IGEM:IMPERIAL/2007/Projects/Cell by date/Design: Difference between revisions

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=Phase 1 - Intial Testing=
 
==Phase 1: Intial Testing==


Phase 1 involves the testing of various simple constructs to confirm that there is gene expression ''in vitro'' and ''in veso''. DNA constructs are as summarized below:
Phase 1 involves the testing of various simple constructs to confirm that there is gene expression ''in vitro'' and ''in veso''. DNA constructs are as summarized below:
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We preferably want to use a constitutive promoter for Cell by Date. If all constitutive promoters are found working then we will need to chose one of them. This decision can be made based on their levels of activity (the higher, the better) and their operating temperature range. This needs to be as close as possible to the CBD operating range of 4&deg;C - 37&deg;C <br>
We preferably want to use a constitutive promoter for Cell by Date. If all constitutive promoters are found working then we will need to chose one of them. This decision can be made based on their levels of activity (the higher, the better) and their operating temperature range. This needs to be as close as possible to the CBD operating range of 4&deg;C - 37&deg;C <br>
==Phase 2: Characterizing specific Construct==






==Phase 1 : Mimicking Printed Sell By Date==


====Introduction====
This first level of complexity hopes to calibrate our system so that it will mimic the function of the Sell By Date printed on the meat packets found in supermarkets, eg. '''Sell By 12/06/07'''


The food industry calculates these Sell By Dates by using a technique called '''Challenge Testing'''.  The Meat we purchase in our local supermarkets was once an animal that was slaughtered, processed and packaged before we pick it up off the shelf at supermarkets.  When the meat is packaged it is already contaminated with bacteria.  Over time these bacteria feed off the meat ,multiply and grow: we perceive this as discoloration or mould formation on our food.  After time the bacteria and grown so much that the food is no longer good to eat. When this happens the food is considered '''spoiled''' or '''off'''.


The time taken for the meat to go off depends upon the environment the meat is placed in, bacteria will grow quicker in higher temperatures for example and so will go off quicker in higher temperatures.  The Sell By Date printed on our meat packets is calculated on the assumption that the meat will be exposed to certain conditions, for example that meat will always be refrigerated at 10<sup>o</sup>C from the time it leaves the processing factory to when we pick it up at the supermarket.  This chain of refrigeration is know as the '''Cold Chain.'''


The Cold Chain of the Food Industry is highly regulated and developed but what happens after we pick it up from the Supermarket Shelf? The Food industry has addressed this problem using conservative estimates of when food will spoil as to avoid people eating off meat.  This means that often the printed cell by date insinuates meat is off when in fact it isn't.


This initial phase of cell by date hopes to improve on the printed Sell by Date that is based on challenge testing. Starting with an intial CFU (representative of initial contamination) & constant temperature we want to look at how long it takes for the OD of the colony, representative of bacterial CFU in meat, to reach a level equivalent to the point where the meat is considered to be spoiled. Using this level and other data collected through preliminary experimentation we hope to set up a model representative of the transient response of our system which if validated will serve as the basis for the later phases of Cell By Date.


====Design Parameters====
Quantitatively our design parameters are:
*Initial CFU : To be taken from Predictive Microbiology Papers / defined on our own terms
*Temperatures : Focus on various temperatures
**4<sup>o</sup>C representing Cold Chain best case scenario (constant refrigeration)
**37<sup>o</sup>C representing Worst Case scenario (constant heating)
**Intermediate temperatures (25<sup>o</sup>C, 10<sup>o</sup>C, 15<sup>o</sup>C, 20<sup>o</sup>C, etc.) to denote the stability of system
*Spoilage Level : Due to time constraints we are unable to wait for the CFU of our colony to be equivalent to the level where the meat is actually spoiled as this would take days to achieve.  Instead we will define our own spoilage limit, it will be the CFU reached in 24hr under Best Case Scenario conditions ,eg. constant 4<sup>o</sup>C, starting with initial CFU taken from papers/FSA.  This means that we are taking the maximum life of our meat to be 24hrs. This spoilage level will have to be determined by preliminary experimentation.
*Units of measurement are taken to be '''GFP synthesized CFU<sup>-1</sup>s<sup>-1</sup>'''


<br clear="all">
=NOT APPLICABLE=


==Phase 2 : Specific Temperature Exposure Device==


=== 2A : Gradient ===
=== 2A : Gradient ===

Revision as of 07:47, 9 August 2007

Cell by Date: Design


Summary

Cell by Date has three levels of complexity and we hope to implement these levels in separate chassis: E. coli, in vitro and in veso. These chassis are being characterized as one of ICGEMs sub-projects. The table below outlines which levels which hope to implement Cell by Date in.

Chassis Phase 1 Phase 2 Phase 3
E.coli X
In-Vitro X X X
In-Veso X X X


Phase 1: Intial Testing

Phase 1 involves the testing of various simple constructs to confirm that there is gene expression in vitro and in veso. DNA constructs are as summarized below:

Test Constructs Notes
Constitutive T7 Promoter
PT7 promoting GFP
PT7 promoting GFP
  • Entire construct is currently available in Biobricks Registry: BBa_E7104 (available), in pSB1A2 (AmpR)
  • pT7 can be used with T7 RNAP in vitro and in veso, and it gives constitutive expression.
Constitutive E.coli Promoter
Ptet</sub promoting GFP
Ptet</sub promoting GFP
  • Entire construct is currently in Biobricks Registry: BBa_I13522 (available), in pSB1A2 (AmpR)
  • pTet can be used with E.coli RNAP in vitro and maybe in veso, and it gives constitutive expression.
Inducible E.coli Promoter
PBad promoting GFP
PBad promoting GFP
  • pBad promoter and GFP available: BBa_J5528 (available), in pSB2K3 (KanR)
  • Need Ara C regulator protein in vitro and in veso. Ara C available with the RBS:BBa_S03550
  • pBad can be used with E.coli RNAP in vitro, and it is inducible by arabinose.
  • To be used in veso, arabinose transport proteins have to be inserted in the phospholipid bilayer of the vesicles.
Constitutive E.coli Promoter
PcI promoting GFP
PcI promoting GFP
  • pcI repressible promoter and GFP avaiable: (put link here)
Constitutive E.coli Promoter
pBad promoting GFP and pcI promoting araC
pBad promoting GFP and pcI promoting araC
  • Available in the registry: ??? (put link here)


Firstly we will test the promoters using the in vitro system. We are not interested in a lot of details, but rather just trying to see the most suitable promoter. The promoter construct, together with the inducer required (if the promoter is inducable) will be mixed together and the levels of fluorescence will be monitored over time.

We preferably want to use a constitutive promoter for Cell by Date. If all constitutive promoters are found working then we will need to chose one of them. This decision can be made based on their levels of activity (the higher, the better) and their operating temperature range. This needs to be as close as possible to the CBD operating range of 4°C - 37°C


Phase 2: Characterizing specific Construct

NOT APPLICABLE

2A : Gradient

Introduction

Having used the data collected from Phase 1 and simulated changes of temperature in the accumulation of reporter gene in model studies, we proceed on to test and validate our findings in Phase 2 where the device is exposed to varying temperature conditions. Since we have already calibrated our device in Phase 1, there is no requirement for further calibration unless a different reporter gene is used. Therefore the higher the exposure to increased temperature conditions, the faster the system will move along a graded colour scheme that has been calibrated in Phase 1. If this can occur without false positives, then it has achieved the objectives that we have set in our specifications.

Design Parameters

  • Temperatures: Focus on varying exposure
    • Ramp analysis
    • Pulse
    • Step
    • Sqaure pulse
  • Testing and validation of our model studies and data analysis
  • Prevention of false positives (indicator reads "off" when it is not)


2B : Discrete Colour Change

  • Builds on 2A by having a discrete colour change when meat is spoilt.
  • Discrete Change occours when meat is exposed to room temperature (25oC) for more than two hours

More info to be added!


Phase 3 : General Meat Heat Exposure Device / Abstraction for re-usability

More info to be added!

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