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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-          <td class="a">
-<p class=MsoNormal>We try to construct a bacterial ‘touch panel’ which is colored by the pressure.</p></td>
-<td class="a" width="3%">　</td>
-        </tr>
-      </table>
+<td class="a" width="2%">
+</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="0%">　</td>
+           <td class="a" width="2%">　</td>
            <td class="a">
-<p class=MsoNormal style='text-indent:10.5pt;mso-char-indent-count:1.0'><span
+<div>Tet promoters is known as a sensitive ones to pressure. (T. Sato et al., 1995)</div><td>
-lang=EN-US>We name it <i>Coli touch.</i></span></p></td>
-<td class="a" width="3%">　</td>
          </tr>
-      </table>
+</table>
+</body>
+</html>
-<table width="100%" border="0" cellspacing="0" cellpadding="0">
+<font size=3>'''Construction'''</font>
+[[Image:Tech_Tet_figure1.jpg|400px|thumb|right|figure2-1. We constructed Ptet-GFP and promoter less-GFP.]]
+<html>
+<body>
+<table width="50%" border="0" cellspacing="0" cellpadding="0">
          <tr>
            <td class="a" width="3%">　</td>
            <td class="a">
-<p class=MsoNormal><span
+<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>
-lang=EN-US>Until iGEM2007, input style was only heat, small molecule and light. These input
+          </tr>
-style have problems. For example, Heat input occur thermal gradient, small
+</table>
-molecule input occur concentration gradient, light input depend on location of a
+</body>
-container. So, we thought input style except these. That is “press”. Press input
+</html>
-has an advantage that input signal travel uniformly in a container.</span></p></td>
-<td class="a" width="3%">　</td>
-        </tr>
-      </table>
+<html>
+ <body>
-<! ---------------------------------------------------------------------------------------------------------->
+</body>
-<p class=MsoNormal><b><u><font size=4>Mechanism</font></u></b></p>
+</html>
-<table width="100%" border="0" cellspacing="0" cellpadding="0">
+{{clear}}
+<font size=3>'''Result -activity of tet promoters- '''</font>
+[[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]]
+<html>
+<body>
+<table width="50%" border="0" cellspacing="0" cellpadding="0">
          <tr>
-           <td class="a" width="0%">　</td>
+           <td class="a" width="2%">　</td>
            <td class="a">
-<p class=MsoNormal style='text-indent:5.25pt;mso-char-indent-count:.5'><span
+<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>
-lang=EN-US>We construct genetic circuits that can respond to pressure signal. Lac promoter was known as a pressure inducible promoter. Lac promoter is repressed by LacI protein. The repression is released by adding IPTG (Isopropyl β-D-1-thiogalactopyranoside). Besides IPTG induction, pressure induction is possible. Under high pressure, the affinity of lac protein for lac operon decreases by transition of Lac protein from tetramer to dimer. Through this mechanism, Lac promoter is induced by pressure. </span></p></td>
-<td class="a" width="3%">　</td>
          </tr>
-      </table>
+</table>
+</body>
+</html>
-<IMG src="https://static.igem.org/mediawiki/2008/b/bd/Test.jpg" alt="[サンプルの絵]" width="300">
-<! ---------------------------------------------------------------------------------------------------------->
-<! strategy>
-<p class=MsoNormal><b><u><font size=4>Strategy</font></u></b></p>
-<table width="100%" border="0" cellspacing="0" cellpadding="0">
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</p>
-        <tr>
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</p>
-          <td class="a" width="0%">　</td>
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</p>
-          <td class="a">
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</p>
-<p class=MsoNormal>We thought two projects, basic project and application project. Basic project consist of two categories. One is construction of pressure responsive genetic (1). The other is creating basic device for a touch display (2). Application project also consist of two categories. One is development of low pressure inducible promoter (3). The other is construction of pressure responsive toggle switch (4). In the future, we want to create the touch display made of E. coli.  </p></td>
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</p>
-          <td class="a" width="3%">　</td>
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</p>
-        </tr>
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</p>
-      </table>
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&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>
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</p>
-<IMG src="https://static.igem.org/mediawiki/2008/c/cf/Test2.jpg" alt="[サンプルの絵]" width="700">
+== <font size=5>'''3. Touch display'''</font> ==
-<table width="100%" border="0" cellspacing="0" cellpadding="0">
+<font size=3>'''Touch display''' (plan)</font>
-        <tr>
-          <td class="a" width="0%">　</td>
-          <td class="a">
-<p class=MsoNormal>We used these equipments (show figure) for pressure experiments. </p></td>
-          <td class="a" width="3%">　</td>
-        </tr>
-      </table>
-<IMG src="https://static.igem.org/mediawiki/2008/a/a3/Tech_kaatu.jpg" align="middle" width="500">
+[[image:Tech Pressure device2.jpg|right|400px|thumb|figure 3-1-b. Design of touch display]]
-<! ---------------------------------------------------------------------------------------------------------->
+<html>
-<p class=MsoNormal><b><u><font size=4>1.Construction</font></u></b></p>
+<body>
+<table width="55%" border="0" cellspacing="0" cellpadding="0">
-<table width="100%" border="0" cellspacing="0" cellpadding="0">
          <tr>
-           <td class="a" width="0%">　</td>
+           <td class="a" width="2%">　</td>
            <td class="a">
-<p class=MsoNormal>We construct these plasmids (show figure). </p></td>
+<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>
+</body>
+</html>
+<p>&nbsp;</p><p>&nbsp;</p>
-<IMG src="https://static.igem.org/mediawiki/2008/6/63/Tech_constraction.jpg" align="middle">
+[[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="45%" border="0" cellspacing="0" cellpadding="0">
-<table width="100%" border="0" cellspacing="0" cellpadding="0">
          <tr>
-           <td class="a" width="0%">　</td>
+           <td class="a" width="2%">　</td>
            <td class="a">
-<p class=MsoNormal>Each part is constructed on low-copy plasmid “pSB6”. </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>
-          <td class="a" width="3%">　</td>
+<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>
+</table>
-<! ---------------------------------------------------------------------------------------------------------->
-<p class=MsoNormal><b><u><font size=4>2. Basic device</font></u></b></p>
+</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>
+<html>
+<body>
 <table width="100%" border="0" cellspacing="0" cellpadding="0">
          <tr>
-           <td class="a" width="0%">　</td>
+           <td class="a" width="2%">　</td>
            <td class="a">
-<p class=MsoNormal>We created pressurizing device for first step of creating touch display. This device has two holes (show figure). Each hole contains tubes and water. Inside tubes E. coli is cultivated. Pressure can travel to inside tubes. One hole (B) is covered with a block made of an acrylic glass. (show figure) Therefore the hole is not pressurized by water. The other (A) is covered with a plastic tape (show figure). Therefore the hole is pressurized.                        </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>
+<td class="a" width="3%">　</td>
          </tr>
        </table>
+</body></html>
-<IMG src="https://static.igem.org/mediawiki/2008/9/9d/Tech_youki.jpg" align="middle" width="500" >
+&nbsp;
-<! --------------------------------------------------------------------------------------------------------->
+[[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]]
-<p class=MsoNormal><b><u><font size=4>3. Development of low pressure inducible promoter </font></u></b></p>
+<html><body>
 <table width="100%" border="0" cellspacing="0" cellpadding="0">
          <tr>
-           <td class="a" width="0%">　</td>
+           <td class="a" width="2%">　</td>
            <td class="a">
-<p class=MsoNormal>In the stage of basic project, very high pressure (30MPa) is required for pressure induction of lac promoter. But lower pressure response is needed for creating touch display. Therefore we develop lower pressure inducible promoter. We performed random mutagenesis to lac promoter and tried to screen promoters with FACS.</p></td>
+<div><b>The touch display successfully regulated GFP expression in <i>E. coli</i> !</b></div></td>
-          <td class="a" width="3%">　</td>
          </tr>
-      </table>
+</table>
-<IMG src="https://static.igem.org/mediawiki/2008/3/3e/Tech_dev1.jpg" align="middle" width="500" >
-<! --------------------------------------------------------------------------------------------------------->
+<a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</a>
-<p class=MsoNormal><b><u><font size=4>4.Genetic toggle switch to implement rewritable function </font></u></b></p>
+<a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</a>
+  </div>
+ </body>
+</html>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+== <font size=5>'''4. Low pressure-inducible promoter'''</font> ==
+[[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 push with the fingers.
+    </div>
+    <div>
+Therefore, we tried to develop low pressure-inducible promoter.
+    </div>
+ </body>
+</html>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><span style='font-family:"ＭＳ 明朝","serif";mso-ascii-font-family:
-Century;mso-hansi-font-family:Century'>・</span><span lang=EN-US> genetic toggle
-switch</span></p>
-<p class=MsoNormal><span lang=EN-US><span
-style='mso-spacerun:yes'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>- Writable
-function</span></p>
-<p class=MsoNormal><span lang=EN-US>P<sub>lac</sub> is induced under 30 MPa
-pressure</span></p>
-<p class=MsoNormal><span lang=EN-US>(P<sub>L </sub>is not induced by 30 MPa
-pressure)</span></p>
-<p class=MsoNormal><span lang=EN-US>Express GFP, CI, and CI represses P<sub>L </sub></span></p>
-<p class=MsoNormal><span lang=EN-US>→ bright !! </span></p>
-<p class=MsoNormal><span lang=EN-US>- Erasable function</span></p>
-<p class=MsoNormal><span lang=EN-US>P<sub>L</sub> is induced by heat</span></p>
-<p class=MsoNormal><span lang=EN-US>Express LacI, P<sub>lac </sub>represses GFP
-</span></p>
-<p class=MsoNormal><span lang=EN-US>→ go out little by little…</span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><span lang=EN-US>Future work</span></p>
-<p class=MsoNormal><span lang=EN-US><span
-style='mso-spacerun:yes'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>- Identify
-pressure model parameter from experimental data</span></p>
-<p class=MsoNormal><span lang=EN-US><span
-style='mso-spacerun:yes'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>- Simulate
-pressure induction by pressure model and estimate </span></p>
-<p class=MsoNormal><span lang=EN-US><span
+<font size=3>'''Methods''' </font>
-style='mso-spacerun:yes'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+<gallery widths="400px" heights="230px" >
-</span>promoter-RBS strength to implement rewritable function</span></p>
+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>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+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.
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+*'''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.
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+*'''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.
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<font size=3>'''Results - Sequence and Characterization -''' </font>
+We finished step 1.
+Fluorescent and non-fluorescent bacteria were sorted and we characterized their promoter.
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<gallery widths="430px" heights="240px" >
+Image:Tokyotech2008Dot plot of cell sorting .png|figure 4-4. Dot plot of cell sorting
+Image:Tokyotech2008Results of sequencing .png|figure 4-5. Results of sequencing
+</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.
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+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.
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+== <font size=5>'''5. Write/Erase cycle'''</font> ==
+[[Image:Write-Erase_cycle.png|thumb|400px|right|figure 5-1. Write/Erase cycle]]
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+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}}
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+=== Genetic toggle switch to implement Write/Erase cycle ===
+[[Image:Toggle_genetic_circuit_model.png|thumb|480px|right|figure 5-2. Genetic toggle switch]]
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+# '''Write-function'''
+## '''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.
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+{{clear}}
-<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US>Mathematical</span></b><b
+=== Mathematical model ===
-style='mso-bidi-font-weight:normal'><span style='font-family:"ＭＳ 明朝","serif";
+==== Why did we use mathematical model? ====
-mso-ascii-font-family:Century;mso-hansi-font-family:Century'>　</span><span
+[[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]]
-lang=EN-US>model <o:p></o:p></span></b></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+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.
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+{{clear}}
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+==== Classical toggle switch model ====
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+<gallery widths = "300px" heights="215px" perrow = "1" align = "right">
+Image:Classical_toggle_switch_model.png| figure 5-4. Classical toggle switch model
+Image:Transfer_Function.png| figure 5-5. Transfer function of P<sub>lac</sub>
+</gallery>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+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>.
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+''' Identification of n<sub>LacI</sub> '''
-<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
+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.
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+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'''.
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+''' Conditions for bistability'''
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+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.
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+[[Image:Conditions_for_bistability.png‎|300px|figure 5-5.'''P<sub>L</sub> - RBS strength range from 2.4 to 7.8''']]
+{{clear}}
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+==== Pressure model ====
+[[Image:Pressure-inducible_toggle_switch_model.png|thumb|300px|right| 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 (&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.
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+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.
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+{{clear}}
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+==== The feasibility of implementation of Write/Erase cycle====
+[[Image:Desirable_condition_94.png|400px|thumb|right|figure 5-7. The domain of appropriate parameters if &alpha;<sub>Plac</sub> = 3.0]]
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+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.
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+[[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 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.
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