Team:NYMU-Taipei/Project/Time Regulation

From 2008.igem.org

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{{:Team:NYMU-Taipei/Header}}
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We, the subteam self-proclaimed as the regulators of time, control time through the use of oscillators.
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We, the subteam self-proclaimed as the regulators of time, control time through the use of a timer and oscillators.
Our goal is to design a subsystem such that it would set to detach after a specified amount of time.
Our goal is to design a subsystem such that it would set to detach after a specified amount of time.
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We are using two very different kinds of oscillators to meet our goal:
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{| border="1" style="width:90%;margin-left:5%;border:1px solid gray;"
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== Motivation ==
 +
To enhance the the effect of the micro dialysis machine in the intestine lumen, the half life of the micro dialysis machine is designed to be prolonged. However, since our micro dialysis machine is made in the chassis of E. coli, there are two possible risks while staying in the intestine too long. One is that the E. coli may be apoptosis or necrosis due to large amount of the metabolite wastes it absorbed. The other is the bacteria balance in the intestine may be disturbed thanks to the E. coli being added. Thus, it's crucial and necessary to have a timer in our micro dialysis machine.
 +
Besides, the timer has many biological and engineering application, such as artificial pacemakers, biological clocks, etc.
 +
 
 +
== Timer Design ==
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[[Image:NYMU_timer.png|900px]]
 +
 
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Our timer consists of three parts: <span style="background:#cfc;">starting the timer</span>, <span style="background:#ffc;">counting the time</span>, and <span style="background:#fcc;">"stopping" the timer</span>.
 +
=== Timer Start ===
 +
We want our timer to start when our BacToKidney attaches to the small intestine. It attaches to the small intestine using the pH Sensor's pNhaA, so we also use the same promoter to start our timer.
 +
=== Counting Time ===
 +
Counting time using oscillators is the part we spent most of our time doing.
 +
For our timer, we have created an "oscillatory interface" which allows one to substitute any oscillator to be used with our timer.
 +
==== Oscillatory Interface ====
 +
Our interface only requires that there exist a promoter in the oscillatory system such that it shows an oscillatory effect due to the oscillator being in the same cell. We call this the '''Oscillatory Promoter'''.
 +
 
 +
For example, in the [[Team:NYMU-Taipei/Project/Time Regulation/Reloxilator|Reloxilator]], the pRE promoter has an oscillatory effect, and is therefore the oscillatory promoter for the Reloxilator.
 +
 
 +
==== Our Oscillators ====
 +
Since any oscillator could be used as long as it confirms to our oscillatory interface, we decided to make two oscillators with very different time periods to fit our purposes:
 +
{| border="1" style="width:90%;margin-left:5%;border:1px solid gray;background:#ffc;"
|-
|-
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| style="width:50%;padding:5px;vertical-align:top;" | [[Team:NYMU-Taipei/Cyanoxilator|Cyanoxilator]] (Cyanobacterial Oscillator)
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| style="width:50%;padding:5px;vertical-align:top;" | '''[[Team:NYMU-Taipei/Project/Time Regulation/Cyanoxilator|Cyanoxilator]]''' (Cyanobacterial Oscillator)
* Uses the three Kai proteins existing in Cyanobacteria (KaiA, KaiB, KaiC) that already naturally has a oscillatory rhythm and moving it over to  ''E. coli''.
* Uses the three Kai proteins existing in Cyanobacteria (KaiA, KaiB, KaiC) that already naturally has a oscillatory rhythm and moving it over to  ''E. coli''.
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| style="width:50%;padding:5px;" | [[Team:NYMU-Taipei/Reloxilator|Reloxilator]] (Relaxation Oscillator)
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* Has a time period of between 14 to 60 hours.
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| style="width:50%;padding:5px;" | '''[[Team:NYMU-Taipei/Project/Time Regulation/Reloxilator|Reloxilator]]''' (Relaxation Oscillator)
* A tuneable intracell-synchronized relaxation oscillator based on the combination of:
* A tuneable intracell-synchronized relaxation oscillator based on the combination of:
** The lysogenic/lysis genetic switch in the phage &lambda; and giving it oscillatory and tuneable properties.
** The lysogenic/lysis genetic switch in the phage &lambda; and giving it oscillatory and tuneable properties.
** The intracellular-synchronizing properties of the ''Vibrio Fischeri'' organism.
** The intracellular-synchronizing properties of the ''Vibrio Fischeri'' organism.
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* Has a time period of around 46 minutes.
|}
|}
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=== Timer Stop ===
 +
{| style="border:0px solid gray;"
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|-
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| [[Image:NYMU_threshold.png|NYMU_threshold.png|300px]]
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| style="border-left:1px solid gray;padding 5px;" | We stop the timer by setting a threshold. As the protein produced by the oscillator promoter accumulates and reaches a certain threshold, the output promoter activity would suddenly change. This output promoter is connected to a protease which would cause the detachment of BacToKidney from the small intestine.
 +
|}
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 +
 +
== Timer Circuit Design ==
 +
{| border="1"
 +
|-
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| [[Image:NYMU_timer_circuit0.png|310px]]
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| [[Image:NYMU_timer_circuit.png|310px]]
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| [[Image:NYMU_timer_circuit_bb.png|310px]]
 +
|}
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* The three figures above show the circuit design for the timer. The first is looking at it from a higher view, the second is in a more logical circuit design view, and the third is the more physical design with biobricks pictures.
 +
* The Legend for the second picture:
 +
** The Oscillatory Promoter comes from the whichever oscillator is being used.
 +
** pX is a repressable promoter (and X is the gene that can repress it).
 +
** The light blue shaded area represent's the circuit's logic AND gate.
 +
** The dark blue gene "Next" expresses a protein that accumulates to be used for the thresholdable promoter.
 +
 +
== Experimental Results ==
 +
So far, we've finished constructing all of the [[Team:NYMU-Taipei/Project/Time Regulation/Cyanoxilator|Cyanoxilator]] and have done a few [[Team:NYMU-Taipei/Project/Time Regulation/Results_of_Cyanoxilator|reporting assays]]. For the [[Team:NYMU-Taipei/Project/Time Regulation/Reloxilator|Reloxilator]], all the separate components (the oscillator, tuner, synchronizer, and reporter) have been constructed, but only the oscillator, tuner and reporter have been successfully ligated together. A few [[Team:NYMU-Taipei/Project/Time Regulation/Results_of_Reloxilator|reporting assays]] were also performed on the oscillator-tuner-reporter construct.
 +
 +
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{{:Team:NYMU-Taipei/Footer}}
{{:Team:NYMU-Taipei/Footer}}

Latest revision as of 23:29, 29 October 2008

NYMU Banner-wider 965px.png

We, the subteam self-proclaimed as the regulators of time, control time through the use of a timer and oscillators. Our goal is to design a subsystem such that it would set to detach after a specified amount of time.

Contents

Motivation

To enhance the the effect of the micro dialysis machine in the intestine lumen, the half life of the micro dialysis machine is designed to be prolonged. However, since our micro dialysis machine is made in the chassis of E. coli, there are two possible risks while staying in the intestine too long. One is that the E. coli may be apoptosis or necrosis due to large amount of the metabolite wastes it absorbed. The other is the bacteria balance in the intestine may be disturbed thanks to the E. coli being added. Thus, it's crucial and necessary to have a timer in our micro dialysis machine. Besides, the timer has many biological and engineering application, such as artificial pacemakers, biological clocks, etc.

Timer Design

NYMU timer.png

Our timer consists of three parts: starting the timer, counting the time, and "stopping" the timer.

Timer Start

We want our timer to start when our BacToKidney attaches to the small intestine. It attaches to the small intestine using the pH Sensor's pNhaA, so we also use the same promoter to start our timer.

Counting Time

Counting time using oscillators is the part we spent most of our time doing. For our timer, we have created an "oscillatory interface" which allows one to substitute any oscillator to be used with our timer.

Oscillatory Interface

Our interface only requires that there exist a promoter in the oscillatory system such that it shows an oscillatory effect due to the oscillator being in the same cell. We call this the Oscillatory Promoter.

For example, in the Reloxilator, the pRE promoter has an oscillatory effect, and is therefore the oscillatory promoter for the Reloxilator.

Our Oscillators

Since any oscillator could be used as long as it confirms to our oscillatory interface, we decided to make two oscillators with very different time periods to fit our purposes:

Cyanoxilator (Cyanobacterial Oscillator)
  • Uses the three Kai proteins existing in Cyanobacteria (KaiA, KaiB, KaiC) that already naturally has a oscillatory rhythm and moving it over to E. coli.
  • Has a time period of between 14 to 60 hours.
Reloxilator (Relaxation Oscillator)
  • A tuneable intracell-synchronized relaxation oscillator based on the combination of:
    • The lysogenic/lysis genetic switch in the phage λ and giving it oscillatory and tuneable properties.
    • The intracellular-synchronizing properties of the Vibrio Fischeri organism.
  • Has a time period of around 46 minutes.

Timer Stop

NYMU_threshold.png We stop the timer by setting a threshold. As the protein produced by the oscillator promoter accumulates and reaches a certain threshold, the output promoter activity would suddenly change. This output promoter is connected to a protease which would cause the detachment of BacToKidney from the small intestine.


Timer Circuit Design

NYMU timer circuit0.png NYMU timer circuit.png NYMU timer circuit bb.png
  • The three figures above show the circuit design for the timer. The first is looking at it from a higher view, the second is in a more logical circuit design view, and the third is the more physical design with biobricks pictures.
  • The Legend for the second picture:
    • The Oscillatory Promoter comes from the whichever oscillator is being used.
    • pX is a repressable promoter (and X is the gene that can repress it).
    • The light blue shaded area represent's the circuit's logic AND gate.
    • The dark blue gene "Next" expresses a protein that accumulates to be used for the thresholdable promoter.

Experimental Results

So far, we've finished constructing all of the Cyanoxilator and have done a few reporting assays. For the Reloxilator, all the separate components (the oscillator, tuner, synchronizer, and reporter) have been constructed, but only the oscillator, tuner and reporter have been successfully ligated together. A few reporting assays were also performed on the oscillator-tuner-reporter construct.


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