Team:Technion-Israel/Project
From 2014.igem.org
The Project
"Success consists of going from failure to failure without loss of enthusiasm."--Winston Churchill
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'
Neetd lots of editing
(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
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 countinue reading about our TaZ experimentation.
Gene Deletion
Failed to delete ackA-pta genes
Neetd lots of editing
Here are all the protocols we used in the project
to view the files you will need Adobe Acrobat Reader or similar
Chemical Trasformation
DNA Kit Plate Instructions
Z-Competent™ cells and Mix&Go
Gel/PCR Extraction
Gel Preparation
Gibson Assembly
Glycerol stock
LB, BA, SOB, SOC
Ligation
Presto Mini-prep
PCR
Phosphorylation and blunt ligation
Restriction enzyme
Electroporation Trasformation
Gene deletion using λ red
Genome extaraction
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.
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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