From 2014.igem.org

Revision as of 13:32, 10 October 2014 by KarenJ (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Safie by Technion-Israel

The idea

Neetd lots of editing

Imagine that with a simple test you could know what your food contains, a moment before you eat
You could test if your ice cream contains nuts
You could test if the water you are drinking contains heavy metals
And you could test if the vegan dish in a restaurant contains eggs.

The Technion iGEM Team has a project that takes us one step closer to a future like this

Over the past year the team has been developing a life saving biological system that uses E. coli bacteria to detect substances at low concentrations in a variety of products (Such as food, drinking water, and industrial products). With the help of this system it is possible to visually detect the presence of the substances quite easily.

One of the principles of the system we have developed is the ability to easily switch the substance to be detected; the same system can be used to detect:
*avi's pictures*

How It works

Neetd lots of editing

Our team is building a proof-of-concept computational network using E. coli cells for the detection of trace amounts of harmful substances, such as allergens in food. Each cell is an independent computational module programmed to execute instructions based on common input. They comprise bypassed toggle switches - mimicking transistors (the basis of any computational system), an interchangeable promoter serves as an input port, and a histidine kinase two-component signaling system fused to a sensor in the periplasmic domain will serve as a converter for other input signals. The independent modules communicate using quorum sensing to produce a unified output signal. We are chemically synthesizing a photo-switching molecule connecting the cells together, creating an artificial biofilm. The result is a network made of independent computational elements that upon detection of a substance, co-operate to provide the user with an output visible to the naked eye.

Gate Construct

Alpha System

The Alpha system is the alpha design version we've planned for a system that can detect various materials in low concentrations.
Unlike other biosensors, our system is meant to be as versatile as possible- with a small change in the genetic level it can detect molecule A instead of molecule B. That saves us the need to construct a different detector for every dangerous material out there.
*Maybe a picture of a bun and a heavy metal frightened because of safie*
The alpha system consists of three gates:
*picture of a cell with the schemes of the three gates*
Each gate has an important role in the detection process inside each cell but the system as a whole is also based on communication between bacteria through production of quorum sensing molecules.

Gate 1

Built and Biobricked

The goal of the first gate is to produce AHL molecules when a material of interest is present in the bacteria's environment.

The construct includes a switchable promoter for the detection of the material of interest. In the example above the promoter is Ptet which is activated by tetR.
When the material is present it will get into the cell, attach to the promoter and the transcription of the LuxI gene will begin. LuxI gene encodes for an AHL synthase. AHL is a signal molecule that can diffuse freely into the cell and out.
The NheI restriction site was added to the construct so that the promoter activating the gate can be easily changed. EcoRI restriction site is found at the beginning of the prefix, and by a simple restriction reaction with the two enzymes we could take the promoter out and replace it with a different one.

But why do we want our bacteria to produce AHL molecules? How does it help us to see if a toxin or an allergen is present in our sample?
Continue reading about gate 2 and 3 to find out

Gate 2

Built and Biobricked

The gate serves as a sensitivity tuner, allowing us to control on the timing of the measurement. When we want to start our measurement we just need to add IPTG to the bacteria solution.

The gate consists of the Plac promoter and the LuxR gene. When IPTG is added to a sample containing our bacteria, LuxR gene is transcribed. LuxR is a transcriptioninal activator that binds directly to the AHL molecules, so that a LuxR-AHL complex is formed.

And what happens in our system when the complex LuxR-AHL is formed?
Read the next sections about gates 3 to find out

Gate 3

Failed to build

Gate 3 enables us to see if our material of interest is present in a random sample by producing GFP and amplifying the signal.

This construct is the final part of our alpha system. The promoter of the gate is the Plux promoter which undergoes activation when the complex LuxR-AHL binds to it. If the promoter is activated we get the production of a reporter gene (GFP) and the LuxI gene.
As been said in the gate 1 section, LuxI transcription results in AHL molecules.

Beta System

Histdin Kinase

Coming soon

Azobenzene

Modeling

New Method

Methods & Protocols

Here are all the protocols we used in the project

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

Atibiotics preparation
DNA Kit Plate
Chemical Trasformation
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

Lab Notebook

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

Gene Deletion & Histedin Kinase

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

Learn More

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.

Learn More

Azobenzene

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

Learn More

Safety

Visit our Facebook page
Design: HTML5 UP & Technion_Israel Team
igem14il@gmail.com

Team:Technion-Israel/Project

From 2014.igem.org

The Project

"Success consists of going from failure to failure without loss of enthusiasm."--Winston Churchill

Neetd lots of editing

The Technion iGEM Team has a project that takes us one step closer to a future like this

Neetd lots of editing

Alpha System

Gate 1

Built and Biobricked

The goal of the first gate is to produce AHL molecules when a material of interest is present in the bacteria's environment.

Gate 2

Built and Biobricked

The gate serves as a sensitivity tuner, allowing us to control on the timing of the measurement. When we want to start our measurement we just need to add IPTG to the bacteria solution.

Gate 3

Failed to build

Gate 3 enables us to see if our material of interest is present in a random sample by producing GFP and amplifying the signal.

Beta System

Coming soon

Here are all the protocols we used in the project

Gene Deletion & Histedin Kinase

Gate Construst

Azobenzene