Valencia/Lab Work

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{{Valencia/Cabecera}}
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<center>For a detailed descrition of the lab work please go to the [[Valencia/Lab Diary| Lab Diary]] section.</center>
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<center>'''For a detailed descrition of the lab work please go to the [[Valencia/Lab Diary| Lab Diary]] section'''</center>
===Construction===
===Construction===
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We aimed for a straight-forward strategy that used all the parts from the registry of parts. Our first achievement was to ligate the repressed promoter with the repressors, so to have two different constructions like this:
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We aimed for a straight-forward strategy that used all the parts from the registry of parts. From the computer work, we knew that in this system, ''time matters'', so we focused on proteins that have a rapid degradation tag. This affects both repressors as well as the fluorescence proteins and this is the way how our system should work on a matter of minutes, not hours...
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This is the comparator a device that permits the modularity of the system, as one can use any desired input or ouput. In our case, we wanted to use fluorescence.
+
Our first achievement was to ligate the repressed promoter with the repressors, so to have two different constructions like this:
 +
 
 +
[[Image:VComparator.jpg|400px|center]]
 +
 
 +
This is the comparator, a device that permits the modularity of the system, as one can use any desired input or ouput. In our case, we wanted to use fluorescence.
We aimed to assemble the fluorescence proteins backwards, those would be our output signals. We would have those constructs:
We aimed to assemble the fluorescence proteins backwards, those would be our output signals. We would have those constructs:
 +
 +
[[Image:VComparator-fluor.jpg|550px|center]]
 +
Now, we had to assemble the sensing part of our system, this would be the osmolarity-sensing promoters pOmpR and pOmpRm. we wanted to assemble them in order to build the E.coliRuler, the promoter calibrator device.
Now, we had to assemble the sensing part of our system, this would be the osmolarity-sensing promoters pOmpR and pOmpRm. we wanted to assemble them in order to build the E.coliRuler, the promoter calibrator device.
 +
 +
We first wantted to assemble pOmpR in both plasmids, so to validate the model and yield some system-specific parameters, then we wanted to compare pOmpR to pOmpRm.
 +
 +
This would be our first system's design:
 +
 +
[[Image:VPompr.jpg|550px|center]]
 +
 +
And, finally, we would like to have compared pOmpR to pOmpRm, like:
 +
 +
[[Image:VPomprm.jpg|550px|center]]
At last, we desired to have both constructs in one single bacterium, that is the reason why we had in mind to change the resistance backbone of one of the constructs to kanamicine. This way we would have a double selection for the finally transformed bacteria.
At last, we desired to have both constructs in one single bacterium, that is the reason why we had in mind to change the resistance backbone of one of the constructs to kanamicine. This way we would have a double selection for the finally transformed bacteria.
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===Experiments===
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===Fluorescence experiments===
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we will first start validating our model. we will use pOmpR as both inputs of the system and asses the dynamic behaviour of the system as well as experiments varying the osmolarity of the medium. this way we hope to define the values of our equations, thus taking the leap from an effective model to a model that describes more correctly the system we work on.
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We planned to do two kinds of experiments.
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then, with this corrected model, we will be able to precisely study the differences between pOmpR and pOmpRm and to calibrate them. This way, we could also use this model to make some good predictions about what parameters we could tune in order to make the system behave the way we want.
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First validating our model, using pOmpR as both inputs. And, then, compare pOmpR with pOmpRm.
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once we have calibrated pOmpR and pOmpRm, we will aim to a third variant of this promoter that will have different strenght.
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You can take a closer look at it on our [[Valencia/Results#Future experiments|future experiments]].

Latest revision as of 15:23, 26 October 2007

VBacteriaAnimada.gif
Summary The idea Promoter calibrator Biological comparator Electronic comparator Biological controller Lab Work Silico Work Results Adversities


For a detailed descrition of the lab work please go to the Lab Diary section

Construction

We aimed for a straight-forward strategy that used all the parts from the registry of parts. From the computer work, we knew that in this system, time matters, so we focused on proteins that have a rapid degradation tag. This affects both repressors as well as the fluorescence proteins and this is the way how our system should work on a matter of minutes, not hours...

Our first achievement was to ligate the repressed promoter with the repressors, so to have two different constructions like this:

VComparator.jpg

This is the comparator, a device that permits the modularity of the system, as one can use any desired input or ouput. In our case, we wanted to use fluorescence.

We aimed to assemble the fluorescence proteins backwards, those would be our output signals. We would have those constructs:

VComparator-fluor.jpg


Now, we had to assemble the sensing part of our system, this would be the osmolarity-sensing promoters pOmpR and pOmpRm. we wanted to assemble them in order to build the E.coliRuler, the promoter calibrator device.

We first wantted to assemble pOmpR in both plasmids, so to validate the model and yield some system-specific parameters, then we wanted to compare pOmpR to pOmpRm.

This would be our first system's design:

VPompr.jpg

And, finally, we would like to have compared pOmpR to pOmpRm, like:

VPomprm.jpg

At last, we desired to have both constructs in one single bacterium, that is the reason why we had in mind to change the resistance backbone of one of the constructs to kanamicine. This way we would have a double selection for the finally transformed bacteria.

Fluorescence experiments

We planned to do two kinds of experiments.

First validating our model, using pOmpR as both inputs. And, then, compare pOmpR with pOmpRm.

You can take a closer look at it on our future experiments.

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