User:Lytao/2

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==<h1>Experiment results of the Basic Design <br/><br/>and Discussion</h1>==
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<html ><a href= "#Verification of ΔcheZ  strain"><u>1.Verification of ΔcheZ  strain<html></u></a> <br/>
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<html><a  href= "#Verification of the function of the constructed Aptamer-cheZ part">&nbsp;<u>2.Verification of the function of the constructed Aptamer-cheZ part</u></a></html><br/>
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<html><a  href= "#Verification of the original Toggle-switch from PKU">&nbsp;<u>3.Verification of the original Toggle-switch from PKU<html></u> </a> <br/>
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<html><a  href= "#Verification of the modified Toggle-switch">&nbsp;<u>4.Verification of the modified Toggle-switch</u> </a> </html><br/>
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<html><a  href= "#Test of Incorporated Aptamer-cheZ part into one side of the original Toggle-switch">&nbsp;<u>5.Test of Incorporated Aptamer-cheZ part into one side of the original Toggle-switch<html></u> </a><br/>
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<html><a  href= "#Test of Incorporated Aptamer-cheZ part into one side of the modified Toggle-switch">&nbsp;<u>6.Test of Incorporated Aptamer-cheZ part into one side of the modified Toggle-switch</u> </a> </html><br/>
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<h1>Modules</h1>
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<html><a name="Verification of ΔcheZ strain" id=""></a></html>
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<html ><a href= "#Riboswitch"><u>1.Riboswitch<html></u></a> <br/>
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<html><a href= "#Toggle-switch">&nbsp;<u>2.Toggle-switch</u></a></html><br/>
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<html><a  href= "#Quorum">&nbsp;<u>3.Quorum Sensing<html></u> </a> <br/>
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<html><a  href= "#Artificial">&nbsp;<u>4.Artificial Innate Immunity System<html></u> </a> <br/>
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<html><a name="Riboswitch" id=""></a></html>
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==Riboswitch==
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<div class="center"><div class="thumb tnone"><div class="thumbinner" ><img alt="Riboswitch.jpg" src="/wiki/images/3/36/Riboswitch.jpg" width="554" height="162" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:Riboswitch.jpg" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png"  alt="" /></a></div></div>Figure 1: Model for how the theophylline-sensitive synthetic riboswitch controls the translation of the CheZ protein.</div></div></div><br />
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==Verification of ΔcheZ  strain==
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<p>&nbsp;&nbsp;&nbsp;&nbsp;The size of colonies of ''E.coli'' strain RP1616 was much smaller than that ''E.coli'' steain RP437 under the same circumstance and after same period of incubation time(about 10h), and the result is shown in Figure1. In colony PCR using the primers of cheZ gene following , RP437 absolutely has much more outcomes than RP1616 and we conclude that RP1616 is actually a ΔcheZ strain.</p>
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[[File:sst().jpg|center|350px| Figure1.]]
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<p align=center class="ppp">Figure1.The result of colony PCR(From left to right, the first lane is the marker, the third and the forth lane is the PCR outcome of strain RP437 and strain RP1616, the sixth and the seventh lane is the PCR outcome of strain RP437 and strain RP1616.)</p>
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<html><a name="Verification of the function of the constructed Aptamer-cheZ part" id=""></a></html>
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==Verification of the function of the constructed Aptamer-cheZ part<html><a  href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Aptamer-cheZ"><font size="4"><u>(protocol1)</u></font></a><a  href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Dose-Dependent"><font size="4"><u>(protocol2)</u></font></a> </html>==
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In the absence of theophylline (left),the mRNA adopts a conformation in which the ribosome binding site is paired and translation of CheZ is inhibited. In the absence of CheZ, the protein CheY-P remains phosphorylated and the cells tumble in place. In the presence of theophylline (shown in red), the mRNA can adopt a conformation in which the ribosome binding site is exposed and CheZ is expressed, thus allowing the cells to run and tumble. (Images from: Shana Topp, Justin P. Gallivan (2006) Guiding Bacteria with Small Molecules and RNA. J.Am.Chem.Soc, 129,6870-6811)
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<p>&nbsp;&nbsp;&nbsp;&nbsp;From the results shown in Figure2. We can be sure that the Aptamer-cheZ part actually works effectively, especially on 0.3% Semi-solid medium.</p>
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<html><a name="Toggle-switch" id=""></a></html>
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[[File:X().jpg|center|650px| ]]
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<p align=center class="ppp">Figure2. The growing state of the reprogrammed bacteria with Aptamer-cheZ part(Left:0.3%agar with 0mM Theophylline, Right:0.3%agar with 0.25mM Theophylline)</p>
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<html><a name="Verification of the original Toggle-switch from PKU" id=""></a></html>
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==Toggle-switch==
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==Verification of the original Toggle-switch from PKU<html><a href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Toggle Switch"><font size="4"><u>(protocol)</u></font></a> </html>==
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[[File:Res14.jpg|300px|center ]]
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<div class="center" ><div class="thumb tnone" style="width:554px;"><div class="thumbinner" style="width:554px;"><a href="/File:Toggle-switch.jpg" class="image"><img alt="Toggle-switch.jpg" src="/wiki/images/7/79/Toggle-switch.jpg" width="554" height="62"></a> <div class="thumbcaption"><div class="magnify"><a href="/File:Toggle-switch.jpg" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Figure 2: Toggle switches incorporate three mechanisms: positive feedback, double-negative feedback, and the repressor binding cooperatively and two different kinds of bacteria differentiate out of homogeneous conditions. (Images from: Chunbo Lou, Xili Liu, Ming Ni.etc (2010) Synthesizing a novel genetic sequential logic circuit: a push-on push-off switch. Molecular Systems Biology, 6:350)</div></div></div></div>
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<p align=center class="ppp">Figure3.A conlony of the bacteria with the original Toggle Switch exhibit two different states(4X Objective) </p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;By calculating with the help of the fluorescence microscope, the ratio between the numbers of colonies with RFP and colonies with GFP ≈ 8:25. It means the original Toggle Switch is not fit for our purpose, so we modify the original by luxPR-cI device.</p>
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<html><a name="Verification of the modified Toggle-switch" id=""></a></html>
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==Verification of the modified Toggle-switch  <html><a href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Toggle Switch"><font size="4"><u>(protocol)</u></font></a> </html>==
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<div class="center" ><div class="thumb tnone" style="width:502px;"><div class="thumbinner" style="width:502px;"><a href="/File:Toggle-switch1.jpg" class="image"><img alt="Toggle-switch1.jpg" src="/wiki/images/thumb/a/ab/Toggle-switch1.jpg/500px-Toggle-switch1.jpg" width="500" height="155" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:Toggle-switch1.jpg" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><font size="2" align="center">Figure 3: Constructions to modulate the ratio of toggle switches. The above bar represents pSB3T5, a low to medium copy BioBrick standard vector, where LuxpR (BBa_R0062) controls the expression of cI repressor which would influence the ratio of toggle switch. (Images by USTC-IGEM with TinkerCell)</div></div></div></div>
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</font><font size="3">
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[[File:Rs14.jpg|550px|center ]]
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<p align=center class="ppp">Figure4. The conlonies of the bacteria with the modified Toggle Switch exhibit two different states or just one state(4X Objective)</p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;By calculating with the help of the fluorescence microscope, the ratio between the numbers of colonies with RFP and colonies with GFP ≈ 6:1. It is useful to our project design.</p>
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<html><a name="Test of Incorporated Aptamer-cheZ part into one side of the original Toggle-switch" id=""></a></html>
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<html><a  name="Quorum" id=""></a></html>
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==Test of Incorporated Aptamer-cheZ part into one side of the original Toggle-switch  <html><a  href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Semi-Solid"><font size="4"><u>(protocol1)</u></font></a> <a  href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Toggle"><font size="4"><u>(protocol2)</u></font></a></html>==
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[[File:Resul.jpg|650px|center ]]
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<p align=center class="ppp">Figure5.The growing state of the reprogrammed bacteria with original Toggle-switch-Aptamer-cheZ Device(Red circle:the range of motion, Blue Triangle:the gradient of thepphylline)</p>
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[[File:Resddul.jpg|300px|center ]]
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<p align=center class="ppp">Figure6.The fluorescent Photo(4X Objective) of the colony shown in Figure5 Left.</p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;According to the results above, by using this device we can not make the reprogrammed bacteria be divided into two different states effectively, and because of the bias to the green fluorescence and moving towards the high concentration of the theophylline, the bacteria trending to stay are very rare.</p>
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<html><a name="Test of Incorporated Aptamer-cheZ part into one side of the modified Toggle-switch" id=""></a></html>
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==Quorum Sensing==
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==Test of Incorporated Aptamer-cheZ part into one side of the modified Toggle-switch <html><a  href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Toggle Switch-Aptamer-cheZ"><font size="4"><u>(protocol)</u></font></a> </html>==
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[[File:Re.jpg|center|480px ]]
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<div class="center"><div class="thumb tnone" style="width:554px;"><div class="thumbinner" style="width:554px;"><a href="/File:Quorum_sensing.jpg" class="image"><img alt="Quorum sensing.jpg" src="/wiki/images/5/5c/Quorum_sensing.jpg" width="554" height="269" /></a> <div class="thumbcaption"><div class="magnify"><a href="/File:Quorum_sensing.jpg" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div></div></div></div></div>
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<p align=center class="ppp">Figure7.</p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;Figure7 shows the fluctuation of expression of the modified Toggle Switch device, the number of bacteria with red fluorescent decreases from right to left.</p>
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[[File:Re3.jpg|900px|center]]
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<p align=center class="ppp">Figure8.</p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;Figure8 shows four different growing states of the reprogrammed bacteria, in which the yellow circle represent the range of motion of reprogrammed bacteria(from the second tube（from right to left) shown in Figure7), and from left to right the concentration of the theophylline increases.</p>
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[[File:Results12.jpg|450px|center ]]
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<p align=center class="ppp">Figure9.The fluorescent Photo(4X Objective）of the colony1 in the fourth image of Figure8, and from left to right the concentration of the theophylline increases.</p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;Figure9 clearly display the dividing line between the reprogrammed bacteria at two different states, and the bacteria mainly express GFP indeed moving towards the high concentration of theophylline. This means the modified Toggle-switch-Aptamer-cheZ actually work as our design.</p>
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</font><font size="2" align="center">Figure 4: The quorum-sensing paradigm in Gram-negative bacteria. a. At low bacterial cell densities AHL molecules are synthesized and accumulate. Depending on the length of the acyl chain, AHLs either diffuse or are pumped out of the cell into the local environment, where the molecules are available for diffusion into, or uptake by, bacterial cells. b. At high bacterial cell densities, a threshold concentration of AHL molecules has accumulated. The R protein forms a complex with its cognate AHL and this complex either activates or represses the transcription of target genes. As the I gene is usually positively regulated by the R protein in complex with the cognate AHL, rapid amplification of the AHL signal results, which facilitates the coordinate transcriptional regulation of multiple genes. (Images from: Andrée M. Lazdunski, Isabelle Ventre and James N. Sturgis(2004) Regulatory circuits and communication in Gram-negative bacteria. Microbiology, Volume 2, July 2004, 581-592)</font><font size="3">
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==Discussion==
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<p>&nbsp;&nbsp;&nbsp;&nbsp;The experiment result mainly depend on the the random fluctuation of expression of the modified Toggle Switch device, so this device has a certain probability to switch from one state to the other, then it may lead to stopping of the moving bacteria like colony2(Figure10.).</P>
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[[File:R2.jpg|350px|center ]]
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<div class="center"><div class="thumb tnone" style="width:463px;"><div class="thumbinner" style="width:463px;"><a href="/File:Quorum_sensing1.jpg" class="image"><img alt="Quorum sensing1.jpg" src="/wiki/images/2/2d/Quorum_sensing1.jpg" width="463" height="407" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:Quorum_sensing1.jpg" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div></div></div></div></div>
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<p align=center class="ppp">Figure10.The fluorescent Photo(4X Objective）of the colony2 in the lower right of Figure8, and from left to right the concentration of the theophylline increases.</p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;To weaken this randomness, we may use the quorum sensing to control the moving of the reprogrammed bacteria, and we construct a model to simulate this proposal by computer ([https://2011.igem.org/Team:USTC-China/Drylab/modeling '''Modeling''']).
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</font><font size="2">Figure 5: Integration of quorum sensing to help grouping. a. LasR/LasI repressible promoter and cI assembled on pSB3T5. In order to increase the ratio of bacteria moving away, we insert aptamer-CheZ  into the other side of toggle switch compared with experiments earlier. With lasI-lasR accumulating, LasR/LasI repressible promoter in bacteria which express CheZ and move along theophylline gradient would get repressed and less cI might push off toggle switch to the other state. b. LasR positively regulated promoter and cI434 assembled on pSB3T5. Another design concerns with a LasR positively regulated promoter, which would be activated and express cI434 in bacteria expressing CheZ and also push off toggle switch to the other state. (Images by USTC-IGEM with TinkerCell)</font>
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<html><a name="Artificial" id=""></a></html>
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==Artificial Innate Immunity System==
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<div class="center"><div class="thumb tnone" style="width:389px;"><div class="thumbinner" style="width:389px;"><a href="/File:V().jpg" class="image"><img alt="350" src="/wiki/images/a/a6/V%28%29.jpg" width="389" height="248" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:V().jpg" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div></div></div></div></div>
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<p>Figure7:By using the quorum sensing, the engineered bacteria can express Pyosin and Lysis proteins in the high AHL concentration condition, then these bacteria will destroy themselves and release the Pyosin to kill the pathogens(Image from Nazanin Saeidi, Choon Kit Wong, Tat-Ming Lo .etc (2011)).</p>
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<div class="center"><div class="thumb tnone" style="width:677px;"><div class="thumbinner" style="width:677px;"><a href="/File:J().jpg" class="image"><img alt="250" src="/wiki/images/f/f8/J%28%29.jpg" width="677" height="217" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:J().jpg" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div></div></div></div></div>
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<p>Figure8:According to the basic design and we assume the small molecule is theophylline, so under the control of this module, the reprogrammed bacteria with green fluorescence will move toward the infection site with a high concentration of theophylline and the expression of Lysis becomes more and more, while the reprogrammed bacteria with red fluorescence will stay at the original site and wait to creat the second wave of the reprogrammed bacteria with green fluorescence, they have been protected from being destroyed by the pyosin from the reprogrammed bacteria with green fluorescence. The reprogrammed bacteria with green fluorescence will destroy themselves and release the Pyosin, which is expressed at the beginning of the process, to kill the pathogens.(Images by USTC-IGEM with TinkerCell)</p>
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Revision as of 12:55, 27 October 2011

Contents

Experiment results of the Basic Design

and Discussion

1.Verification of ΔcheZ strain
 2.Verification of the function of the constructed Aptamer-cheZ part
 3.Verification of the original Toggle-switch from PKU
 4.Verification of the modified Toggle-switch
 5.Test of Incorporated Aptamer-cheZ part into one side of the original Toggle-switch
 6.Test of Incorporated Aptamer-cheZ part into one side of the modified Toggle-switch


Verification of ΔcheZ strain

    The size of colonies of E.coli strain RP1616 was much smaller than that E.coli steain RP437 under the same circumstance and after same period of incubation time(about 10h), and the result is shown in Figure1. In colony PCR using the primers of cheZ gene following , RP437 absolutely has much more outcomes than RP1616 and we conclude that RP1616 is actually a ΔcheZ strain.

Figure1.

Figure1.The result of colony PCR(From left to right, the first lane is the marker, the third and the forth lane is the PCR outcome of strain RP437 and strain RP1616, the sixth and the seventh lane is the PCR outcome of strain RP437 and strain RP1616.)

Verification of the function of the constructed Aptamer-cheZ part(protocol1)(protocol2)

    From the results shown in Figure2. We can be sure that the Aptamer-cheZ part actually works effectively, especially on 0.3% Semi-solid medium.

X().jpg

Figure2. The growing state of the reprogrammed bacteria with Aptamer-cheZ part(Left:0.3%agar with 0mM Theophylline, Right:0.3%agar with 0.25mM Theophylline)

Verification of the original Toggle-switch from PKU(protocol)

Res14.jpg

Figure3.A conlony of the bacteria with the original Toggle Switch exhibit two different states(4X Objective)

    By calculating with the help of the fluorescence microscope, the ratio between the numbers of colonies with RFP and colonies with GFP ≈ 8:25. It means the original Toggle Switch is not fit for our purpose, so we modify the original by luxPR-cI device.

Verification of the modified Toggle-switch (protocol)

Rs14.jpg

Figure4. The conlonies of the bacteria with the modified Toggle Switch exhibit two different states or just one state(4X Objective)

    By calculating with the help of the fluorescence microscope, the ratio between the numbers of colonies with RFP and colonies with GFP ≈ 6:1. It is useful to our project design.

Test of Incorporated Aptamer-cheZ part into one side of the original Toggle-switch (protocol1) (protocol2)

Resul.jpg

Figure5.The growing state of the reprogrammed bacteria with original Toggle-switch-Aptamer-cheZ Device(Red circle:the range of motion, Blue Triangle:the gradient of thepphylline)

Resddul.jpg

Figure6.The fluorescent Photo(4X Objective) of the colony shown in Figure5 Left.

    According to the results above, by using this device we can not make the reprogrammed bacteria be divided into two different states effectively, and because of the bias to the green fluorescence and moving towards the high concentration of the theophylline, the bacteria trending to stay are very rare.

Test of Incorporated Aptamer-cheZ part into one side of the modified Toggle-switch (protocol)

Re.jpg

Figure7.

    Figure7 shows the fluctuation of expression of the modified Toggle Switch device, the number of bacteria with red fluorescent decreases from right to left.

Re3.jpg

Figure8.

    Figure8 shows four different growing states of the reprogrammed bacteria, in which the yellow circle represent the range of motion of reprogrammed bacteria(from the second tube（from right to left) shown in Figure7), and from left to right the concentration of the theophylline increases.

Results12.jpg

Figure9.The fluorescent Photo(4X Objective）of the colony1 in the fourth image of Figure8, and from left to right the concentration of the theophylline increases.

    Figure9 clearly display the dividing line between the reprogrammed bacteria at two different states, and the bacteria mainly express GFP indeed moving towards the high concentration of theophylline. This means the modified Toggle-switch-Aptamer-cheZ actually work as our design.

Discussion

    The experiment result mainly depend on the the random fluctuation of expression of the modified Toggle Switch device, so this device has a certain probability to switch from one state to the other, then it may lead to stopping of the moving bacteria like colony2(Figure10.).

R2.jpg

Figure10.The fluorescent Photo(4X Objective）of the colony2 in the lower right of Figure8, and from left to right the concentration of the theophylline increases.

    To weaken this randomness, we may use the quorum sensing to control the moving of the reprogrammed bacteria, and we construct a model to simulate this proposal by computer (Modeling).

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