Team:Tsinghua-A/Project/echo

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<P><FONT FACE="Arial, sans-serif"><FONT SIZE=5 STYLE="font-size: 20pt"><SPAN LANG="en-US">Project
<P><FONT FACE="Arial, sans-serif"><FONT SIZE=5 STYLE="font-size: 20pt"><SPAN LANG="en-US">Project
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</SPAN></FONT></FONT></FONT><A HREF="https://2011.igem.org/Team:Tsinghua-A/Project/col"><FONT COLOR="#99284c"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">Collaboration
</SPAN></FONT></FONT></FONT><A HREF="https://2011.igem.org/Team:Tsinghua-A/Project/col"><FONT COLOR="#99284c"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">Collaboration
with other teams</SPAN></FONT></FONT></FONT></A></SPAN></FONT></P>
with other teams</SPAN></FONT></FONT></FONT></A></SPAN></FONT></P>
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<h2>Contents</h2>
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<li class="toclevel-1 tocsection-1"><a href="#NAME_source"><span class="tocnumber">1.1</span> <span class="toctext">Why ECHO?</span></a></li>
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<li class="toclevel-1 tocsection-2"><a href="#Group"><span class="tocnumber">1.2</span> <span class="toctext">Act as group</span></a></li>
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<li class="toclevel-1 tocsection-3"><a href="#pre-pre"><span class="tocnumber">1.3</span> <span class="toctext">How does it work</span></a></li>
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<li class="toclevel-1 tocsection-4"><a href="#homo"><span class="tocnumber">1.4</span> <span class="toctext">Homochronous?</span></a></li>
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<li class="toclevel-1 tocsection-5"><a href="#delay"><span class="tocnumber">1.5</span> <span class="toctext">Delay?</span></a></li>
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<li class="toclevel-1 tocsection-6"><a href="#enhance"><span class="tocnumber">1.6</span> <span class="toctext">Enhancement</span></a></li>
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<li class="toclevel-1 tocsection-7"><a href="#design"><span class="tocnumber">2</span> <span class="toctext">Project Design</span></a></li>
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<li class="toclevel-1 tocsection-8"><a href="#high"><span class="tocnumber">3</span> <span class="toctext">Project Highlights</span></a></li>
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<P STYLE="margin-bottom: 0cm"><FONT COLOR="#800000"><FONT FACE="Arial, sans-serif"><FONT SIZE=5 STYLE="font-size: 20pt"><SPAN LANG="en-US">1.
<P STYLE="margin-bottom: 0cm"><FONT COLOR="#800000"><FONT FACE="Arial, sans-serif"><FONT SIZE=5 STYLE="font-size: 20pt"><SPAN LANG="en-US">1.
Project Idea</SPAN></FONT></FONT></FONT></P>
Project Idea</SPAN></FONT></FONT></FONT></P>
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</SPAN><I>Why we name after &lsquo;ECHO&rsquo;?</I></SPAN></FONT></FONT></FONT></P>
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</SPAN><I>Why we name after &lsquo;ECHO&rsquo;?</h1></I></SPAN></FONT></FONT></FONT></P>
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</SPAN><I>How can bacteria act as group?</I></SPAN></FONT></FONT></FONT></P>
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</SPAN><I>What does &lsquo;homochronous&rsquo; refer?</I></SPAN></FONT></FONT></FONT></P>
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<I>What more can we achieve?</I></SPAN></FONT></FONT></FONT></P>
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<I>What more can we achieve?</h1></I></SPAN></FONT></FONT></FONT></P>
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<P STYLE="margin-bottom: 0cm"><FONT COLOR="#6b2394"><FONT FACE="Arial, sans-serif"><FONT SIZE=5 STYLE="font-size: 20pt"><SPAN LANG="en-US">2.
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Once C6 is less than the threshold, tetR stops repression, and C12 is
Once C6 is less than the threshold, tetR stops repression, and C12 is
generated again. All in all, the oscillation begins.</SPAN></FONT></FONT></FONT></P>
generated again. All in all, the oscillation begins.</SPAN></FONT></FONT></FONT></P>
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<P STYLE="margin-bottom: 0cm"><FONT COLOR="#6b9994"><FONT FACE="Arial, sans-serif"><FONT SIZE=5 STYLE="font-size: 20pt"><SPAN LANG="en-US"><h1 id="high">3.
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Project Highlights</h1></SPAN></FONT></FONT></FONT></P>
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<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=3><SPAN LANG="en-US">Several oscillation systems have been established by scientists, and our ECHO is not alone. But we must say that, ECHO is special and, to some extent, unique. So, besides the basic functions of an oscillator, what else can our system achieve? </SPAN></FONT></FONT></FONT>
 +
</P><br>
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<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=3><SPAN LANG="en-US">In our expectation, ECHO is not just a single oscillator. Parameters that determine the property of an oscillation is the period and the phase, and we hope to maneuver each of the three independently. </SPAN></FONT></FONT></FONT>
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</P><br>
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<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=3><SPAN LANG="en-US">The period of the cycle is up to the reaction rate. Suggest we want to elongate the period, so we need to slow down the rate. In our system, we use aTc to generate a delay. The molecule aTc will bind with TetR once it’s added into the culture, which results in the anti-repression of Line B2, thus, much more time is needed for the procedure. In that way, we realize a longer period.</SPAN></FONT></FONT></FONT>
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</P><br>
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<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=3><SPAN LANG="en-US">As for the phase adjustment, we focus on the accumulation of AHLs and try to change the degradation. We design an extra gene, pBad, to generate AHLs under commands. When promoter pBad is activated by arabinose, C12 is produced to counteract the degradation. That delay a certain cycle and oscillation will follow another pace, therefore, we change the phase. Just like the time difference we feel in our life. During the moment when somebody flies from Beijing to Boston, the plane orients the opposite direction of Earth’s rotation (like the counteraction to degradation), and consequently, he has to subtract 12 hours in his watch and get used to the new time (follow another pace and change the phase).</SPAN></FONT></FONT></FONT>
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Latest revision as of 12:28, 28 October 2011

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Project Section

Operation ECHO | Collaboration with other teams



1. Project Idea


1.1 Why we name after ‘ECHO’?


In Greek myths, there is a story of the love between nymph Echo and Narcissus. The voice of Echo remains hovering in the valley in the end of the story, from then on, ‘echo’ starts to have a meaning as reverberation or response of sound. In the field of automation, we use ‘feedback’ to refer the process of response in a system, that is, the process in which part of the output is returned to its input in order to regulate its further output, just as an echo.


Analogously, ‘echo’ plays a significant role in our design. CELL-A generates AHLs, sending a message to CELL-B, and after B expresses the particular gene, B will ‘echo’ the message to A as a response. Therefore, the pre-state of A has a regulating influence on the next-state of A, restricting A to fluctuate in a certain behavior within a certain range. The ECHO feedback part contributes to the communication and well-function of the system.


1.2 How can bacteria act as group?


As the passages state above, CELL-A & B communicate by AHLs both within their own populations and with the other. To understand this principle, we need to take a further look at the quorum sensing (QS).


QS is the ability of populations of bacteria to communicate and coordinate their behavior via inter-cellular and inter-species signaling molecules. AHL is one example of these molecules. AHLs produced by a certain group of E.coli (as Group I) accumulate, and once the concentration passes the threshold, the receptors in another group (as Group II) are activated, and the specific gene is regulated to express. Thus, bacteria will act as two groups, in which I induces II under the quorum sensing.


1.3 How does the system work?


In ecology, the predator-prey system often serves as a wonderful example to describe population dynamics. In the system, a predator feeds on its prey, resulting in the decline of the prey population, while the lack of prey will lead to the death of predators those who depend on preys to survive, consequentially an opportunity for preys to reproduce. This system indicates the co-existence of nature, and to give a more detailed description of the up and down of the populations, we call it oscillation. Another oscillating device is known in digital electronic circuits, named Ring Oscillator, which is composed of an odd number of NOT gates attaching in a chain, with the last gate is fed back into the first.



Figure: a predator-prey system



Figure: ring oscillator



Though in different field, these two oscillations do have some commonality, that is, the feedback (or ‘echo’).


When the idea is applied in the synthetic biology, we use two populations of E.coli to construct the system, in which CELL-B represses A and A induces B, just as predators feed on preys and preys feed predators. Of course this system have a feedback network. Then, theoretically, the system will oscillate, and light in red (RFP in CELL-A) and green (GFP in B).


Then, how’s the interrelationship between RED and GREEN, is RED negated to GREEN, or just the very same? It seems contradictory when we take the first glance of the interaction, that is, A increases the population of B while B decreases A. But after taking the delay of biological reaction into consideration, it’s easy to get the answer, which has already been stated in the title: homochronous.



1.4 What does ‘homochronous’ refer?


The prefix ‘homo’ represents ‘same’, while ‘chrono’ means ‘time-related’. As a jargon of Information, the word ‘homochronous’ describes the relationship between two signals that their corresponding significant instants are displaced by a constant interval of time.


Here we use this term to depict the relationship between CELL-A & B. That is to say, in the process of oscillating, the AHL expression rate of CELL-A and that of B (also RED and GREEN fluorescent signals) share the same period, or fluctuate in a same pace. In addition, the interval of two signals remains constant, so the RED signal comes out while GREEN fades away at the same time, and vice versa. Consequently, time goes, RED and GREEN flare alternately.


1.5 Why we need a delay?


Oscillation is more than just a feedback. In order to keep oscillating, the system should introduce a delay part to avoid a steady result.

Suggest a two-node system without a delay part, and write ordinary differential equations (ODEs) to describe the interaction as follows:


dAdt=α∙Bn1k1n1+Bn1-μ∙A …...(1)

dBdt=β∙k2n2k2n2+An2-μ∙B …...(2)


set:

dAdt=0 …...(3)

dBdt=0......(4)


then we get a simplified equation:

1μ∙B-1β-1β∙k2n2∙αμ∙Bn1Bn1+k1n1n2=0......(5)


with (A0, B0) as a steady-state solution, the system is incapable to oscillate.


Figure: the improved network


Now we realize the importance of delay. Therefore, an auxiliary state B2 is introduced to the improved system, as a delay part. Moreover, B2 enables an access to maneuver the system.



1.6 What more can we achieve?


In our expectation, ECHO is not just a single oscillator. Three parameters that determine the property of an oscillation is the amplitude, the period and the phase, and we hope to maneuver each of the three independently.


The amplitude represents the intensity of the fluorescence. It’s not a hard work to amplify it by increasing the expressing rate of the GFP/ RFP gene. The period of the cycle is up to the accumulation of AHLs, so both the producing and the degrading will help. As for the phase, we design an extra circuit to generate AHLs under commands, in order to accelerate the process of accumulation before reaching the threshold, shorten a certain cycle and thus change the phase.





2. Project_Design






Two E.coli populations (CELL-A & B) regulate each other to realize oscillation under the QS modules, LuxI/LuxR together with LasI/LasR.

The constitutive promoter p1 in CELL-A produces LasR, a transcriptional regulator, who maintains its high concentration within the cell.


Then, an acyl-homoserine latone (AHL), 3OC12HSL from LasI in CELL-B will bind with LasR, lighting the green fluorescence and inducing the expression of LuxI, who synthesize 3OC6HSL. C6 accumulates in the culture and permeates into CELL-B, where it binds with LuxR. This process activates the expression of TetR and CFP, and therefore, represses LasI to produce C12.


Let’s have a review of the principle of oscillation, that is, A induces B, B represses B2, and B2 induces A, forming a feedback network with a delay part. Corresponding to the network is the three lines of gene, based on the interactions of C6/Lux, C12/Las and TetR/aTc.


When the concentration of C12 is high and C6 is low in the very beginning, luxI is intended to produce C6. As C6 mounts up, tetR starts to restrain the expression of C12. Then, under a certain rate of degradation, C12 decreases, resulting in the same decrease of C6. Once C6 is less than the threshold, tetR stops repression, and C12 is generated again. All in all, the oscillation begins.


3. Project Highlights





Several oscillation systems have been established by scientists, and our ECHO is not alone. But we must say that, ECHO is special and, to some extent, unique. So, besides the basic functions of an oscillator, what else can our system achieve?


In our expectation, ECHO is not just a single oscillator. Parameters that determine the property of an oscillation is the period and the phase, and we hope to maneuver each of the three independently.


The period of the cycle is up to the reaction rate. Suggest we want to elongate the period, so we need to slow down the rate. In our system, we use aTc to generate a delay. The molecule aTc will bind with TetR once it’s added into the culture, which results in the anti-repression of Line B2, thus, much more time is needed for the procedure. In that way, we realize a longer period.


As for the phase adjustment, we focus on the accumulation of AHLs and try to change the degradation. We design an extra gene, pBad, to generate AHLs under commands. When promoter pBad is activated by arabinose, C12 is produced to counteract the degradation. That delay a certain cycle and oscillation will follow another pace, therefore, we change the phase. Just like the time difference we feel in our life. During the moment when somebody flies from Beijing to Boston, the plane orients the opposite direction of Earth’s rotation (like the counteraction to degradation), and consequently, he has to subtract 12 hours in his watch and get used to the new time (follow another pace and change the phase).