From 2007.igem.org

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Testing and Discussion

Investigation properties of system under isothermal scenarios

In order to investigate the properties of our system we began by looking at isothermal scenarios in order to determine primarily the activation energy of our sytem follow Giannuzzi's work with beef. Again this is useful because as per Taoukis' research for a TTI to function correctly its activation energy needs to be close to the target product. In Figure 1 above we see that under isothermal conditions a steady rate of fluoresence was observed, corresponding to a steady rate of protein production.

Investigation System properties under Dynamic Temperature Conditions

With some understanding of how Pseuodmonas spoils aerobically stored ground beef it is really important for us to gain some understanding of how our sytem behaves in similar scenarios to the models developed by Koutsoumanis and Giannuzi in order to extract the same parameters.

For Isothermal conditions we have been able to devlope some simple models of the gene expression of our sytem with a strong dependance on energy depletion as will feel this is the major limiting factor of our system. As seen above in figures 3 and 4 with energy depletion as our major limiting factor we expect the concentration of our reporter gene to increase to a peak and then as energy runs out expressoin is curbed and decay of our reporter takes over giving an exponential like decay.

Validation and Conclusion

Property	Value	Achieved ?
Inputs	Isothermal conditions between 0 & 40 C	No
	Dynamic conditions eg. steps & ramps	hmm
Outputs	System should give a visual signal to indicate level of thermal exposure where beef is off	No we were unable to use a visual reporter
Activation Energy	System needs to have an activation Energy 30 +/- 20 kJ/mol	No activation energy of system was calculated to be 1.5kJ/mol
Health Regulations	System must not be living replicating bacteria	Yes through use of cell free chassis
Response Time	System needs to have a response time under 1 hour	hmm tbd
Lifespan	System must have a shelf life of 7 days	No with current packaging methods evaporation meant that our system 'died' after 2 days

Previous work has been carried out to model the spoilage of ground beef by living organisms. Above in figure 1 is is a model developed by Koutsoumanis in 2006 which colesly fit the behaviour of the spoilge organism we are interested in, Pseudomonas, under dynamic temperature conditions. One of the Key conclusions drawn from this model is the almost instantaneous repsone time of the Pseudomonas' growth parameter. The result of this is that our system needs to have a quick respone time to correclty report the temperature history of the beef.

Koutsoumanis' and also one Giannuzzi developed in 1998 are both based on the Gompertz model. This model allows some insight into the mechanisms of ground beef spoilage. In particular through manipulation of the Gompertz Parameters and assuming a Arrhenius type relationship between Pseudomonas' growth parameter and temperature we can infer the Activation energy of the spoilage reaction. This is shown in figure 2 in which a stongly linear behaviour allows us to continue with our Arrhenius assumption and extract that Activatoin energy of the spoilge reaction. As given in our specifications the Activation energy for ground meat seem to be around 30kJ/mol. The result of this as per Taoukis' work is that our system needs to have a similar activation energy. I hope to determine our system activation energy in the same way Giannuzzi determined Pseudomonas'.