Valencia/The idea
From 2007.igem.org
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Contents |
Basis
The potential and flexibility of the comparator idea have been of crescent importance to choose it as our project.
This idea fits perfectly in the iGEM project philosophy of modularity as it could be a very useful piece for more sofisticated projects in future. This was the main reason to select it as our project. Because of the simplicity in the structure of the project it will be possible to carry out wet lab experiments in the limited time period of the competition. Furthermore, as a consequence of its versatility, we can increase the complexity of our project as much as we want if we see that we have time to do it.
Starting with a simple, robust circuit we think that it could be of interest to the scientific community who have been working on sensing bacterias.
The basic structure of the project can be explained as follows. Initially if we have an equal amount of external parameters such as any chemical compound, the expression level of both the repressor will be equal and which in turn will results in equal level of GFP and RFP (or other fluorescence proteins), which are essentially the reporters for the external variables or parameters.
Then, one of the two sensor devices (let's say sensor 1) starts to have more strengh and, so, it's repressor 1 and its reporter 1 are transcripted in some more concentration. This repressor 1 inhibits the promoter from the other construction (let's say 2), so we have less repressor 2 and reporter 2 from the construction 2.
Finally, we have an increase on the first fluorescence and a decrease on the second one: that way we have compared two sensing strenghts and amplified the stronger one.
What to detect
If we have a comparator, we have to decide what to compare, and try to do that on the time span that iGEM has.
We have decided to post here whatever ideas come up for the sensing promoters:
- heavy metals promoters: we could use some promoters that detect Cadmium and Mercury (for example)
- human lipofillic hormone detector: some hormones can difuse through the membrane, if we find some promoters that respond to the levels of two given hormones, we could implement an hormone-level detector. but, we may need to put a new protein on our system (the protein that takes the hormone and binds to the DNA) --this may be too complex for the two months we have left.
- flavour detector: using last year's idea, we could take two different flavours and have different transduction signals to detect relative levels of both. for example, vanillin and banana... --idem, this could be far too complicated.
- osmolarity: last year projest started as a modular detector. one of the aspects we aimed to sense was the osmolarity. the idea is preety simple: E. coli has osmolarity sensing devices, one of them is EnvZ-OmpR two-component system. we have some experience working with pOmpR promoter and we will compare pOmpR and pOmpRm in order to find the real parameters of our system.
Usefulness
The idea of the comparator is a robust idea that, at the same time, is flexible: this device can be implemented in a lot of diferent systems. With the idea of comparing levels of inputs and tune levels of outputs, we can play with a lot of differents inputs and outputs.
Some examples of applications of the idea can be:
Promoter calibrator: our project
Using this comparator over a defined substrate we can measure the relative strenght of a promoter. The comparator can be used to compare the relative strenght of diffetent promoters activated by the same substance. Or, also, to describe the different rates of two promoters and their constants...
The precise quantification of a given device is a task yet to be addressed by the iGEM community, and we have decided to aim for it as an example of our comparator. So, if we can achieve the experimental work of this project, we will be able to get values for the parameters of our system (like transcription&traduction rate, Hill coeficient, decay rate, monomers per cell, etc.)... This could help us to get some data from our system and start using real parameters, not only literature ones.
- a project that could root from this one could be to have the same promoters and varying a repressor protein and its target promoter, but the modularity of this side-effect project is not that good...
Controllers: Theoretical development
A controller is a device which maintain the value of a magnitud in a prefixed value. They have a wide range of application in engineering that goes from maintaining the temperature of a house costant to lead the performance of very complex processes in industrial applications in a very accurated way.
Proportional-integral-derivative controller (PID controller) is a generic control loop feedback mechanism that have a wide difussion. The Proportional value determines the reaction to the current error, the Integral determines the reaction based on the sum of recent errors and the Derivative determines the reaction to the rate at which the error has been changing. The weighted sum of these three actions is outputted to a control the desired magnitud.
Anyway depending of the variable to control sometimes it is enough using a PI or even a P controller.
If we play a little bit with the comparator device we can see that mixing both of them we can obtain a rough P controller. Coupling two different comparators, with inverted signals, we could be able of maintaining a given level of some protein, metabolite or phisiologcal parameter (pH).
Another interesting application of this controller is relate with bacterial replication. If we can find a sensing device for some mitotic factor, we could be able of determine when and how our bacteria should replicate.
Discrete level detector
Coupling many different comparators, with sensing devices of different strenght and sensibility, we could be able of building some kind of detector with as many 'steps' as different 'sensibilities' we can achieve. Mutational studies on the sensing promoters should be of huge importance here.
Analogic to Digital signal converter (ADC)
Nowadays is quite common in electronics to work with digital signals (the information is sent in terms of binary code) than with analogic signals (the information is used as it comes). The main reason to work with digital signals is because it is less sensible to noise and other interferences than analogic signals. Therefore the systems are more controlable and it's easier to work with them.
This way of working, that has demostrated its usefulness when we are working with electric signals, also could be very interesting while working with concentrations.
There is a type of ADC in electronics that are built using comparators and their engineering can be adapted directly to our device design.
more info about ADC can be found in [http://en.wikipedia.org/wiki/Analog-to-digital_converter here]
real GEL comparator
Coupling an AND gate to the output of the comparator, we could achieve a three-fluorescence-output.
This is useful in order to detect whenever the sensing strenghts are equal. So, in this case, we would have equal amounts of proteins AND1 and AND2, those proteins would activate the AND promoter and, then, RFP would start to glow.
We could build it this way:
- pLacI-RBS-TetR-RBS-CFP-RBS-AND1-T
- pTetR-RBS-LacI-RBS-YFP-RBS-AND2-T
- pAND-RBS-RFP-T
Filters
Borrowing another electronic idea, we thought that a high pass low pass or band pass filter (HPF, LPF, BPF) can also be implemented with our comparator.
In electronics a filter is a device which deletes some undesirable frequencies of a signal. For instance, the electric signal of the net (fixed frecuencies of 50 hz or 60 hz) in some cases could produce some interferences in another electromagnetic signal. If we work with an engine that uses those signals, those interferences are undesirable. So, installing the filter (BPF) in the engine this interference could be avoided. In some other tecnical applications it is useful to delete the signals with a higher or lower frecuency than certain level, this is performed by the HPF and LPF filters.
If we talk now in terms of concentration signals instead of electrical signals, this kind of devices could be also quite useful in biology. Let's say we have a bacteria that has a basal production of a chemical compound and we have a metabolic circuit implemented that is activated by this compound. This filter device can be used to avoid interferences of our circuit with the bacterial metabolims. How would this work? we add in our circuit a LPF that deletes the signal sent to our promoter by the bacterial natural production.
The implementation of this idea is quite simple as we basically need to use our comparator as a concentration comparator between a basal signal and the real signal. If the real signal is higher than the basal one, instead of using a reporter as output of our device, we add our metabolite producing circuit. If the real signal is lower than the basal one, then there is no production of our desired metabolite.
