects a substance it changes color by producing green luciferase


(2) The light emitted from the bacterium causes the azobenzene molecules to change conformation to a "sticky" form


(3) The azobenzene molecules cause the bacteria to aggregate by forming bonds through azobenzene, allowing fast diffusion of quorum sensing molecules and the rest of the bacteria turn green as well

Histidine Kinase

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

Protocols
Lab Notebook

to view the files you will need Adobe Acrobat Reader or similar

Gene Deletion & Histidine Kinase

This is Rebecca's and Karen's lab notebook for gene deletion attempts and TaZ biobrick building.

Gate Construst

These are a few notebooks arranged together of all gate constructs. Lab work done by Tal, Rica, Ronen, Shira, Noa, Alex and Ittai.

Azobenzene

This is the lab notebook of all Azobenzene lab work done by Faris, our chemist. This notebook is all chemistry.

Safety



One of the biggest concerns regarding synthetic biology in the general public is "Will the genetically modified organism be safe for me? What happens when you release the organism you designed into the environment? What if you create something you cannot control?"
These are valid questions that need to be answered when creating genetically modified bacteria. We tackled the important safety aspect in the project in three different ways:

(1) Teaching and trying to understand synthetic biology.
(2) Safety in the lab.
(3) Our system's safety.

Synthetic Biology Education


We understand the fear and concern of the public about GMOs. Therefore we wanted to expose the public to synthetic biology.
We gave lectures to teens and adults from different backgrounds about synthetic biology, its great potential and safety concerns.
We also emphasized the importance of safe lab work as part of our lab activity for children.


Safety in the Lab


We took all the necessary precautions such as lab coats, safety goggles when using liquid Nitrogen and always woregloves.
No food was allowed in the lab and there was a separate area for computer work.
The dress code was also strict- when working in the lab we wore closed shoes and long pants/skirt.
While working in the lab we used Ethidium Bromide (EB) for using gel electrophoresis and analysis. This substance is a potent mutagen that is used as a nucleic acid stain.
Therefore, we took special precautions such as working with EB only in the chemical hood, and having separate disposal for EB. We also had have a separate area on the bench where we ran the gels. This area has its own equipment such as tips, pipettors and gloves.
When performing gel extraction, we were exposed to UV light for short periods of time. To minimize the exposure we used protection equipment such as face protection shields and full body lab coats.

Non-biological Safety
Another safety aspect of our project is the chemical one.
The Azobenzene production was done using a few chemicals that needed special caution such as 70% Nitric Acid. This substance is hazardous when it comes in contact with skin. Therefore, a face shield, full suit and all the appropriate protection was worn.
Another substance was chloroform which is carcinogenic. All the safety measures was taken, including personal protection and exhaust ventilation in the chemical hood. We also used AgO, THF and Zinc dust. AgO is irritating to the eyes and respiratory system, THF is hazardous when it comes in contact with skin and has carcinogenic effects, Zinc dust is an irritant when it comes in contact with skin. Therefore, the use of all these substances was under the guidance of our mentors and every step was evaluated by experienced chemists (We consulted Ruth Goldschmidt from Professor Livney's lab and also Emma Gerts, who is in charge of organic chemistry labs in the Technion) who advised us on all the necessary safety measures needed to be taken. The whole process was always done according to all safety precautions, in a chemical hood and the disposal was according to MSDS of the reagents.
Other chemical materials we used in the production of Azobenzene were not hazardous.
The Azobenzene as a product is not hazardous and is biologically safe. The product is not volatile and is not hazardous when it comes in contact with skin (according to Woolley Group, Department of Chemistry in the University of Toronto, Canada).
Our project combines both synthetic biology and chemistry. We think it's important to have a safety program in the iGEM competition for chemistry, not only for biology, which will allow the iGEM HQ to supervise chemical lab work as well.


Our System's Safety


Now that we discussed the general safety, the safety of our project needs to be assessed.
In our project we used E. coli strains as model organisms for our systems.
We chose this bacteria since it is common in laboratory use and the strains we used (BL21, Top 10-DH10β, DH5αz1, JW3367-3, BW25113) are non-pathogenic and safe to work with.
In the future, when we finish testing the whole system, adding a kill switch into the system would be a MUST to ensure a safe use of the bacteria as a detector.

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