Team:Groningen/Project

From 2012.igem.org

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=== Theory ===
=== Theory ===
 +
'''Functionality'''
 +
<br>
 +
The monitor would be realized as a self-contained unit containing the sensing and reporting mechanisms.
 +
The sensing mechanism should allow monitoring of multiple foodstuffs at temperatures down to 4°C while having a response rate between 2 to 5 hours. The chassis must be able to survive long periods of inactivity.
 +
The reporter would utilize a color-coded system of identification, i.e. green is edible, red is not. Ideally, this should happen in a matter of minutes, but a combined detection/reporting response within 2 to 5 hours is sufficient.
 +
<br>
 +
<br>
 +
'''Sensing Mechanism'''
 +
<br>
 +
'''''Chassis'''''
 +
<br>
 +
The main factors in choosing a chassis are a prolonged dormant phase, ease of use, and the ability to survive in a cold environment. For this reason bacillus subtilis was selected. While it is not psychrotrophic (i.e. able to grow at <7oC), it has a dormant phase and is easy to use. It also allows for some novelty of using eukaryotic receptors in a prokaryotic chassis. Given a longer timeframe, the project methodology may be applied to detoxified strain of b. cereus, which is a psychrotrophic bacterium.
 +
<br>
 +
'''''Targets'''''
 +
{|
 +
| Amines || Trace amine-associated receptor 5 (TAAR5) - a eukaryotic G-coupled protein receptor
 +
|-
 +
| pH || Diffusion through the outer membrane
 +
|-
 +
| Ammonium || 1. NrgA ion channel at low concentrations 2. Diffusion through the outer membrane at high concentrations 
 +
|}
 +
<br>
 +
'''Reporters'''
 +
<br>
 +
'''''Indication methods'''''
 +
<br>
 +
There are a number of possibilities for changing the color of a bacterium, but the most suitable is the carotenoid pathway, either on its own or augmented with the tuning system developed for e.chromi by the 2009 Imperial College of London iGEM team. The carotenoid pathway provides a latched gradient from red to orange to yellow through the use of pigment.
 +
<br>
 +
<br>
 +
'''Enhancements'''
 +
<br>
 +
'''''Reaction time'''''
 +
# Amplify the production of the precursor and using an engineered enzymatic switch.
 +
# Adapt the fast-response mechanisms of the London yoghurt to e.chromi.
 +
'''''Thresholds and Sensitivity'''''
 +
<br>
 +
Histamine receptors need to calibrated to the total volume of meat (and volume of bacteria) within the package as histamine is always present.
 +
<br>
 +
A comprehensive set of experiments is necessary to determine the relationship between histamine concentration and spoilage rate. Once the relationship is known then the sensor mechanism can be tuned (if using e.chromi)
 +
Color progression
 +
<br>
 +
A switch is required to halt the red-to-orange and orange-to-yellow reactions The color change will use the color of the containment unit as a starting point (green). The indicator only need cover the base color as the indicator pigment is opaque.
 +
<br>
 +
<br>
 +
'''Challenges'''
 +
# Eukaryotic receptor in prokaryotic cell.
 +
# Tuning of the signaling and carotenoid pathways and the receptors threshold to levels appropriate for the quantity and geometry of the meat in the package.
 +
# Coupling the receptors to the carotenoid pathway.
 +
# Modification of the carotenoid pathway to disable the red-to-orange and orange-to-yellow transitions.
-
==== Research ====
 
==== Modeling ====
==== Modeling ====
===== Objectives =====
===== Objectives =====

Revision as of 17:26, 10 May 2012

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Contents

Overall project

Tell us more about your project. Give us background. Use this is the abstract of your project. Be descriptive but concise (1-2 paragraphs)


Results

Video/animation and diagram of the critter in action

Model Analysis

Experimental Analysis

Project Details

Theory

Functionality
The monitor would be realized as a self-contained unit containing the sensing and reporting mechanisms. The sensing mechanism should allow monitoring of multiple foodstuffs at temperatures down to 4°C while having a response rate between 2 to 5 hours. The chassis must be able to survive long periods of inactivity. The reporter would utilize a color-coded system of identification, i.e. green is edible, red is not. Ideally, this should happen in a matter of minutes, but a combined detection/reporting response within 2 to 5 hours is sufficient.

Sensing Mechanism
Chassis
The main factors in choosing a chassis are a prolonged dormant phase, ease of use, and the ability to survive in a cold environment. For this reason bacillus subtilis was selected. While it is not psychrotrophic (i.e. able to grow at <7oC), it has a dormant phase and is easy to use. It also allows for some novelty of using eukaryotic receptors in a prokaryotic chassis. Given a longer timeframe, the project methodology may be applied to detoxified strain of b. cereus, which is a psychrotrophic bacterium.
Targets

Amines Trace amine-associated receptor 5 (TAAR5) - a eukaryotic G-coupled protein receptor
pH Diffusion through the outer membrane
Ammonium 1. NrgA ion channel at low concentrations 2. Diffusion through the outer membrane at high concentrations


Reporters
Indication methods
There are a number of possibilities for changing the color of a bacterium, but the most suitable is the carotenoid pathway, either on its own or augmented with the tuning system developed for e.chromi by the 2009 Imperial College of London iGEM team. The carotenoid pathway provides a latched gradient from red to orange to yellow through the use of pigment.

Enhancements
Reaction time

  1. Amplify the production of the precursor and using an engineered enzymatic switch.
  2. Adapt the fast-response mechanisms of the London yoghurt to e.chromi.

Thresholds and Sensitivity
Histamine receptors need to calibrated to the total volume of meat (and volume of bacteria) within the package as histamine is always present.
A comprehensive set of experiments is necessary to determine the relationship between histamine concentration and spoilage rate. Once the relationship is known then the sensor mechanism can be tuned (if using e.chromi) Color progression
A switch is required to halt the red-to-orange and orange-to-yellow reactions The color change will use the color of the containment unit as a starting point (green). The indicator only need cover the base color as the indicator pigment is opaque.

Challenges

  1. Eukaryotic receptor in prokaryotic cell.
  2. Tuning of the signaling and carotenoid pathways and the receptors threshold to levels appropriate for the quantity and geometry of the meat in the package.
  3. Coupling the receptors to the carotenoid pathway.
  4. Modification of the carotenoid pathway to disable the red-to-orange and orange-to-yellow transitions.

Modeling

Objectives
Implementation
Models
Simulations
Data

Lab work

Chassis and Vectors

Functional Modules

Protocols

Standard Operating Protocols

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