IGEM:IMPERIAL/2007/Cell By Date/Testing

Cell by Date

• Introduction
• Specifications
• Design
• Modelling
• Fabrication
• Testing
• Validation
• Notes
• References

--Anthony Lazzaro 08:04, 8 October 2007 (CDT) Hey Guys finally pulled the finger out and am writing up cell by date experiments

1.Determining System Capabilities

Having determined that our construct works in cell extract we set out to determine our system's expression level has as a function of temperature and time.

As can be seen in the above plot our results showed that for isothermal conditions the level of fluoresence and hence expression increased linearly with time. When comparing different expression levels at different temperatures we see that our sytem produces more at higher temperatures which was expected.

By linearly approximating the rate of fluoresence we have been to look at whether we can use an arrhenius type relationship to show how the rate of fluoresence increase with temperature. Doing so has shown that an arrhenius type relationship seems to fit and also our system seems to have an activation energy of 1.5kJ/mol

2.Determining Effect of sampling time on system capabilities

3.Determining Effect of DNA concentration on system capabilities

4.Determining effect of packaging on system capabilities

5.Determining System Capabilities in Vivo

Characterisation idea:

We're probably not going to present both projects so can use cell by date as a means to characterise the cell extract as a chassis. This would involve:

More experiments to determine the effect DNA concentration effects expression levels

More expreiments to determine the effect of temperature on the effect of expression eg. where does it turn off.

The logic behind this is that because we are using a constituitive promoter which is widely used this characterisation will prove useful to many other people when deciding whether to use cell extract as a chassis.

So for example in vivio 37 may be optimal (need to do literature research) but in may be optimal there may be a polynomail relationship in vitro but a different one in vivo

Proof of Principle TTI for beef spoilage

We can't do experiments for more than a day because of this coupling our system to the spoilge of meat is not possible. Our only hope for doing this is to not use the fluorometer and have a visible reporter, DsRed is proving to be problematic so another plan is needed.

This plan would be a proof of principle in which we only do experiments over the course of 6 hours using the fluorometer. In these six hours would could characterise the system and try to show it could be use for beef spoilage. There would be four experiements :

1.Isothermal for 6 hours at 10 C

2.Isothermal for 6 hours at 20 C

3.Step up from 10 to 20 to capture response time

4.Step down from 20 to 10 to capture positive history effect

We are using these temperatures because I have found the spoilge time for beef at these temperatures. Further computational work needs to be done to show how beef spoilge will occour in the step scenarios. I have found a paper that does just this and have contacted the author asking for his computer code, i have recieved no response yet so we will proably have to develope our own code.

Forget the Beef idea :

An alternative route would be to forget about beef and just focus on a break in the cold chain eg. product independant.

This would involve looking up the specifications of a generic cold chain or look at what our system can do and pick a cold chain that is similar so for example we know our system is off at 4 degrees C (need to tripple check) .

With this done we could then hypothesise that evaporation won't be a problem with perfect packagaing and that our system will only express when is taken out of the cold chain eg. above 4 degrees C. so our system is no longer a TTI but merely a switch which tell us if there has been a breakdown in the cold chain.