Team:Tokyo Tech

From 2008.igem.org

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Latest revision as of 05:25, 30 October 2008

Main	Protcol	Parts Submitted to the Registry	Our Team	Acknowledgements

1 1. Our project
2 2. Pressure induction
3 3. Touch display
4 4. Low pressure-inducible promoter
5 5. Write/Erase cycle
- 5.1 Genetic toggle switch to implement Write/Erase cycle
- 5.2 Mathematical model

1. Our project

Our project is creation of "Coli Touch"!!

What is "Coli touch"?

“Coli Touch” has a pressure sensitive display composed of an E. coli lawn. When you touch its display, touched section is colored.

Next I'll tell you about “Coli Touch” work system. Display of “Coli Touch” contains many E. coli. When you touch this display, pressure applies to E. coli in this display. Pressure applied E. coli expresses GFP.

Why pressure?

“Coli Touch” uses pressure as input. Why do we use pressure? Past input methods (small molecules, heat and light) are difficult to induce uniformly. Pressure can induce uniformly.

2. Pressure induction

Introduction

Tet promoters is known as a sensitive ones to pressure. (T. Sato et al., 1995)

Construction

figure2-1. We constructed Ptet-GFP and promoter less-GFP.

We chose the tet promoter (Ptet) as a pressure-inducible promoter. We constructed two plasmids - one is Ptet-GFP on pSB6. The other is promoter less-GFP on pSB6 as a negative control. We measured activity of tet promoters under 0.1 MPa and 30 MPa without repressor protein.

Result -activity of tet promoters-

figure2-2. Pressure response of the tet promoter without repressor protein. Tet promoters' activity increased under 30 MPa

The result shows that the tet promoter activity under 30 MPa, without repressor protein, is about 3-fold stronger than the tet promoter activity under 0.1 MPa. Therefore, we confirmed that the tet promoter was induced under 30 MPa.

3. Touch display

Touch display (plan)

figure 3-1-b. Design of touch display

Touch display which we planned has many holes, and E. coli is in these holes.(figure 3-1 a)

As the first step of creating the touch display, we created Prototype touch display. figure 3-1 b)

figure 3-1-a. Plan to create touch display. We created two-holes display as the first step.

Prototype touch display

figure 3-2. Prototype touch display. The display has two kinds of holes and two kinds of covers.

We created a Prototype touch display made of acrylic glasses. This touch display has two kinds of holes(show figure 3-2). Each hole contains culture medium and E. coli is cultivated in these holes. One hole (A) can be pressurized, because the hole is covered with only a plastic tape. Water pressure conducts into the hole.

The other (B) is not pressurized, because the hole is covered with a block made of acrylic glasses. Water pressure doesn’t conduct into the hole.

figure 3-3. Touch display. We pour culture medium into the holes.

Result ~ E. coli in the touch display ~

After incubation, we observed the E. coli by a fluorescence microscope.

figure 3-5. Images from fluorescence microscope. E. coli on left image is more bright than right one

The touch display successfully regulated GFP expression in E. coli !

4. Low pressure-inducible promoter

Figure 4-1. - Pressure response of Plac

It is known that lac promoter is induced under 30 MPa (T. Sato et al., 1995). However, 30 MPa is too high to push with the fingers.

Therefore, we tried to develop low pressure-inducible promoter.

Methods

Figure 4-2 - PCR random mutagenesis

Figure 4-3 - Screening scheme with flow cytometry

We tried to develop a low pressure inducible promoter by PCR random mutagenesis to lac promoter. Then we screened an E. coli library, with flow cytometry, for promoters that are induced under low pressure. This scheme is based on the ability to separate bacteria, with a flow cytometer, in response to expression, or lack of expression, of a fluorescent marker.

Step 1 - Fluorescent bacteria without repressor protein were collected by a flow cytometer. This sorted pool contains bacteria bearing both constitutive and low pressure-inducible promoter.

Step 2 - This sorted pool contains bacteria bearing both constitutive and pressure-inducible promoter fusions. False positives are removed with repressor protein. All non-fluorescent bacteria are sorted.

Step 3 - A final passage. We sorted bacteria under pressure with repressor protein. Fluorescent bacteria are sorted. False negatives are removed. Therefore bacteria bearing promoter that are low pressure-inducible are isolated.

Results - Sequence and Characterization - We finished step 1. Fluorescent and non-fluorescent bacteria were sorted and we characterized their promoter.

figure 4-4. Dot plot of cell sorting

figure 4-5. Results of sequencing

We sorted fluorescent (A) and non-fluorescent bacteria (B) with a flow cytometer. Then, we analyze these base sequences.

A have mutations in LacI binding site or non-functional DNA.
B have mutations in CAP binding site, -35 or non-functional DNA.

Therefore, fluorescent bacteria have no mutation in CAP binding site, -35 or -10.

We have successfully demonstrated that it is possible to collect promoters desired functions by PCR random mutagenesis and screening with a flow cytometry. This results indicate that we can screen low pressure-inducible lac promoter mutant with this methods.

5. Write/Erase cycle

figure 5-1. Write/Erase cycle

While we can implement write-function, we want to implement additionally erase-function and memory-function. Erase-function enables us to erase the painted picture, and memory-function enables us to keep the picture after we stop induction. We call these functions "Write/Erase cycle". In order to implement Write/Erase cycle, we tried to construct genetic toggle switch.

Genetic toggle switch to implement Write/Erase cycle

figure 5-2. Genetic toggle switch

Write-function
1. P_lac is under 30 MPa pressure.
2. P_lac expresses CI and GFP.
3. CI represses P_L and decreases LacI expression.
4. Low LacI expression increases P_lac activity. ⇒ Bright!!
Erase-function
1. The heat activates P_L.
2. P_L expresses LacI.
3. LacI represses Plac.
4. Therefore, GFP expression decreases.

Mathematical model

Why did we use mathematical model?

figure 5-3. Left If P_L is not activated or is a bit, write-function is available. Right If P_L is activated too much, write-function is not available

As mentioned above, it is known that P_lac is activated 94-fold under 30 MPa while we didn't know the increase of P_L strength under 30 MPa. If P_L is activated too much and P_lac activity is weaker than P_L activity, we can't implement write-function. So, how much is the range of the increase of P_L activity under 30 MPa so as to become advantageous to that of P_lac? To know this range, we need to use mathematical model.

Classical toggle switch model

figure 5-4. Classical toggle switch model

figure 5-5. Transfer function of P_lac

Our mathematical model under 0.1 MPa is equal to a classical toggle switch model shown in figure 5-4. n_CI is the cooperativity of repression of the lambda promoter, n_LacI is the cooperativity of repression of the lac promoter, α_PL is the effective rate of LacI synthesis and α_Plac is the effective rate of CI synthesis. n_CI and n_LacI are called "Hill coefficient". α_PL and α_Plac depend on strength of promoter-RBS, and are adjustable. Identifying value of n_CI and n_LacI are required for the modeling. But we know n_CI = 3.0 (T. Tian et al., 2006). So, we measured fluorescence intensity various IPTG concentration to identify n_LacI.

Identification of n_LacI

By testing how LacI represses the lac promoter, Hill coefficient of lac promoter should be decided. In order to adjust effective concentration of LacI, IPTG was added.

GFP fluorescence intensity was enhanced in an IPTG-dose dependent manner. It indicates that the LacI repression was getting weaker by adding IPTG. The characteristics of the lac promoter were calculated by fitting Hill function to the plots shown in figure 5-5, Finally, we obtained n_LacI = 2.2.

Conditions for bistability

We calculated the range of α_PL in which a toggle switch model is bistability. Here, we set α_Plac = 3.0. The result is below.

Pressure model

figure 5-6. Pressure-inducible genetic toggle switch model

We proposed pressure-inducible genetic toggle switch model which has additional parameters to the classic toggle switch model. These parameters show the increase of activity by pressure (β_(p)Plac or β_(p)PL). Under atmospheric pressure (0.1 MPa), β_(0.1)Plac = 1.0 and β_(0.1)PL = 1.0. On the other hand, under 30 MPa, β_(30)Plac = 94 and β_(30)PL was not known.

In order to implement write-function, the transit of the system from bistability to monostable (P_lac is stronger than P_L) by 30 MPa pressure is required. Therefore, we calculated the range of β_(30)PL which satisfies the above conditions.

The feasibility of implementation of Write/Erase cycle

figure 5-7. The domain of appropriate parameters if α_Plac = 3.0

According to the result of simulation, we found that we can implement Write/Erase function even if P_L is activated 2.5-fold when α_PL = 7.8.

figure 5-8. Pressure-response ability of P_L

We identified β_(30)PL = 1.4 by our wet experiment under 30MPa pressure (figure 5-6). Therefore, we can implement Write/Erase cycle if we choose an appropriate P_L - RBS strength.

Retrieved from "http://2008.igem.org/Team:Tokyo_Tech"

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 !align="center" width=20% style="text-align:center"|[[Team:Tokyo_Tech|Main]]
+!align="center" width=20% style="text-align:center"|[[Team:Tokyo_Tech/Protocol|Protcol]]
 !align="center" width=20% style="text-align:center"|[[Team:Tokyo_Tech/Parts|Parts Submitted to the Registry]]
 !align="center" width=20% style="text-align:center"|[[Team:Tokyo_Tech/Team|Our Team]]
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-== <font size=5>'''1 Our project''' </font>==
+== <font size=5>'''1. Our project''' </font>==
 <html>
   <body>
+   <table width="100%" border="0" cellspacing="0" cellpadding="0" >
+<tr>
+<td class="a" width="2%">
+</td>
+<td class="a">
+<div>
+<b>
+<font size=4>Our project is creation of "<i style='mso-bidi-font-style:normal'>Coli</i> Touch"!!</font>
+</b>
+</div>
+</td>
+<td>
+<img src="https://static.igem.org/mediawiki/2008/f/f8/Tech_figure2.jpg" width="400" align="right">
+</td>
+</tr>
+   </table>
 <table width="100%" border="0" cellspacing="0" cellpadding="0" >
-        <tr>
+<tr>
-          <td class="a" width="2%">　</td>
-          <td class="a">
+<td>
-<div><b><font size=4>Our project is to create "<i style='mso-bidi-font-style:normal'>Coli</i> Touch"!!</font></b></div>
+<img src="https://static.igem.org/mediawiki/2008/5/57/Tech_Coli_Touch.jpg" width="300" align="left">
 </td>
-     <td>
-<img src="https://static.igem.org/mediawiki/2008/d/df/Tech_Design_of_%22coli_touch%22.jpg" width="400" align="right"></td>
-        </tr>
+<td class="a" width="2%">
-      </table>
+</td>
+<td class="a">
+<p><div><b>What is "<i style='mso-bidi-font-style:normal'>Coli</i> touch"?</b></div></p>
+<div>“<i style='mso-bidi-font-style:normal'>Coli</i> Touch” has a pressure sensitive display composed of an <i style='mso-bidi-font-style:normal'>E. coli</i> lawn. When you touch its display, touched section is colored.</div>
+<div>Next I'll tell you about “<i style='mso-bidi-font-style:normal'>Coli</i> Touch” work system. Display of “<i style='mso-bidi-font-style:normal'>Coli</i> Touch” contains many <i style='mso-bidi-font-style:normal'>E. coli</i>. When you touch this display, pressure applies to <i style='mso-bidi-font-style:normal'>E. coli</i> in this display. Pressure applied <i style='mso-bidi-font-style:normal'>E. coli</i> expresses GFP.
+</div><p><div><b>Why pressure?</b></div></p>
+<p><div>“<i style='mso-bidi-font-style:normal'>Coli</i> Touch” uses pressure as input. Why do we use pressure? Past input methods (small molecules, heat and light) are difficult to induce uniformly.
+Pressure can induce uniformly.
+</div></p>
+</td>
+</tr>
+   </table>
+</body>
+</html>
+== <font size=5>'''2. Pressure induction'''</font> ==
+<font size=3>'''Introduction'''</font>
+<html>
+<body>
 <table width="100%" border="0" cellspacing="0" cellpadding="0">
          <tr>
            <td class="a" width="2%">　</td>
            <td class="a">
- <p><div>What is "<i style='mso-bidi-font-style:normal'>Coli</i> touch"?</div></p>
+<div>Tet promoters is known as a sensitive ones to pressure. (T. Sato et al., 1995)</div><td>
- <div>“<i style='mso-bidi-font-style:normal'>Coli</i> Touch” has a pressure sensitive display composed of an <i style='mso-bidi-font-style:normal'>E. coli</i> lawn. When you touch its display, touched section is colored.
-<div>Next I'll tell you about “<i style='mso-bidi-font-style:normal'>Coli</i> Touch” work system.“<i style='mso-bidi-font-style:normal'>Coli</i> Touch” ‘s display has many <i style='mso-bidi-font-style:normal'>E. coli</i>. When you touch this display, pressure input travel to <i style='mso-bidi-font-style:normal'>E. coli</i> in this display.
-And pressure applied <i style='mso-bidi-font-style:normal'>E. coli</i> expresses GFP.
-</div>
-</div>
- <p><div>Why pressure?</div></p>
- <p><div>“<i style='mso-bidi-font-style:normal'>Coli</i> Touch” use pressure as input. Then why we use pressure input? Past input way (small molecule, heat and light) are difficult to induce uniformly.
-Pressurize can induce unifomly.
-It's prospect of technological application in confirmatory experiment.</div></p>
-</td>
-<td class="a" width="3%">　</td>
          </tr>
-      </table>
+</table>
 </body>
 </html>
-== <font size=5>'''2 Pressure induction'''</font> ==
 <font size=3>'''Construction'''</font>
-[[Image:Tech_Const1.jpg|400px|thumb|rigth|figure2-1. We constructed Ptet-GFP and promoter less-GFP for confirmatory experiment]]
+[[Image:Tech_Tet_figure1.jpg|400px|thumb|right|figure2-1. We constructed Ptet-GFP and promoter less-GFP.]]
 <html>
 <body>
@@ Line 67: / Line 97: @@
            <td class="a" width="3%">　</td>
            <td class="a">
-<div>For confirming pressure-response ability of pressure-inducible promoter, we experimented under 0.1 MPa and 30 MPa pressure. We chose TetR promoter(Ptet) as pressure-inducible promoter and we constructed two plasmids - one is Ptet-GFP on <a href="http://partsregistry.org/Part:BBa_I739201">pSB6</a>, the other is promoter less-GFP on <a href="http://partsregistry.org/Part:BBa_I739201">pSB6</a> as a negative control.</div></td>
+<div> We chose the tet promoter (Ptet) as a pressure-inducible promoter. We constructed two plasmids - one is Ptet-GFP on <a href="http://partsregistry.org/Part:BBa_I739201">pSB6</a>. The other is promoter less-GFP on <a href="http://partsregistry.org/Part:BBa_I739201">pSB6</a> as a negative control. We measured activity of tet promoters under 0.1 MPa and 30 MPa without repressor protein.</div></td>
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 </table>
@@ Line 81: / Line 111: @@
 {{clear}}
-<font size=3>'''Result ~ activity of Ptet ~ '''</font>
+<font size=3>'''Result -activity of tet promoters- '''</font>
-[[Image:Tech_disp_fig1.jpg|450px|thumb|right|figure2-2. ]]
+[[Image:Tech Pressure response of tet promoter1.jpg|450px|thumb|right|figure2-2. Pressure response of the tet promoter without repressor protein. Tet promoters' activity increased  under 30 MPa]]
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-<div>The result shows that Ptet activity under 30 MPa pressure is about 3 fold stronger than Ptet activity under 0.1 MPa pressure. Therefore, we confirmed that Ptet was induced under 30 MPa pressure.</div><td>
+<div>The result shows that the tet promoter activity under 30 MPa, without repressor protein, is about 3-fold stronger than the tet promoter activity under 0.1 MPa. Therefore, we confirmed that the tet promoter was induced under 30 MPa.</div><td>
          </tr>
 </table>
@@ Line 110: / Line 141: @@
 <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</p>
-== <font size=5>'''3 Touch display'''</font> ==
+== <font size=5>'''3. Touch display'''</font> ==
 <font size=3>'''Touch display''' (plan)</font>
-[[image:Tech_display1.JPG|center|500px|]]
+[[image:Tech Pressure device2.jpg|right|400px|thumb|figure 3-1-b. Design of touch display]]
-<font size=3>'''Basic touch display'''</font>
 <html>
- <body>
+<body>
-  <div class=Section1 style='layout-grid:18.0pt'>
+<table width="55%" border="0" cellspacing="0" cellpadding="0">
-<table width="100%" border="0" cellspacing="0" cellpadding="0">
-        <tr>
-          <td class="a" width="25%">　</td>
-          <td class="a" width="50%">
-<a href="https://static.igem.org/mediawiki/2008/e/e6/Tech_aqulyl2.JPG"><IMG src="https://static.igem.org/mediawiki/2008/e/e6/Tech_aqulyl2.JPG
-" align="middle" width="500"></a></td>
-<td class="a" width="40%"></td>
-        </tr>
-      </table>
-<table width="100%" border="0" cellspacing="0" cellpadding="0">
          <tr>
            <td class="a" width="2%">　</td>
            <td class="a">
-<p class=MsoNormal>We created a basic touch display made of acrylic glasses.This touch display has two kinds of holes.This device made of Acrylic glasses and has two holes (show figure). Each hole contains culture medium and E. coli is cultivated in those holes.One hole (A) can be pressurized, because the hole is covered with only a plastic tape, water pressure conducts into the hole.The other (B) is not pressurized, because the hole is covered with a block made of acrylic glass, water pressure doesn’t conduct into the hole.</font></p>
+<div>Touch display which we planned has many holes, and <i>E. coli</i> is in these holes.(figure 3-1 a)<div>
-<td class="a" width="3%">　</td>
+<div>As the first step of creating the touch display, we created Prototype touch display. figure 3-1 b)</div>
+</p></td>
          </tr>
 </table>
-<table width="100%" border="0" cellspacing="0" cellpadding="0">
-        <tr>
-          <td class="a" width="10%">　</td>
-          <td class="a" width="2%"><IMG src="https://static.igem.org/mediawiki/2008/a/a5/Tech_aqulyl1.jpg
-" align="middle"></td>
-        </tr>
-      </table>
 </body>
 </html>
+<p>&nbsp;</p><p>&nbsp;</p>
-<font size=3>'''How to press “touch display”'''</font>
+[[image:Tech Pressure device,jpg.jpg|left|400px|thumb|figure 3-1-a. Plan to create touch display. We created two-holes display as the first step.]]
+{{clear}}
+<font size=3>'''Prototype touch display'''</font>
+[[image:Tech_aqulyl2.JPG|right|450px|thumb|figure 3-2. Prototype touch display. The display has two kinds of holes and two kinds of covers. ]]
 <html>
 <body>
-<table width="100%" border="0" cellspacing="0" cellpadding="0">
+<table width="45%" border="0" cellspacing="0" cellpadding="0">
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            <td class="a">
-<p>Dilute culture medium by 1% by adding fresh medium and suitable antibiotic (ampicilin; 50㎍/ml). Next, infuse this culture medium into holes of the display with oxygen-saturated Fluorinert (25% volume of medium). Put this tube into pressure vessel filled with water (1). Next cap the vessel (2). And then apply pressure to the vessel by pressure device (4). Finally, start incubation at 37℃ immediately at each the vessel for 16h (5).</p></td>
+<div class=MsoNormal>We created a Prototype touch display made of acrylic glasses. This touch display has two kinds of holes(show figure 3-2). Each hole contains culture medium and <i>E. coli</i> is cultivated in these holes. One hole (A) can be pressurized, because the hole is covered with only a plastic tape. Water pressure conducts into the hole.</div>
+<div>The other (B) is not pressurized, because the hole is covered with a block made of acrylic glasses. Water pressure doesn’t conduct into the hole.</font></div>
+<td class="a" width="3%">　</td>
          </tr>
 </table>
-</body>
-</html>
-[[Image:Tech_pressure.jpg|center|800px]]
+</body></html>
+[[image:Tech_aqulyl1.jpg|left|thumb|800px|figure 3-3. Touch display. We pour culture medium into the holes.]]
+<p>&nbsp;</p>
+<p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</p>
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</p>
+<font size=3>'''Result ~ <i>E. coli</i> in the touch display ~'''</font>
-<font size=3>'''Result ~ E. coli in the touch display ~'''</font>
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-<p class=MsoNormal>After pressurized the display, we observed the E. coli in the touch display by a fluorescence microscope.</p></td>
+<p class=MsoNormal>After incubation, we observed the <i>E. coli</i> by a fluorescence microscope.</p></td>
 <td class="a" width="3%">　</td>
          </tr>
        </table>
+</body></html>
-<table width="100%" border="0" cellspacing="0" cellpadding="0">
+&nbsp;
-        <tr>
-          <td class="a" align="center">
+[[image:Tech_apulyl_pic1.jpg|thumb|center|600px|figure 3-5. Images from fluorescence microscope. <i>E. coli</i> on left image is more bright than right one]]
-<a href="https://static.igem.org/mediawiki/igem.org/0/08/Tech_apulyl_pic1.jpg"><IMG src="https://static.igem.org/mediawiki/igem.org/0/08/Tech_apulyl_pic1.jpg
+<html><body>
-" align="middle" width="600"></a></td>
-</table>
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            <td class="a">
-<div><b>The touch display successfully regulated GFP expression in E. coli.!</b></div></td>
+<div><b>The touch display successfully regulated GFP expression in <i>E. coli</i> !</b></div></td>
          </tr>
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@@ Line 201: / Line 228: @@
 </html>
-== <font size=5>'''4 Low pressure-inducible promoter'''</font> ==
+== <font size=5>'''4. Low pressure-inducible promoter'''</font> ==
-[[Image:tokyotech2008pressure response of plac.png|thumb|right|405px|box|Figure 4-1. - Pressure response of Plac]]
+[[Image:tokyotech2008Previous study - Pressure response of Plac.png|thumb|right|350px|box|Figure 4-1. - Pressure response of Plac]]
 <html>
   <body>
      <div>
-It is known that lac promoter is induced under 30 MPa (T. Sato et al., 1995). However, 30 MPa is too high to use as input.
+It is known that lac promoter is induced under 30 MPa (T. Sato et al., 1995). However, 30 MPa is too high to push with the fingers.
      </div>
      <div>
-Therefore, we are trying to develop low pressure inducible promoter.
+Therefore, we tried to develop low pressure-inducible promoter.
      </div>
   </body>
@@ Line 216: / Line 243: @@
-==== Method ====
-===== Principle =====
-LacI binds to lacI binding site and repress lac promoter.
-In addition, pressure of 30 MPa activated the lac promoter.
-We propose a hypothesis that 30 MPa induce a conformational change in lacI reprssure and change the affinity for lacI binding site weaker. Therefore, lacI can't repress lac promoter under 30 MPa.
-===== Strategy =====
-<gallery widths="400px" heights="280px" >
-Image:tokyotech08pcr mutagenesis.png|Figure 4-2 - RCP random mutagenesis
-Image:tokyotech08screening system.png|Figure 4-3 - Screening system with flow cytometer
-</gallery>
-We are trying to develop low pressure inducible promoter by PCR random mutagenesis to lac promoter in order to reduce affitity to lacI.
-And we are screening an E. coli library for promoters that are induced under low pressure using a fluorescence activated cell sorter (FACS).
-This scheme is based on the ability to separate bacteria with a FACS in response to expression, or lack of expression, of a fluorescent marker.
-*'''step 1''' - Fluorescent bacteria without repressor protein were collected by FACS. This sorted pool contains bacteria bearing both constitutive and low pressure-inducible promoter.
-*'''step 2''' - Constitutive promoter are removed with repressor protein and sorting all non-fluorescent bacteria.
-*'''step 3''' - A final passage through under pressure with repressor protain and sorting for fluorescent bacteria removes false negatives and enriches for bacteria bearing promoter that are low pressure inducible.
-==== Results ====
-We finished step 1.
-Fluorescent and non-fluorescent bacteria were sorted and we characterized their promoter.
-===== Sequence and Characterization =====
-<gallery widths="430px" heights="220px" >
-Image:Dev 3.JPG|Figure 4-4 - FACS
-Image:Tech dev 4.JPG|Figure 4-5 - Sequensing
-</gallery>
-We sorted fluorescent (A) and non-fluorescent bacteria (B) with a FACS.
-Then, we analyze these base sequences.
-*A have mutations in lacI binding site or non-functional DNA.
-*B have mutations in CAP binding site, -35 or non-functional DNA.
-==== Conclusion ====
-We have successfully demonstrated that it is possible to collect objective promoter by PCR random mutagenesis and screening with a FACS.
-So we clearly believe that we can screen low pressure inducible lac promoter mutant with this strategy.
-== <font size=5>'''5 Write/Erase cycle'''</font> ==
-[[Image:Write-Erase_cycle.png|thumb|350px|right|figure5-1. Write/Erase cycle]]
-While we can implement write-function, we want to implement additionally erase-function and memory-function. Erase-function enables us to erase the painted picture, and memory-function enables us to keep the picture when we stop induction. We call these functions "Write/Erase cycle".
-In order to implement Write/Erase cycle, we tried to construct genetic toggle switch.
-{{clear}}
-=== Genetic toggle switch ===
-[[Image:Toggle_genetic_circuit_model.png|thumb|500px|right|figure 5-2. Genetic toggle switch]]
-'''1. Write-function'''
-* 30 MPa pressure activates '''P<sub>lac</sub>'''
-* P<sub>lac</sub> expresses CI and GFP
-* CI represses P<sub>L</sub> and causes low LacI expression
-* Low LacI expression increases P<sub>lac</sub> activity ⇒ '''Bright!!'''
-'''2. Erase-function'''
-* The heat activates '''P<sub>L</sub>'''
-* P<sub>L</sub> expresses LacI
+<font size=3>'''Methods''' </font>
+<gallery widths="400px" heights="230px" >
+Image:Tokyotech2008PCR random mutagenesis.png|Figure 4-2 - PCR random mutagenesis
+Image:Tokyotech2008Screening scheme with flow cytometer.png|Figure 4-3 - Screening scheme with flow cytometry
+</gallery>
-* LacI represses Plac
+We tried to develop a low pressure inducible promoter by PCR random mutagenesis to lac promoter.
+Then we screened an ''E. coli'' library, with flow cytometry, for promoters that are induced under low pressure.
+This scheme is based on the ability to separate bacteria, with a flow cytometer, in response to expression, or lack of expression, of a fluorescent marker.
+*'''Step 1''' - Fluorescent bacteria without repressor protein were collected by a flow cytometer. This sorted pool contains bacteria bearing both constitutive and low pressure-inducible promoter.
-* Therefore, GFP expression decreases
+*'''Step 2''' - This sorted pool contains bacteria bearing both constitutive and pressure-inducible promoter fusions. False positives are removed with repressor protein. All non-fluorescent bacteria are sorted.
+*'''Step 3''' - A final passage. We sorted bacteria under pressure with repressor protein. Fluorescent bacteria are sorted. False negatives are removed. Therefore bacteria bearing promoter that are low pressure-inducible are isolated.
+<font size=3>'''Results - Sequence and Characterization -''' </font>
+We finished step 1.
+Fluorescent and non-fluorescent bacteria were sorted and we characterized their promoter.
-=== Mathematical model ===
+<gallery widths="430px" heights="240px" >
-<!--<gallery widths="200px" heights="150px" perrow="1" align="right" caption="Differential equation models">
+Image:Tokyotech2008Dot plot of cell sorting .png|figure 4-4. Dot plot of cell sorting
-Image:Room_pressure_model.png|figure 5-3. Atmospheric pressure model. It is known that n<sub>P<sub>L</sub></sub> = 3.0 from earlier studies
+Image:Tokyotech2008Results of sequencing .png|figure 4-5. Results of sequencing
-Image:30MPa_pressure_model2.png|figure 5-4. 30 MPa pressure model
 </gallery>
+We sorted fluorescent (A) and non-fluorescent bacteria (B) with a flow cytometer.
+Then, we analyze these base sequences.
+*A have mutations in LacI binding site or non-functional DNA.
+*B have mutations in CAP binding site, -35 or non-functional DNA.
+Therefore, fluorescent bacteria have  no mutation in CAP binding site, -35 or -10.
-We used two differential equation models and we call them 'atmospheric pressure model' and '30MPa pressure model'.
+We have successfully demonstrated that it is possible to collect promoters desired functions by PCR random mutagenesis and screening with a flow cytometry.
-In order to implement rewritable function, we decided to use the difference of nullclines of each system so as to satisfy the following condition
+This results indicate that we can screen low pressure-inducible lac promoter mutant with this methods.
-* Converge to high CI concentration state under 30 MPa pressure (writable)
+== <font size=5>'''5. Write/Erase cycle'''</font> ==
-* Bistable under atmospheric pressure (erasable)
+[[Image:Write-Erase_cycle.png|thumb|400px|right|figure 5-1. Write/Erase cycle]]
+While we can implement write-function, we want to implement additionally erase-function and memory-function. Erase-function enables us to erase the painted picture, and memory-function enables us to keep the picture after we stop induction. We call these functions "Write/Erase cycle".
+In order to implement Write/Erase cycle, we tried to construct genetic toggle switch.
+{{clear}}
-[[Image:Desirable_condition.png|700px|thumb|left|figure 5-5. Desirable condition. left:under atmospheric pressure (nullclines of figure 5-2), right:under 30MPa pressure (nullclines of figure 5-4).]]-->
+=== Genetic toggle switch to implement Write/Erase cycle ===
+[[Image:Toggle_genetic_circuit_model.png|thumb|480px|right|figure 5-2. Genetic toggle switch]]
-==== Why did we use mathematical model ? ====
+# '''Write-function'''
-[[Image:Conditions_for_Write-function.png|thumb|500px|right| figure 5-3. Left If P<sub>L</sub> is not activated or is a bit, write-function is available. Right If P<sub>L</sub>  is activated too much, write-function is not unavailable]]
+## '''P<sub>lac</sub>''' is under 30 MPa pressure.
+## P<sub>lac</sub> expresses CI and GFP.
+## CI represses P<sub>L</sub> and decreases LacI expression.
+## Low LacI expression increases P<sub>lac</sub> activity. ⇒ '''Bright!!'''
+# '''Erase-function'''
+## The heat activates '''P<sub>L</sub>'''.
+## P<sub>L</sub> expresses LacI.
+## LacI represses Plac.
+## Therefore, GFP expression decreases.
+{{clear}}
+=== Mathematical model ===
+==== Why did we use mathematical model? ====
+[[Image:Conditions_for_Write-function.png|thumb|500px|right| figure 5-3. Left If P<sub>L</sub> is not activated or is a bit, write-function is available. Right If P<sub>L</sub>  is activated too much, write-function is not available]]
-As mentioned above, it is known that P<sub>lac</sub> is activated '''94.0'''-fold under 30 MPa while we don't know the increase of P<sub>L</sub> strength under 30 MPa. If P<sub>L</sub> is activated too much, P<sub>lac</sub> activity may be weaker than P<sub>L</sub> activity and we can't implement write-function. So, how much is the range of the increase of P<sub>L</sub> activity under 30 MPa so as to become advantageous to P<sub>lac</sub>? To know this range, we need to use mathematical model.
+As mentioned above, it is known that P<sub>lac</sub> is activated '''94'''-fold under 30 MPa while we didn't know the increase of P<sub>L</sub> strength under 30 MPa. If P<sub>L</sub> is activated too much and P<sub>lac</sub> activity is weaker than P<sub>L</sub> activity, we can't implement write-function. So, how much is the range of the increase of P<sub>L</sub> activity under 30 MPa so as to become advantageous to that of P<sub>lac</sub>? To know this range, we need to use mathematical model.
@@ Line 314: / Line 329: @@
 <gallery widths = "300px" heights="215px" perrow = "1" align = "right">
-Image:Classical_toggle_switch_model.png| figure. 5-4 Classical toggle switch model
+Image:Classical_toggle_switch_model.png| figure 5-4. Classical toggle switch model
-Image:Transfer_Function.png| figure 5-5. Hill function
+Image:Transfer_Function.png| figure 5-5. Transfer function of P<sub>lac</sub>
 </gallery>
-Our mathematical model under atmospheric pressure is equal to a classical toggle switch model shown in figure 5-4. Where n<sub>CI</sub> and n<sub>LacI</sub> are hill coefficients, and &alpha;<sub>PL</sub> and &alpha;<sub>Plac</sub> are strength of promoter-RBS which are adjustable. We need to identify value of n<sub>CI</sub> and n<sub>LacI</sub> respectively. But we fortunatly know '''n<sub>CI</sub> = 3.0''' (T. Tian et al., 2006). So, we experimented IPTG assay aimed to identify n<sub>LacI</sub>.
+Our mathematical model under 0.1 MPa is equal to a classical toggle switch model shown in figure 5-4. n<sub>CI</sub> is the cooperativity of repression of
+the lambda promoter, n<sub>LacI</sub> is the cooperativity of repression of the lac promoter, &alpha;<sub>PL</sub> is the effective rate of LacI synthesis
+and &alpha;<sub>Plac</sub> is the effective rate of CI synthesis.
+n<sub>CI</sub> and n<sub>LacI</sub> are called "Hill coefficient".
+&alpha;<sub>PL</sub> and &alpha;<sub>Plac</sub> depend on strength of promoter-RBS, and are adjustable.
+Identifying value of n<sub>CI</sub> and n<sub>LacI</sub> are required for the modeling. But we know '''n<sub>CI</sub> = 3.0''' (T. Tian et al., 2006). So, we measured fluorescence intensity various IPTG concentration to identify n<sub>LacI</sub>.
-''' IPTG assay '''
+''' Identification of n<sub>LacI</sub> '''
 By testing how LacI represses the lac promoter, Hill coefficient of lac promoter should be decided. In order to adjust effective concentration of LacI, IPTG was added.
-GFP fluorescence intensity was enhanced in an IPTG-dose dependent manner. It indicates that the LacI repression was getting weaker by adding IPTG. We formulated Hill function fitting the data shown in figure 5-5, and the characteristics of the lac promoter expressed in Hill function was determined. Finally, we obtained '''n<sub>LacI</sub> = 2.2'''.
+GFP fluorescence intensity was enhanced in an IPTG-dose dependent manner. It indicates that the LacI repression was getting weaker by adding IPTG. The characteristics of the lac promoter were calculated by fitting Hill function to the plots shown in figure 5-5, Finally, we obtained '''n<sub>LacI</sub> = 2.2'''.
 ''' Conditions for bistability'''
-We simulated the range of &alpha;<sub>PL</sub> in which a toggle switch model is bistability. Here, we set &alpha;<sub>Plac</sub> = 3.0. The result is below.
+We calculated the range of &alpha;<sub>PL</sub> in which a toggle switch model is bistability. Here, we set &alpha;<sub>Plac</sub> = 3.0. The result is below.
@@ Line 338: / Line 358: @@
 ==== Pressure model ====
-[[Image:Pressure-inducible_toggle_switch_model.png|thumb|300px|right| firuger 5-6. Pressure model]]
+[[Image:Pressure-inducible_toggle_switch_model.png|thumb|300px|right| figure 5-6. Pressure-inducible genetic toggle switch model ]]
-We proposed pressure model which has additional parameters to the classic toggle switch model. These parameters are the increase of activity by pressure (&beta;<sub>(p)Plac</sub> or &beta;<sub>(p)PL</sub>). Under atmospheric pressure (0.1 MPa), &beta;<sub>(0.1)Plac</sub> = 1.0 and &beta;<sub>(0.1)PL</sub> = 1.0. On the other hand, under 30 MPa, &beta;<sub>(30)Plac</sub> = 94 and &beta;<sub>(30)PL</sub> is not known.
+We proposed pressure-inducible genetic toggle switch model which has additional parameters to the classic toggle switch model. These parameters show the increase of activity by pressure (&beta;<sub>(p)Plac</sub> or &beta;<sub>(p)PL</sub>). Under atmospheric pressure (0.1 MPa), &beta;<sub>(0.1)Plac</sub> = 1.0 and &beta;<sub>(0.1)PL</sub> = 1.0. On the other hand, under 30 MPa, &beta;<sub>(30)Plac</sub> = 94 and &beta;<sub>(30)PL</sub> was not known.
-In order to implement write-function, we need the transit of the system from bistability to monostable (P<sub>lac</sub> is stronger than P<sub>L</sub>) by 30 MPa pressure. Therefore, we calculated the range of &beta;<sub>(30)PL</sub> which satisfies the above condition.
+In order to implement write-function, the transit of the system from bistability to monostable (P<sub>lac</sub> is stronger than P<sub>L</sub>) by 30 MPa pressure is required. Therefore, we calculated the range of &beta;<sub>(30)PL</sub> which satisfies the above conditions.
 {{clear}}
@@ Line 348: / Line 368: @@
 [[Image:Desirable_condition_94.png|400px|thumb|right|figure 5-7. The domain of appropriate parameters if &alpha;<sub>Plac</sub> = 3.0]]
-According to the result of simulation, we found that we can implement Write/Erase function even if P<sub>L</sub> is activated 7-fold when &alpha;<sub>PL</sub> = 3.0.
+According to the result of simulation, we found that we can implement Write/Erase function even if P<sub>L</sub> is activated 2.5-fold when &alpha;<sub>PL</sub> = 7.8.
 [[Image:PL_pressure_response.png‎|200px|none|thumb|left|figure 5-8. Pressure-response ability of P<sub>L</sub>]]
-We identified '''&beta;<sub>(30)PL</sub> = 1.4''' by our experiment under 30MPa pressure(figure 5-6). Therefore, we can implement Write/Erase cycle if we choose an appropriate P<sub>L</sub> - RBS strength.
+We identified '''&beta;<sub>(30)PL</sub> = 1.4''' by our wet experiment under 30MPa pressure (figure 5-6). Therefore, we can implement Write/Erase cycle if we choose an appropriate P<sub>L</sub> - RBS strength.
-== <font size=5>'''Our Team'''</font> ==
-<div align='right'>
-[[Team:Tokyo_Tech/Home|see more]]
-</div>
-== <font size=5>'''Acknowledgements'''</font> ==
-<div align='right'>
-[[Team:Tokyo_Tech/Acknowledgements|see more]]
-</div>