Team:Tsinghua-A/Project/echo

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Project Section

Operation ECHO | Collaboration with other teams

Project_Idea

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.

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.

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.

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.

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.

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.

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.

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+<BODY LANG="zh-CN" BGCOLOR="#ffffff" DIR="LTR">
+<P><FONT FACE="Arial, sans-serif"><FONT SIZE=5 STYLE="font-size: 20pt"><SPAN LANG="en-US">Project
+Section</SPAN></FONT></FONT></P>
+<P ALIGN=CENTER><FONT FACE="Times New Roman, serif"><SPAN LANG="en-US"><FONT COLOR="#99284c"><FONT FACE="Arial, sans-serif"><FONT SIZE=5><SPAN LANG="en-US">Operation
+ECHO</SPAN></FONT></FONT></FONT><FONT COLOR="#6b4794"><FONT FACE="Times New Roman, serif"><SPAN LANG="en-US">
+</SPAN></FONT></FONT><FONT COLOR="#6b4794"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">|
+</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>
+<P ALIGN=CENTER><BR><BR>
+</P>
+<P ALIGN=CENTER><BR><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=5 STYLE="font-size: 20pt"><SPAN LANG="en-US">Project_Idea</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 16pt"><SPAN LANG="en-US"><I>Why
+we name after &lsquo;ECHO&rsquo;?</I></SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-bottom: 0cm; page-break-before: always"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">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, &lsquo;echo&rsquo; starts to have a
+meaning as reverberation or response of sound. In the field of
+automation, we use &lsquo;feedback&rsquo; 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. </SPAN></FONT></FONT></FONT>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm; page-break-before: always">
+<BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">Analogously,
+&lsquo;echo&rsquo; 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 &lsquo;echo&rsquo; 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.</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 16pt"><SPAN LANG="en-US"><I>How
+can bacteria act as group?</I></SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-bottom: 0cm; page-break-before: always"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">As
+the passages state above, CELL-A &amp; 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).
+</SPAN></FONT></FONT></FONT>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm; page-break-before: always">
+<BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">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.</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 16pt"><SPAN LANG="en-US"><I>How
+does the system work?</I></SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">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. </SPAN></FONT></FONT></FONT>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><IMG SRC="http://bioinfo.au.tsinghua.edu.cn/member/IGEM/pro_pngs/1.png" NAME="graphics5" ALIGN=BOTTOM WIDTH=392 HEIGHT=316 BORDER=0></P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">Figure:
+a predator-prey system</SPAN></FONT></FONT></FONT></P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><IMG SRC="http://bioinfo.au.tsinghua.edu.cn/member/IGEM/pro_pngs/2.png" NAME="graphics1" ALIGN=BOTTOM WIDTH=519 HEIGHT=142 BORDER=0></P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">Figure:
+ring oscillator</SPAN></FONT></FONT></FONT></P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">Though
+in different field, these two oscillations do have some commonality,
+that is, the feedback (or &lsquo;echo&rsquo;).</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">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).</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">Then,
+how&rsquo;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&rsquo;s easy to get the
+answer, which has already been stated in the title: homochronous. </SPAN></FONT></FONT></FONT>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 16pt"><SPAN LANG="en-US"><I>What
+does &lsquo;homochronous&rsquo; refer?</I></SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">The
+prefix &lsquo;homo&rsquo; represents &lsquo;same&rsquo;, while
+&lsquo;chrono&rsquo; means &lsquo;time-related&rsquo;. As a jargon of
+Information, the word &lsquo;homochronous&rsquo; describes the
+relationship between two signals that their corresponding significant
+instants are displaced by a constant interval of time. </SPAN></FONT></FONT></FONT>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">Here
+we use this term to depict the relationship between CELL-A &amp; 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.</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 16pt"><SPAN LANG="en-US"><I>Why
+we need a delay?</I></SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">Oscillation
+is more than just a feedback. In order to keep oscillating, the
+system should introduce a delay part to avoid a steady result.</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">Suggest
+a two-node system without a delay part, and write ordinary
+differential equations (ODEs) to describe the interaction as follows:</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">dAdt=&alpha;&#8729;Bn1k1n1+Bn1-&mu;&#8729;A
+&hellip;...(1) </SPAN></FONT></FONT></FONT>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">dBdt=&beta;&#8729;k2n2k2n2+An2-&mu;&#8729;B
+&hellip;...(2)</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">set:</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">dAdt=0
+&hellip;...(3)</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">dBdt=0......(4)</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">then
+we get a simplified equation:</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">1&mu;&#8729;B-1&beta;-1&beta;&#8729;k2n2&#8729;&alpha;&mu;&#8729;Bn1Bn1+k1n1n2=0......(5)</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT FACE="Times New Roman, serif"><SPAN LANG="en-US"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt">with
+(A</FONT></FONT></FONT><FONT COLOR="#000000"><SUB><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt">0</FONT></FONT></SUB></FONT><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt">,
+B</FONT></FONT></FONT><FONT COLOR="#000000"><SUB><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt">0</FONT></FONT></SUB></FONT><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt">)
+as a steady-state solution, the system is incapable to oscillate.</FONT></FONT></FONT></SPAN></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><IMG SRC="http://bioinfo.au.tsinghua.edu.cn/member/IGEM/pro_pngs/3.png" NAME="graphics2" ALIGN=BOTTOM WIDTH=294 HEIGHT=310 BORDER=0></P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">Figure:
+the improved network</SPAN></FONT></FONT></FONT></P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">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. </SPAN></FONT></FONT></FONT>
+</P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 16pt"><SPAN LANG="en-US"><I>What
+more can we achieve?</I></SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">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.</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">The
+amplitude represents the intensity of the fluorescence. It&rsquo;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.</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=5><SPAN LANG="en-US">Project_Design</SPAN></FONT></FONT></FONT></P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><IMG SRC="http://bioinfo.au.tsinghua.edu.cn/member/IGEM/pro_pngs/4.png" NAME="graphics3" ALIGN=BOTTOM WIDTH=574 HEIGHT=219 BORDER=0></P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><IMG SRC="http://bioinfo.au.tsinghua.edu.cn/member/IGEM/pro_pngs/5.png" NAME="graphics4" ALIGN=BOTTOM WIDTH=632 HEIGHT=220 BORDER=0></P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P ALIGN=CENTER STYLE="margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">Two
+E.coli populations (CELL-A &amp; B) regulate each other to realize
+oscillation under the QS modules, LuxI/LuxR together with LasI/LasR. </SPAN></FONT></FONT></FONT>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">The
+constitutive promoter p1 in CELL-A produces LasR, a transcriptional
+regulator, who maintains its high concentration within the cell. </SPAN></FONT></FONT></FONT>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">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.</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">Let&rsquo;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. </SPAN></FONT></FONT></FONT>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><FONT COLOR="#000000"><FONT FACE="Arial, sans-serif"><FONT SIZE=4 STYLE="font-size: 15pt"><SPAN LANG="en-US">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.</SPAN></FONT></FONT></FONT></P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P STYLE="margin-left: 1.75cm; margin-bottom: 0cm"><BR>
+</P>
+<P ALIGN=LEFT STYLE="margin-left: 0.56cm; margin-bottom: 0cm"><BR>
+</P>
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+</HTML>