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From 2012.igem.org

Contents 1 Overall project 2 Results 2.1 Video/animation and diagram of the critter in action 2.2 Model Analysis 2.3 Experimental Analysis 3 Project Details 3.1 Theory 3.1.1 Modeling 3.1.1.1 Objectives 3.1.1.2 Implementation 3.1.1.3 Models 3.1.1.4 Simulations 3.1.1.5 Data 3.2 Lab work 3.2.1 Chassis and Vectors 3.2.2 Functional Modules 3.2.3 Protocols 4 Standard Operating Protocols 4.1 General 4.2 Lab Equipment and Materials 4.3 Methodology

Overall project

More detailed abstract to come once the theory has been proven experimentally. For now, the homepage provides a broad overview.

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.

Limitations due to time constraints

1. Food packages cannot be packed with a nitrogen atmosphere (no vacuum-packing). B. subtilis is aerobic.
2. Principles will be tested at temperatures above 7°C as working with B. cereus is too time consuming.

Modeling

Objectives

Implementation

Models

Simulations

Data

Lab work

Chassis and Vectors

Functional Modules

Protocols

Standard Operating Protocols

In an effort to apply business concepts to the iGEM project we have agreed to conduct the project according to the following protocols. The specifics of these sections (such as the actual protocols or equipment listing) are contained within the SOP binder in the laboratory. This binder will be digitized for next year’s iGEM teams.

General

1. How to set up an experiment.
   • Fully describe the experiment in a document before it is scheduled to be performed (document is described in point 2).
   • After the experiment is documented, it should be reviewed by at least one other iGEM member.
   • Upon completion of the experiment there should be a short discussion/interpretation of the results and a short outlook for subsequent experiments.
2. Experiment Template
   • Insert outline of the template here
3. Acronyms.
   • Each iGEM member is assigned an acronym: e.g. Marius Uebel will be MU.
4. Data management.
   • Each filename should contain the original creator, date, and type of file. E.g. MU_20120412_igem12_sop_proposal.doc
5. Ordering of material.
   • A single person should be responsible for ordering, this prevents multiple orders of an item and a more controlled inventory.

Lab Equipment and Materials

1. Equipment.
   • Every piece of lab equipment should be accompanied by the manufacturer’s manual and a short how-to manual.
   • Overview and location of all our equipment should be written down in this part
2. Protocols
   • All lab protocols should be consolidated into a central location in the lab in print form.

Methodology

1. Culture
   • Contains general information and cultivation requirements of the chassis
2. Assay
   • Short overview of any assay kits changes undertaken to suit the actual experiment.
   • All information on original assays