Difference between revisions of "Team:NTU-LIHPAO-Taiwan/Notebook"

 
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/*-------------------Media-------------------*/
 
/*-------------------Media-------------------*/
 
/*-------------------------------------------*/
 
/*-------------------------------------------*/
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<div class="Slidemenu">
 
<div class="Slidemenu">
 
<ul>
 
<ul>
−
<li><div class=width_small><div id=Position_Now><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan">Home</a></div></div>
+
<li><div class=width_small><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan">Home</a></div>
 
</li>
 
</li>
  
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<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Basic_Part">Basic Parts</a></li>
 
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Basic_Part">Basic Parts</a></li>
 
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Composite_Part">Composite Parts</a></li>
 
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Composite_Part">Composite Parts</a></li>
−
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Part_Collection">Part Collection</a></li>
 
 
</ul>
 
</ul>
 
</li>
 
</li>
 
 
−
<li><div class=width_small><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Notebook">Notebook</a></div>
+
<li><div class=width_small><div id=Position_Now><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Notebook">Notebook</a></div></div>
 
</li>
 
</li>
 
          
 
          
Line 806: Line 800:
 
<ul class="main-Content">
 
<ul class="main-Content">
 
<li>
 
<li>
−
<span class="title">Protocols</span>
+
<span class="title">Months</span>
 
<ul class="sub-Content">
 
<ul class="sub-Content">
−
<li><a href="#First1">Preparation of Competent Cells (<i>E.coli</i>)</a></li>
+
<li><a href="#First1">July</a></li>
−
<li><a href="#First2">DNA Dissolution</a></li>
+
<li><a href="#First2">August</a></li>
−
<li><a href="#First3">Agarose Gel Electrophoresis</a></li>
+
<li><a href="#First3">September</a></li>
−
<li><a href="#First4">Gel Extraction</a></li>
+
−
<li><a href="#First5">DNA Ligation</a></li>
+
−
<li><a href="#First6">Plasmid DNA Extraction Using Alkaline Lysis Method (<i>E.coli</i>)</a></li>
+
−
<li><a href="#First7">Transformation(<i>E.coli</i>)</a></li>
+
−
<li><a href="#First8">Protein Extraction</a></li>
+
−
<li><a href="#First9">SDS-PAGE</a></li>
+
−
<li><a href="#First10">Western Blot</a></li>
+
−
<li><a href="#First11">Immunostaining</a></li>
+
−
<li><a href="#First12">Thawing Caco-2 cells</a></li>
+
−
<li><a href="#First13">Freezing Caco-2 cells</a></li>
+
−
<li><a href="#First14">Caco2 Cell passage</a></li>
+
−
<li><a href="#First15">Seeding in transwell</a></li>
+
−
<li><a href="#First16">Measure TEER</a></li>
+
 
</ul>
 
</ul>
 
</li>
 
</li>
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var sub_Content = main_Contents[i].querySelector(".sub-Content");
 
var sub_Content = main_Contents[i].querySelector(".sub-Content");
 
if (i === index) {
 
if (i === index) {
−
sub_Content.style.height = '20rem';
+
sub_Content.style.height = '12rem';
 
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} else {
 
sub_Content.style.height = '0';
 
sub_Content.style.height = '0';
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<div class="ContentBox">
 
<div class="ContentBox">
 
<div class="ContentHolder">
 
<div class="ContentHolder">
 +
<div class="Container_Top1"></div>
  
−
<div class="Text1" id="First1"><Red>Preparation of Competent Cells (<i>E.coli</i>)</Red></div>
+
<div class="Container2" id="First1"><div class="Text2">7/4</div></div>
−
<div class="Text2">
+
−
<Green>Materials and Reagents :</Green>
+
−
</div>
+
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>Manufacture <i>CP<Super>+</Super></i> plate</Green>
−
<li><i>Escherichia coli</i> DH5α</li>
+
−
<li>LB broth</li>
+
−
<li>TB buffer</li>
+
−
<li>DMSO</li>
+
−
<li>Liquid nitrogen</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
+
 
−
<Green>Equipment :</Green>
+
 
−
</div>
+
<div class="Container2"><div class="Text2">7/6</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Red>Learn how to transform</Red>
−
<li>Ice</li>
+
−
<li>Shaker</li>
+
−
<li>Spectrometer</li>
+
−
<li>Centrifuge</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
 
−
<Green>Procedure :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Green>Suicide Part:</Green>
−
<li>Inoculate 10μL <i>Escherichia coli</i> DH5α into 10mL LB broth (1:1000)</li>
+
−
<li>Grow for 12-16 hours at 37℃ with shaking</li>
+
−
<li>Inoculate 500μL <i>Escherichia coli</i> DH5α into 50mL LB broth (1:100)</li>
+
−
<li>Grow for 2 hours at 37℃ with shaking to O.D.<Sub>600</Sub>=0.4-0.6</li>
+
−
<li>Split the cell into two Falcon tubes, both contain 25 mL <i>Escherichia coli</i> DH5α</li>
+
−
<li>Centrifuge at 4℃, 3000rpm(800G) for 10 minutes</li>
+
−
<li>Discard the supernatant</li>
+
−
<li>Resuspend the cell pellet by gently adding 8.5mL TB buffer (1/3 V)</li>
+
−
<li>On ice for 10 minutes</li>
+
−
<li>Centrifuge at 4℃, 3000rpm(800G) for 10 minutes</li>
+
−
<li>Discard the supernatant using pipetman</li>
+
−
<li>Resuspend the cell pellet by gently adding 2mL TB buffer (1/12.5 V)</li>
+
−
<li>Add 150μL DMSO (7%)</li>
+
−
<li>On ice for 10 minutes</li>
+
−
<li>Transfer the cell to new eppendorfs with 60μL per tube</li>
+
−
<li>Freeze the cell in liquid nitrogen</li>
+
−
<li>Store the cell at -80℃</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text1" id="First2"><Red>DNA Dissolution</Red></div>
+
<div class="Text3_Sub">
−
<div class="Text2">
+
Transformation of:
−
<Green>Materials and Reagents :</Green>
+
</div>
−
</div>
+
<div class="Text3_SubSub">
 +
[RBS] (BBa_B0034), [C2] (BBa_C0053)
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">7/7</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Red>Learn how to manufacture competent cell</Red>
−
<li>DNA (BioBricks/synthesized)</li>
+
−
<li>ddH<Sub>2</Sub>O</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
 
−
<Green>Procedure :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Blue>Manufacture competent cell</Blue>
−
<li>Add 10μL ddH<Sub>2</Sub>O</li>
+
−
<li>Wait for 1 minute until the DNA dissolve</li>
+
−
<li>Pipet several times and transfer the DNA to an eppendorf</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text1" id="First3"><Red>Agarose Gel Electrophoresis</Red></div>
 
−
<div class="Text2">
 
−
<Green>Materials and Reagents :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>PYY part:</Green>
−
<li>Agarose</li>
+
</div>
−
<li>1X TAE</li>
+
<div class="Text3_Sub">
−
<li>Tracking dye</li>
+
Transformation of:
−
<li>Marker (1kb/100bp)</li>
+
</div>
−
<li>EtBr</li>
+
<div class="Text3_SubSub">
−
<li>ddH<Sub>2</Sub>O</li>
+
[promoter] (BBa_J23100), [signal peptide] (BBa_K554002), [terminator] (BBa_B0015)
−
</ol>
+
</div>
−
</div>
+
−
<div class="Text2">
+
−
<Green>Equipment :</Green>
+
−
</div>
+
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>Suicide Part:</Green>
−
<li>Gel tray</li>
+
−
<li>Well comb</li>
+
−
<li>Electrophoresis tank</li>
+
−
<li>UV detector</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
+
<div class="Text3_Sub">
−
<Green>Procedure :</Green>
+
Transformation of:
−
</div>
+
</div>
 +
<div class="Text3_SubSub">
 +
[pC1] (BBa_R0051), [pC2] (BBa_R0053), [endolysin] (BBa_K112806), [C1] (BBa_C0051)
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">7/8</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Red>Learn how to extract the plasmid</Red>
−
<div class="Text_TitleUnderline">Casting an Agarose Gel (m/v = 1.0% or 1.5%)</div>
+
−
<li>Measure out appropriate mass of agarose powder into a serum bottle</li>
+
−
<li>Add appropriate volume of 1X TAE</li>
+
−
        <li>Let agarose solution cool down to acceptable temperature for bare hands</li>
+
−
        <li>Pour the solution into a gel tray with the well comb in place, and push the bubbles away with a pipette tip</li>
+
−
        <li>Let sit at room temperature for 30 minutes, until it has completely solidified</li>
+
−
<div class="Text_TitleUnderline">Loading Samples and Running the Gel</div>
+
−
        <li>Place the gel as well as its tray into the electrophoresis tank containing 1X TAE  </li>
+
−
        (Make sure that the surface is higher than the top of the gel and not overflow)
+
−
        <li>Mix the samples with tracking dye (1/10 V) sufficiently</li>
+
−
        <li>Load the samples into the each well</li>
+
−
        <li>Load marker (usually in the first and last lane)</li>
+
−
        <li>Set an appropriate voltage (full/half) and run the gel for 15-20 minutes</li>
+
−
        <div class="Text_TitleUnderline">Imaging the Gel</div>
+
−
        <li>Put the gel into a container filled with 1X TAE and EtBr, staining for 5 minutes</li>
+
−
        <li>Replace EtBr solution with water and destain for 3 minutes</li>
+
−
        <li>Put the gel in an UV detector and record the result</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text1" id="First4"><Red>Gel Extraction</Red></div>
 
−
<div class="Text2">
 
−
<Green>Materials and Reagents :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Red>Learn how to conduct the gel extraction and electrophoresis</Red>
−
<li>Gel/PCR buffer</li>
+
</div>
−
<li>W1 buffer</li>
+
−
<li>Wash buffer</li>
+
−
<li>ddH<Sub>2</Sub>O</li>
+
−
</ol>
+
−
</div>
+
−
<div class="Text2">
+
−
<Green>Equipment :</Green>
+
−
</div>
+
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>Suicide Part:</Green>
−
<li>Cutter knife</li>
+
−
<li>Eppendorf</li>
+
−
<li>Vortex mixer</li>
+
−
<li>Dry bath incubator</li>
+
−
        <li>DF Column & Collection tube</li>
+
−
        <li>Mini Centrifuge</li>
+
−
        <li>Microcentrifuge</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
+
<div class="Text3_Sub">
−
<Green>Procedure :</Green>
+
Extract the plasmid:
−
</div>
+
</div>
 +
<div class="Text3_SubSub">
 +
[RBS] (BBa_B0034), [C2] (BBa_C0053)
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RBS] cleave by EcoRI, [C2] cleave by EcoRI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RBS] ----- Result: Failed (Smeared band was too blurred)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[C2] ----- Result: Succeeded
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pC1] (BBa_R0051), [pC2] (BBa_R0053), [endolysin] (BBa_K112806) <b>again</b>
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">7/9</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Green>Suicide Part:</Green>
−
        <div class="Text_TitleUnderline">Gel Dissociation</div>
+
</div>
−
<li>Excise the agarose gel slice containing relevant DNA fragments and remove any extra agarose to minimize the size of the gel slice (<300μL)</li>
+
<div class="Text3_Sub">
−
<li>Transfer the gel slice to an eppendorf</li>
+
Extract the plasmid:
−
<li>Add 500μL Gel/PCR buffer to the sample and mix by vortex</li>
+
</div>
−
        <li>Incubate at 60℃ for 10-15 minutes to ensure the gel slice has been completely dissolved (invert the tube every 2-3 minutes)</li>
+
<div class="Text3_SubSub">
−
        <li>Cool the dissolved sample mixture to room temperature</li>
+
[RBS] (BBa_B0034), [C1] (BBa_C0051), [endolysin] (BBa_K112806)
−
        <div class="Text_TitleUnderline">DNA Binding</div>
+
</div>
−
        <li>Place the DF Column in a 2mL Collection tube</li>
+
<div class="Text3_Sub">
−
        <li>Transfer 800μL of the sample mixture to the DF Column</li>
+
Gel electrophoresis:
−
        <li>Discard the flow-through and place the DF Column back in the Collection tube</li>
+
</div>
−
        (If the sample mixture is more than 800μL, repeat the DNA binding step)
+
<div class="Text3_SubSub">
−
        <div class="Text_TitleUnderline">Wash</div>
+
[RBS] ----- Result: Failed (Smeared band was too blurred)
−
        <li>Add 400μL W1 buffer into the DF Column</li>
+
</div>
−
        <li>Spin down for approximately 20 seconds</li>
+
<div class="Text3_SubSub">
−
        <li>Discard the flow-through and place the DF Column back in the Collection tube</li>
+
[C1] ----- Result: Succeeded
−
        <li>Add 600μL wash buffer (contains ethanol) into the DF Column</li>
+
</div>
−
        <li>Let stand for 1 minute at room temperature</li>
+
<div class="Text3_SubSub">
−
        <li>Spin down for approximately 30 seconds</li>
+
[endolysin] ----- Result: Failed (Smeared band was too blurred)
−
        <li>Discard the flow-through and place the DF Column back in the Collection tube</li>
+
</div>
−
        <li>Centrifuge at 12000 rpm for 3 minutes to dry the column matrix</li>
+
<div class="Text3">
−
        (Can be done twice)
+
<Green>PYY part:</Green>
−
        <li>Discard the flow-through</li>
+
</div>
−
        <div class="Text_TitleUnderline">DNA Elution</div>
+
<div class="Text3_Sub">
−
        <li>Transfer the dried DF Column to a new eppendorf (cap cut)</li>
+
Extract the plasmid:
−
        <li>Add 30μL of 37℃ ddH2O into the center of the column matrix</li>
+
</div>
−
        <li>Let stand for at least 2 minutes to ensure that ddH2O is completely absorbed</li>
+
<div class="Text3_SubSub">
−
        <li>Centrifuge at 12000 rpm for 2 minutes to elute the purified DNA</li>
+
[signal peptide] (BBa_K554002), [terminator] (BBa_B0015)
−
        <li>Re-add the subnatant into the center of the column matrix</li>
+
−
        <li>Centrifuge at 12000 rpm for 2 minutes to elute the purified DNA</li>
+
  
−
</ol>
 
 
</div>
 
</div>
−
<div class="Text1" id="First5"><Red>DNA Ligation</Red></div>
+
<div class="Text3_Sub">
−
<div class="Text2">
+
Gel electrophoresis:
−
<Green>Materials and Reagents :</Green>
+
</div>
−
</div>
+
<div class="Text3_SubSub">
 +
[signal peptide] ----- Result: Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[terminator] ----- Result: Succeeded
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">7/10</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Blue>Manufacture <i>CP<Super>+</Super></i> plate</Blue>
−
<li>DNA (insert & vector)</li>
+
</div>
−
<li>Ligation high ver.2</li>
+
−
</ol>
+
−
</div>
+
−
<div class="Text2">
+
−
<Green>Equipment :</Green>
+
−
</div>
+
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Blue>Manufacture <i>Amp<Super>+</Super></i> plate</Blue>
−
<li>Cooling dry bath incubator</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
 
−
<Green>Procedure :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Green>Suicide Part:</Green>
−
<li>
+
</div>
−
Table
+
<div class="Text3_Sub">
−
<table>
+
Gel electrophoresis:
−
<tr>
+
</div>
−
<th>Components</th>
+
<div class="Text3_SubSub">
−
<th>Volume (μL)</th>
+
[RBS] (BBa_B0034) ----- Result: Failed (Smeared band was too blurred)
−
</tr>
+
</div>
−
<tr>
+
<div class="Text3_SubSub">
−
<td>Insert</td>
+
[C2] (BBa_C0053) ----- Result: Succeeded
−
<td>x</td>
+
</div>
−
</tr>
+
<div class="Text3_Sub">
−
<tr>
+
Gel extraction:
−
<td>Vector</td>
+
</div>
−
<td>y</td>
+
<div class="Text3_SubSub">
−
</tr>
+
[C2]
−
<tr>
+
−
<td>Ligation High</td>
+
−
<td>1</td>
+
−
</tr>
+
−
<tr class="alt">
+
−
<td>Total</td>
+
−
<td>7</td>
+
−
</tr>
+
−
</table>
+
−
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Molar ratio: insert/vector = 3/1
+
−
<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; x + y = 6</br>
+
−
</li>
+
−
<li>Gently mix the solution by pipetting up and down</li>
+
−
<li>Incubate
+
−
<ol class="part3">
+
−
<li>at 16℃ for 2 hours, or</li>
+
−
<li>at 37℃ for 1 hour, or</li>
+
−
<li>at 4℃ overnight</li>
+
−
</ol>
+
−
</li>
+
−
<li>Proceed with bacterial transformation</li>
+
−
</ol>
+
 
</div>
 
</div>
  
−
<div class="Text1" id="First6"><Red>Plasmid DNA Extraction Using Alkaline Lysis Method (<i>E. coli</i>)</Red></div>
+
 
−
<div class="Text2">
+
<div class="Container2"><div class="Text2">7/11</div></div>
−
<Green>Materials and Reagents :</Green>
+
−
</div>
+
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>Suicide Part:</Green>
−
<li><i>Escherichia coli</i> DH5α (with plasmid)</li>
+
</div>
−
<li>LB broth</li>
+
<div class="Text3_Sub">
−
<li>Resuspension solution (MPI, with RNAase)</li>
+
Extract the plasmid:
−
<li>Lysis solution (MPII)</li>
+
</div>
−
<li>Neutralizing solution (MPIII)</li>
+
<div class="Text3_SubSub">
−
<li>Isopropanol</li>
+
[RBS] (BBa_B0034), [C1] (BBa_C0051), [endolysin] (BBa_K112806)
−
<li>70% ethanol</li>
+
</div>
−
<li>ddH<Sub>2</Sub>O</li>
+
−
</ol>
+
−
</div>
+
−
<div class="Text2">
+
−
<Green>Equipment :</Green>
+
−
</div>
+
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>PYY part:</Green>
−
<li>Shaker</li>
+
−
<li>Centrifuge</li>
+
−
<li>Vortex mixer</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
+
<div class="Text3_Sub">
−
<Green>Procedure :</Green>
+
Transformation of:
−
</div>
+
</div>
 +
<div class="Text3_SubSub">
 +
[promoter] (BBa_J23101)
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">7/12</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Green>Suicide Part:</Green>
−
        <li>Grow bacteria in LB broth with appropriate antibiotics at 37℃ overnight with shaking</li>
+
−
<li>Transfer 1.5mL culture to an eppendorf</li>
+
−
<li>Centrifuge at 4℃, 5000rpm for 5 minutes</li>
+
−
<li>Discard the supernatant</li>
+
−
        <li>Add 150μL of resuspension solution (MPI, with RNAase) into each tube, pipet several times, and vortex to completely resuspend the cell pellet</li>
+
−
        <li>Add 150μL of lysis solution (MPII), gently invert the tubes about 20 times, and then let the sample mixture stand at room temperature for 2 minutes</li>
+
−
        <li>Add 150μL of neutralizing solution (MPIII) and mix by inverting the tubes about 20 times. Bacterial chromosomal DNA and proteins can be seen as white precipitates</li>
+
−
        <li>Centrifuge at 4℃, 15000rpm for 15 minutes</li>
+
−
        <li>Carefully transfer 400μL of the supernatant to a new eppendorf</li>
+
−
        (Step 8&9 can be done twice)
+
−
        <li>Add 400μL (same volume as the supernatant) isopropanol, and shake up the tubes as vigorously as one can</li>
+
−
        <li>Centrifuge at 4℃, 15000rpm for 15 minutes</li>
+
−
        <li>Discard the supernatant</li>
+
−
        <li>Add 200μL of 70% ethanol to wash out the salts</li>
+
−
        <li>Centrifuge at 4℃, 15000rpm for 5 minutes</li>
+
−
        <li>Discard the supernatant and remove the remains as much as possible with pipetman</li>
+
−
        <li>Air dry for about 30 minutes</li>
+
−
        <li>Resuspend the DNA pellet with 20μL ddH<Sub>2</Sub>O</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text1" id="First7"><Red>Transformation (<i>E. coli</i>)</Red></div>
+
<div class="Text3_Sub">
−
<div class="Text2">
+
Extract the plasmid:
−
<Green>Materials and Reagents :</Green>
+
</div>
−
</div>
+
<div class="Text3_SubSub">
 +
[pC1] (BBa_R0051)
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">7/13</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>PYY part:</Green>
−
<li>Competent cell (<i>Escherichia coli</i> DH5α)</li>
+
</div>
−
<li>LB plate (Amp+/CP+)</li>
+
<div class="Text3_Sub">
−
</ol>
+
Extract the plasmid:
−
</div>
+
</div>
−
<div class="Text2">
+
<div class="Text3_SubSub">
−
<Green>Equipment :</Green>
+
[promoter] (BBa_J23101)
−
</div>
+
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis (two white colonies & two red colonies):
 +
</div>
 +
<div class="Text3_SubSub">
 +
[promoter] ------ Result: Failed (white colonies didn’t have band, red colonies had band but not the correct size, we speculated that was pollution)
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">7/20</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Red>Discuss about total synthesis</Red>
−
<li>-80℃ refrigerator</li>
+
−
<li>Ice</li>
+
−
<li>37℃ incubator</li>
+
−
<li>Super optimal broth (SOB) (37℃)</li>
+
−
<li>Shaker</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
+
 
−
<Green>Procedure :</Green>
+
 
−
</div>
+
<div class="Container2"><div class="Text2">7/13</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Green>PYY part:</Green>
−
        <li>Take out the competent cells from -80℃ refrigerator</li>
+
</div>
−
        <li>Thaw the cells on ice for 5 minutes</li>
+
<div class="Text3_Sub">
−
        <li>Add 1μL plasmid DNA into the competent cells, and mix gently by pipetting</li>
+
Transformation of:
−
        <li>On ice for 10 minutes</li>
+
</div>
−
        <li>Heat shock at 37℃ for 3 minutes</li>
+
<div class="Text3_SubSub">
−
        <li>On ice for 2 minutes</li>
+
[terminator] (BBa_B0015)
−
        <li>Add 150μL 37℃ SOB into the mixture</li>
+
</div>
−
        <li>Place the tube at 37℃ with shaking for 1 hour(Amp+)/4 hours(CP+) respectively</li>
+
−
        <li>Spread the cells onto the LB plates (Amp+/ CP+)</li>
+
−
        <li>Incubate the plates at 37℃ with shaking for 12-16 hours</li>
+
  
−
</ol>
+
 
 +
<div class="Container2"><div class="Text2">7/24</div></div>
 +
<div class="Text3">
 +
<Red>Discuss our project with professor</Red>
 
</div>
 
</div>
−
<div class="Text1" id="First8"><Red>Protein Extraction</Red></div>
 
−
<div class="Text2">
 
−
<Green>Materials and Reagents :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>PYY part:</Green>
−
<li>plasmid/ BL21 competent cell</li>
+
−
<li>LB plate</li>
+
−
<li>LB broth</li>
+
−
<li>lysis buffer</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
+
<div class="Text3_Sub">
−
<Green>Equipment :</Green>
+
Extract the plasmid:
−
</div>
+
</div>
 +
<div class="Text3_SubSub">
 +
[terminator] (BBa_B0015)
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[terminator] ----- Result: Failed
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">7/25</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Red>Discuss our problems with Instructor</Red>
−
<li>15/ 50ml conial tube</li>
+
−
<li>Shaking incubator</li>
+
−
<li>Erlenmeyer flask</li>
+
−
<li>Spectrophotometer</li>
+
−
<li>Centrifuge</li>
+
−
<li>Sonicator</li>
+
−
<li>Eppendorf</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
+
 
−
<Green>Procedure :</Green>
+
 
−
</div>
+
<div class="Container2"><div class="Text2">7/26</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Green>PYY part:</Green>
−
<li>Transform plamid into BL21 (see transformation)</li>
+
−
<li>Pick up a single colony and incubate into 2ml LB/CP medium, then agitate them in a shaking incubator at 37℃ overnight</li>
+
−
<li>Pour the culture into 100ml LB/CP medium, then agitate until it reach A<sub>600</sub> of 0.6</li>
+
−
<li>Pour the culture into 50ml conical tube, and centrifuge at 8000rpm for 10 min</li>
+
−
<li>Store the supernate and the pellets in the centrifuge tubes in a -80℃ freezer</li>
+
−
<li>Use 6ml lysis buffer(NP-10) to resuspend the cell pellet and transfer into a 15ml centrifuge tube</li>
+
−
<li>Lyse the cell with sonication</li>
+
−
<li>Centrifuge 15ml conial tube at 9000rpm for 10 min</li>
+
−
<li>Store the supernate as coarse extract</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text1" id="First9"><Red>SDS-PAGE</Red></div>
+
<div class="Text3_SubSub">
−
<div class="Text2">
+
Cultured [terminator] again
−
<Green>Materials and Reagents :</Green>
+
</div>
−
</div>
+
 
 +
 
 +
<div class="Container2"><div class="Text2">7/27</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>PYY part:</Green>
−
<li>Solution A (acrylamide/ bis-acrylamide (29:1) 30% solution)</li>
+
</div>
−
<li>Solution B (Tris (Tris base, 1.5 M) 90.8 gm; TEMED 1.8 ml; pH = 8.8/ 500ml)</li>
+
<div class="Text3_Sub">
−
<li>Solution C (Tris (Tris base, 0.5 M) 6.0 gm; TEMED 0.4 ml; pH = 6.7/ 100ml)</li>
+
Extract the plasmid:
−
<li>10% SDS (1kb/100bp)</li>
+
</div>
−
<li>ddH<Sub>2</Sub>O</li>
+
<div class="Text3_SubSub">
−
<li>APS</li>
+
[terminator] (BBa_B0015)
−
<li>Running buffer (1M Tris 12.5 ml; glysine 7.7g/ 500ml)</li>
+
</div>
−
<li>Sample buffer(5X) (Tris base, 125mM X5; EDTA2•Na, 2mM X 5; SDS(2% X2); β-mercaptoethanol, 5% X5)</li>
+
<div class="Text3_Sub">
−
<li>Protein marker</li>
+
Gel electrophoresis:
−
</ol>
+
</div>
−
</div>
+
<div class="Text3_SubSub">
−
<div class="Text2">
+
[terminator] ----- Result: Succeeded
−
<Green>Equipment :</Green>
+
</div>
−
</div>
+
 
 +
 
 +
<div class="Container2" id="First2"><div class="Text2">8/2</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>Suicide Part:</Green>
−
<li>Gel Caster</li>
+
−
<li>Accessories for each gel</li>
+
−
<li>&nbsp;&nbsp;&nbsp;&nbsp;(1) One glass plate</li>
+
−
<li>&nbsp;&nbsp;&nbsp;&nbsp;(2) One aluminum notched plate</li>
+
−
        <li>&nbsp;&nbsp;&nbsp;&nbsp;(3) Two 0.75-mm thick spacers</li>
+
−
        <li>&nbsp;&nbsp;&nbsp;&nbsp;(4) One 0.75-mm thick 10-well comb</li>
+
−
        <li>&nbsp;&nbsp;&nbsp;&nbsp;(5) One well-locating decal</li>
+
−
<li>Power supply</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
+
<div class="Text3_Sub">
−
<Green>Procedure :</Green>
+
Extract the plasmid:
−
</div>
+
</div>
 +
<div class="Text3_SubSub">
 +
[RFP](BBa_E1010)
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RFP] cleave by EcoRI and SpeI
 +
</div>
 +
        <div class="Text3_SubSub">
 +
[Cl] cleave by XbaI and PstI
 +
</div>
 +
        <div class="Text3_SubSub">
 +
[pSB1A3] cleave by EcoRI and PstI
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">8/5</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Blue>Passaged L.c.393</Blue>
−
<li>Assemble one glass plate and one alumimum plate with  two spacers for each gel module</li>
+
−
<li>Prepare the SDS gel solution (see the table below). Two 0.75mm mini gels need approximately 10ml separation gel solution and 5ml stacking solution. Add APS solution at the final step</li>
+
−
        <li>Mix the separation gel solution, and add the solution into the gel module until reaching the designated level. Then add isopropanol on the top of the gel. It takes about 40 min to complete the polymerization step</li>
+
−
        <li>Remove the isopropanol by using a small strip of filter paper. Fill up the stacking gel solution until reaching the top. Insert comb into the gel solution as soon as possible. It takes 10 min to complete the polymerization step</li>
+
−
        <li>Remove the comb and set the gel in the cell buffer dam. Pour the running buffer into the inner chamber and keep pouring after overflow until the buffer surface reaches the required level in the outer chamber</li>
+
−
        <li>Use a micropipette to clean up every single well with the running buffer</li>
+
−
        <li>Mix protein samples with 1/5 volumes of 5X sample buffer. Heat the mixture at 100oC for 10 min. Let it cool down to room temperature</li>
+
−
        <li>Load protein sample and the protein marker into the well</li>
+
−
        <li>Run the electrophoresis at 70V for 20 min, and then 150V for 55 min</li>
+
−
        <li>Remove the two spacer and lift the glass plate, and remove the stacking gel and the remaining gel is ready for CBR staining and Western blot</li>
+
−
</ol>
+
 
</div>
 
</div>
 
<div class="Text3">
 
<div class="Text3">
−
<table class="Table_Width">
+
<Blue>Manufacture MRS plate</Blue>
−
<tr>
+
−
<th>(Unit: ml)</th>
+
−
<th>Separation gel</th>
+
−
<th></th>
+
−
<th></th>
+
−
<th></th>
+
−
<th></th>
+
−
<th></th>
+
−
<th>Stacking gel</th>
+
−
</tr>
+
−
<tr>
+
−
<td>Percentage</td>
+
−
<td>5%</td>
+
−
<td>7.5%</td>
+
−
<td>10%</td>
+
−
<td>12.5%</td>
+
−
<td>15%</td>
+
−
<td>20%</td>
+
−
<td>4%</td>
+
−
</tr>
+
−
<tr>
+
−
<td>Solution A</td>
+
−
<td>16.5</td>
+
−
<td>2.5</td>
+
−
<td>3.35</td>
+
−
<td>4.15</td>
+
−
<td>5</td>
+
−
<td>6.5</td>
+
−
<td>0.66</td>
+
−
</tr>
+
−
<tr>
+
−
<td>Solution B</td>
+
−
<td>2.5</td>
+
−
<td>2.5</td>
+
−
<td>2.5</td>
+
−
<td>2.5</td>
+
−
<td>2.5</td>
+
−
<td>2.5</td>
+
−
<td>-</td>
+
−
</tr>
+
−
<tr>
+
−
<td>Solution C</td>
+
−
<td>-</td>
+
−
<td>-</td>
+
−
<td>-</td>
+
−
<td>-</td>
+
−
<td>-</td>
+
−
<td>-</td>
+
−
<td>1.24</td>
+
−
</tr>
+
−
<tr>
+
−
<td>10% SDS</td>
+
−
<td>0.1</td>
+
−
<td>0.1</td>
+
−
<td>0.1</td>
+
−
<td>0.1</td>
+
−
<td>0.1</td>
+
−
<td>0.1</td>
+
−
<td>0.05</td>
+
−
</tr>
+
−
<tr>
+
−
<td>dH<sub>2</sub>O</td>
+
−
<td>5.7</td>
+
−
<td>4.85</td>
+
−
<td>4</td>
+
−
<td>3.2</td>
+
−
<td>2.35</td>
+
−
<td>0.85</td>
+
−
<td>2.95</td>
+
−
</tr>
+
−
<tr>
+
−
<td>APS(10%)</td>
+
−
<td>0.05</td>
+
−
<td>0.05</td>
+
−
<td>0.05</td>
+
−
<td>0.05</td>
+
−
<td>0.05</td>
+
−
<td>0.05</td>
+
−
<td>0.1</td>
+
−
</tr>
+
−
<tr class="alt">
+
−
<td>Total Volume</td>
+
−
<td>10</td>
+
−
<td>10</td>
+
−
<td>10</td>
+
−
<td>10</td>
+
−
<td>10</td>
+
−
<td>10</td>
+
−
<td>5</td>
+
−
</tr>
+
−
</table>
+
 
</div>
 
</div>
−
<div class="Text1" id="First10"><Red>Western Blot</Red></div>
 
−
<div class="Text2">
 
−
<Green>Materials and Reagents :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>PYY part</Green>
−
<li>Transfer buffer ( Tris 25mM; glycine 0.192M)</li>
+
</div>
−
<li>Methanol</li>
+
<div class="Text3_Sub">
−
<li>10 X TBST (1M Tris pH =8.0; Tween 20, 50ml; NaCl 43.5g)</li>
+
Redissoluted DNA of total synthesis
−
</ol>
+
</div>
−
</div>
+
<div class="Text3_Sub">
−
<div class="Text2">
+
Restriction enzyme:
−
<Green>Equipment :</Green>
+
</div>
−
</div>
+
        <div class="Text3_SubSub">
 +
[signalpeptide -PYY] cleave by EcoRI and PstI
 +
</div>
 +
        <div class="Text3_Sub">
 +
Ligation:
 +
</div>
 +
        <div class="Text3_SubSub">
 +
[signalpeptide-PYY]+ [pSB1C3]
 +
</div>
 +
        <div class="Text3_Sub">
 +
Transformation
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">8/6</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Blue>Passaged L.c.393</Blue>
−
<li>Semi-dry trnasfer cell</li>
+
−
<li>PVDF membrane</li>
+
−
<li>Nitrocellulose paper</li>
+
−
<li>Square Petri dish</li>
+
−
<li>Rotary shaker</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
 
−
<Green>Procedure :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Green>PYY part</Green>
−
        <li>Equilibrate the nitrocellulose paper and SDS-PAGE gel by soaking them with transfer buffer</li>
+
−
<li>Rinse the PVDF membrane with 100% methanol for a few second and equilibrate it with transfer buffer</li>
+
−
<li>Place the elements in the following order onto the semi-dry transfer cell from bottom to top: nitrocellulose paper, PVDF membrane, SDS-PAGE gel, nitrocellulose paper</li>
+
−
<li>Set up the current at 0.09A and run for 75 min</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text1" id="First11"><Red>Immunostaining</Red></div>
+
<div class="Text3_Sub">
−
<div class="Text2">
+
Restriction enzyme:
−
<Green>Materials and Reagents :</Green>
+
</div>
−
</div>
+
<div class="Text3_SubSub">
 +
[PYY] cleave by EcoRI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] cleave by EcoRI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Y2R] cleave by KpnI and XhoI
 +
</div>
 +
<div class="Text3_Sub">
 +
Ligation:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY]+ [pSB1C3]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY]+ [pSB1C3]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Y2R]+[pcDNA]
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">8/7</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>PYY part</Green>
−
<li>3% milk</li>
+
</div>
−
<li>Antibody solution</li>
+
<div class="Text3_Sub">
−
<li>Secondary antibody (anti-mouse HRP)</li>
+
Gel electrophoresis:
−
<li>TBST</li>
+
</div>
−
<li>Western HRP substrate</li>
+
<div class="Text_underline">
−
</ol>
+
Result:
−
</div>
+
</div>
−
<div class="Text2">
+
<div class="Text3_SubSub">
−
<Green>Equipment :</Green>
+
signalpeptide-PYY] ----- Failed (Smeared band was too blurred)
−
</div>
+
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Failed (Smeared band was too blurred)
 +
</div>
 +
<div class="Text3_Sub">
 +
Transform again
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">8/10</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Blue>Manufacture CP<sup>+</sup> plate</Blue>
−
<li>Square petri dish</li>
+
−
<li>Rotary shaker</li>
+
−
<li>UVP Biospectrum</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
 
−
<Green>Procedure :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Green>Suicide part</Green>
−
<li>Put the membrane in the square petri dish block the membrane with the 3% milk/TBST at room temperature for 1hr</li>
+
</div>
−
<li>Discard the milk and replace it with antibody solution(mouse His-probe Antibody) at 4℃ overnight</li>
+
<div class="Text3_Sub">
−
        <li>Recycle the antibody solution. Wash the membrane with TBST three times, 5 min each</li>
+
Gel electrophoresis:
−
        <li>Soak membrane into anti-mouse HRP solution for 1 hr</li>
+
</div>
−
        <li>Discard the anti-mouse HRP solution and wash it with TBST three times, 5 min each</li>
+
<div class="Text_underline">
−
        <li>Add HRP substrate on the membrane for 1 min</li>
+
Result:
−
        <li>Soak the membrane into ddH<sub>2</sub>O to remove the substrate</li>
+
</div>
−
        <li>Observe the result with UVP Biospectrum</li>
+
<div class="Text3_SubSub">
−
</ol>
+
[RFP+Terminator] ----- Failed (Confirmed it was pollution)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Cl+Terminator] ----- Failed
 +
</div>
 +
<div class="Text3_Sub">
 +
Clean up:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RFP+Terminator]
 
</div>
 
</div>
−
<div class="Text1" id="First12"><Red>Thawing Caco-2 cells</Red></div>
 
−
<div class="Text2">
 
−
<Green>Materials and Reagents :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>PYY part</Green>
−
<li>1 ml frozen Caco-2 cells in cryovial</li>
+
</div>
−
<li>Cell culture medium (MEM (minimum essential medium) (GIBCOTM #41500-034)/2.2g NaHCO3/ 0.11 g sodium pyruvate/20% FBS/1% PSA) </li>
+
<div class="Text3_Sub">
−
<li>70% ethanol</li>
+
Clean up:
−
<li>Trypan blue solution</li>
+
</div>
−
</ol>
+
<div class="Text3_SubSub">
−
</div>
+
[signalpeptide-PYY]+ [pSB1A3]
−
<div class="Text2">
+
</div>
−
<Green>Equipment :</Green>
+
<div class="Text3_SubSub">
−
</div>
+
[PYY]+ [pSB1A3]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY]+ [pSB1A3]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Y2R]+[pcDNA]
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">8/11</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>Suicide part</Green>
−
<li>Laminar flow hood</li>
+
</div>
−
<li>Auto pipette</li>
+
<div class="Text3_Sub">
−
<li>Centrifuge</li>
+
Clean up:
−
<li>15 ml centrifuge tubes</li>
+
</div>
−
<li>10 cm dish</li>
+
<div class="Text3_SubSub">
−
<li>Hemocytometer</li>
+
[Cl+Terminator]
−
<li>Optical microscope</li>
+
</div>
−
<li>Suction machine</li>
+
<div class="Text3_Sub">
−
</ol>
+
Gel electrophoresis
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RFP+Terminator]----- Failed (Smeared band was too blurred)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Cl+Terminator] ----- Failed (Smeared band was too blurred)
 
</div>
 
</div>
−
<div class="Text2">
 
−
<Green>Procedure :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Green>PYY part</Green>
−
<li>Pre-warm medium in 37˚C water bath</li>
+
−
<li>Open a laminar flow hood</li>
+
−
        <li>Clean materials, reagents and equipments before moving them into the hood</li>
+
−
        <li>Thawing Caco-2 cells in 37˚C water bath and transfer the cells into a 15ml centrifuge tube</li>
+
−
        <li>Add 9 ml cell culture medium very slowly</li>
+
−
        <li>Centrifuge at 1000 rpm for 5 min</li>
+
−
        <li>Decant the supernate and resuspend cells with 10 ml cell culture medium</li>
+
−
        <li>Mix 10 ul cell suspension and 10 μl trypan blue solution and count total cell number with a hemocytometer</li>
+
−
        <li>Add all cell suspension to a 10 cm dish</li>
+
−
        <li>Write date, cell line and passage number</li>
+
−
        <li>Put the dish into incubator</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text1" id="First13"><Red>Freezing Caco-2 cells</Red></div>
+
<div class="Text3_Sub">
−
<div class="Text2">
+
Extract the plasmid:
−
<Green>Materials and Reagents :</Green>
+
</div>
−
</div>
+
<div class="Text3_SubSub">
 +
[TAT-PYY], [Y2R]
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">8/12</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>Suicide part</Green>
−
<li>Caco-2 cell in 10 cm dish</li>
+
</div>
−
<li>Cell culture medium (MEM (minimum essential medium) (GIBCOTM #10370-021)/10% FBS/ 2 mM glutamine</li>
+
<div class="Text3_Sub">
−
<li>1X PBS (phosphate buffer saline)</li>
+
Redissoluted DNA of total synthesis
−
<li>0.5% trypsin</li>
+
</div>
−
<li>Trypan blue solution</li>
+
<div class="Text3_Sub">
−
<li>70% ethanol</li>
+
Restriction enzyme:
−
<li>Cell freezing media (50% FBS, 40% cell culture medium, 10% DMSO)</li>
+
</div>
−
</ol>
+
<div class="Text3_SubSub">
−
</div>
+
[Lac-RBS] cleave by EcoRI and PstI
−
<div class="Text2">
+
</div>
−
<Green>Equipment :</Green>
+
<div class="Text3_SubSub">
−
</div>
+
[NucA-Terminator] cleave by EcoRI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[NIsI] cleave by EcoRI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[LcSPddhol] cleave by EcoRI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Ligation:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Lac-RBS]+ [pSB1C3]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[NucA-Terminator]+ [pSB1C3]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[NIsI]+ [pSB1C3]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[LcSPddhol]+ [pSB1C3]
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation
 +
</div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>PYY part</Green>
−
<li>Laminar flow hood</li>
+
−
<li>Auto pipette</li>
+
−
<li>Centrifuge</li>
+
−
<li>15 ml centrifuge tubes</li>
+
−
<li>10 cm dish</li>
+
−
<li>Hemocytometer</li>
+
−
<li>Optical microscope</li>
+
−
<li>Suction machine</li>
+
−
<li>Cryovials</li>
+
−
<li>Freezing apparatus</li>
+
−
<li>-80˚C refrigerator</li>
+
−
<li>Liquid nitrogen tank</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
+
<div class="Text3_Sub">
−
<Green>Procedure :</Green>
+
Gel electrophoresis:
−
</div>
+
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY]----- Failed(Confirmed it was pollution)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Cll] ----- Succeeded
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">8/13</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Green>Suicide part</Green>
−
<li>Pre-warm medium in 37˚C water bath</li>
+
</div>
−
<li>Open a laminar flow hood</li>
+
<div class="Text3_Sub">
−
        <li>Clean materials, reagents and equipments before moving them into the hood</li>
+
Extract the plasmid:
−
        <li>Take a 10 cm dish with Caco-2 cell</li>
+
</div>
−
        <li>Discard the medium with a suction machine</li>
+
<div class="Text3_SubSub">
−
        <li>Wash the cells with 5 ml PBS</li>
+
[Lac-RBS]+ [pSB1C3]
−
        <li>Add 1 ml 0.5% trypsin and incubate at 37˚C for 7 minutes</li>
+
</div>
−
        <li>Wash out all the cells from the surface of the dish by pipetting the fresh complete culture medium (9 ml) all over the surface</li>
+
<div class="Text3_SubSub">
−
        <li>Transfer the cell suspension to a 15 ml centrifuge tube</li>
+
[NucA-Terminator]+ [pSB1C3]
−
        <li>Mix 10 μl cell suspension and 10 ul trypan blue solution and count total cell number with a hemocytometer</li>
+
</div>
−
        <li>Add about 1x10<sup>6</sup> cells per cryovial, and add cell freezing media</li>
+
<div class="Text3_SubSub">
−
        <li>Freeze the cryovials in a controlled rate freezing apparatus, decreasing the temperature approximately 1°C per minute in a -80°C refrigerator for more than 3 hour, then move them into liquid nitrogen tank</li>
+
[NIsI]+ [pSB1C3]
−
</ol>
+
</div>
 +
<div class="Text3_Sub">
 +
Clean up:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Cl], [terminator], [RFP]
 +
</div>
 +
<div class="Text3_Sub">
 +
Ligation:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Cl]+ [terminator]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RFP]+ [terminator]
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Cl]+ [terminator] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RFP]+ [terminator] ----- Succeeded
 
</div>
 
</div>
−
<div class="Text1" id="First14"><Red>Caco2 Cell passage</Red></div>
 
−
<div class="Text2">
 
−
<Green>Materials and Reagents :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>PYY part</Green>
−
<li>Caco-2 cell in 10 cm dish</li>
+
</div>
−
<li>Cell culture medium (MEM (minimum essential medium) (GIBCOTM #10370-021)/10% FBS/ 2 mM glutamine</li>
+
<div class="Text3_Sub">
−
<li>1X PBS (phosphate buffer saline)</li>
+
Extract the plasmid:
−
<li>0.5% trypsin</li>
+
</div>
−
<li>Trypan blue solution</li>
+
<div class="Text3_SubSub">
−
<li>70% ethanol</li>
+
[LcSPddhol]+ [pSB1C3]
−
</ol>
+
</div>
−
</div>
+
<div class="Text3_Sub">
−
<div class="Text2">
+
Clean up:
−
<Green>Equipment :</Green>
+
</div>
−
</div>
+
<div class="Text3_SubSub">
 +
[TAT-PYY]
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">8/14</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Blue>DNA sequencing:</Blue>
−
<li>Laminar flow hood</li>
+
</div>
−
<li>Auto pipette</li>
+
<div class="Text3_Sub">
−
<li>Centrifuge</li>
+
[SP-PYY], [PYY], [Y2R], [Lac-RBS], [NucA-Terminator], [NIsI], [LcSPddhol]
−
<li>15 ml centrifuge tubes</li>
+
−
<li>10 cm dish</li>
+
−
<li>Hemocytometer</li>
+
−
<li>Optical microscope</li>
+
−
<li>Suction machine</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
 
−
<Green>Procedure :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Green>Suicide part</Green>
−
<li>Pre-warm medium in 37˚C water bath</li>
+
−
<li>Open a laminar flow hood</li>
+
−
        <li>Clean materials, reagents and equipments before moving them into the hood</li>
+
−
        <li>Take a 10 cm dish with Caco-2 cell</li>
+
−
        <li>Discard the medium with a suction machine</li>
+
−
        <li>Wash the cells with 5 ml PBS</li>
+
−
        <li>Add 1 ml 0.5% trypsin and incubate at 37˚C for 7 minutes</li>
+
−
        <li>Wash out all the cells from the surface of the dish by pipetting the fresh complete culture medium (9 ml) all over the surface</li>
+
−
        <li>Transfer the cell suspension to a 15 ml centrifuge tube</li>
+
−
        <li>Mix 10 ul cell suspension and 10 μl trypan blue solution and count total cell number with a hemocytometer</li>
+
−
        <li>Transfer appropriate amounts of cell suspension to new dishes (1/4 X)</li>
+
−
        <li>Write date, cell line and passage number</li>
+
−
        <li>Put the dish into incubator</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text1" id="First15"><Red>Seeding in transwell</Red></div>
+
<div class="Text3_Sub">
−
<div class="Text2">
+
Ligation:
−
<Green>Materials and Reagents :</Green>
+
</div>
−
</div>
+
<div class="Text3_SubSub">
 +
[Cl]+ [terminator]
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Lac-RBS] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[NucA-Terminator] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[NIsI] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[LcSPddhol] ----- Succeeded
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:  
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RFP-terminator]
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">8/15</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Blue>Passaged L.c.393</Blue>
−
<li>Caco-2 cell in 10 cm dish</li>
+
</div>
−
<li>Cell culture medium (MEM (minimum essential medium) (GIBCOTM #41500-034)/2.2 g NaHCO3/ 0.11 g sodium pyruvate/20% FBS/1% PSA</li>
+
−
<li>1X PBS (phosphate buffer saline)</li>
+
−
<li>0.5% trypsin</li>
+
−
<li>Trypan blue solution</li>
+
−
<li>70% ethanol</li>
+
−
</ol>
+
−
</div>
+
−
<div class="Text2">
+
−
<Green>Equipment :</Green>
+
−
</div>
+
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Green>Suicide part</Green>
−
<li>Laminar flow hood</li>
+
</div>
−
<li>Auto pipette</li>
+
<div class="Text3_Sub">
−
<li>Centrifuge</li>
+
Extract the plasmid:
−
<li>15 ml centrifuge tubes</li>
+
</div>
−
<li>10 cm dish</li>
+
<div class="Text3_SubSub">
−
<li>Hemocytometer</li>
+
[Cl-terminator]
−
<li>Optical microscope</li>
+
</div>
−
<li>Suction machine</li>
+
<div class="Text3_Sub">
−
<li>6-well plate</li>
+
Gel electrophoresis:
−
<li>Millicell plate insert (PIHP03050)</li>
+
</div>
−
</ol>
+
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RFP-terminator] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Cl-terminator] ----- Succeeded
 
</div>
 
</div>
−
<div class="Text2">
 
−
<Green>Procedure :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Green>PYY part</Green>
−
<li>Open 6-well plate, add 1.5 mL medium to each large well (6 wells) which are going to be used</li>
+
−
<li>Add 2 ml PBS/well to other wells</li>
+
−
        <li>Put transwell in larger well</li>
+
−
        <li>Take a 10 cm dish with Caco-2 cell</li>
+
−
        <li>Discard the medium with a suction machine</li>
+
−
        <li>Wash the cells with 5 ml PBS</li>
+
−
        <li>Add 1 ml 0.5% trypsin and incubate at 37˚C for 7 minutes</li>
+
−
        <li>Wash out all the cells from the surface of the dish by pipetting the fresh complete culture medium (9 ml) all over the surface</li>
+
−
        <li>Transfer the cell suspension to a 15 ml centrifuge tube</li>
+
−
        <li>Mix 10 ul cell suspension and 10 μl trypan blue solution and count total cell number with a hemocytometer</li>
+
−
        <li>Cell count: 70/ 2 * orginal volume *100 (*10<sup>4</sup>)= 35 X 10<sup>4</sup> cell/mL (Seeding 42 x 10<sup>4</sup> cell/well)</li>
+
−
        <li>Write date, cell line and passage number</li>
+
−
        <li>Put the dish into incubator</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text1" id="First16"><Red>Measure TEER</Red></div>
+
<div class="Text3_Sub">
−
<div class="Text2">
+
Extract the plasmid:
−
<Green>Materials and Reagents :</Green>
+
</div>
−
</div>
+
<div class="Text3_SubSub">
 +
[TAT-PYY]
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [TAT-PYY]
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] cleave by EcoRI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Succeeded
 +
</div>
 +
 
 +
 
 +
<div class="Container2"><div class="Text2">8/16</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Blue>Passaged L.c.393</Blue>
−
<li>Caco-2 cells seeded in transwell</li>
+
</div>
−
<li>70% ethanol</li>
+
−
</ol>
+
−
</div>
+
−
<div class="Text2">
+
−
<Green>Equipment :</Green>
+
−
</div>
+
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part1">
+
<Blue>Manufacture LB broth</Blue>
−
<li>Laminar flow hood</li>
+
−
<li>Millicell ERS</li>
+
−
</ol>
+
 
</div>
 
</div>
−
<div class="Text2">
 
−
<Green>Procedure :</Green>
 
−
</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
<ol class="part2">
+
<Blue>Manufacture MRS broth</Blue>
−
<li>Prepare laminar hood for experiment</li>
+
</div>
−
<li>To sterilize electrode of Millicell ERS, soak electrode in 70% alcohol for 15 min, and air dry for 15 sec</li>
+
<div class="Text3">
−
        <li>Measure TEER value of each well (n=3)</li>
+
<Green>Suicide part</Green>
−
</ol>
+
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Cl-Terminator] cleave by EcoRI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RFP-Terminator] cleave by EcoRI and PstI
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [SP-PYY], [PYY], [TAT-PYY]
 
</div>
 
</div>
  
  
 +
<div class="Container2"><div class="Text2">8/17</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Ligation:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-RFP-T], [plac-Cl-T]
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [plac-RFP-T], [plac-Cl-T]
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Culture [SP-PYY], [PYY], [TAT-PYY] in BL21 Competent
 +
</div>
 +
<div class="Text3_Sub">
 +
<i>E. coli</i> and Rosetta Competent <i>E. coli</i>
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/18</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Culture [plac-RFP], [plac-Cl], [NucA] in LB broth
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-RFP], [plac-Cl]
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-RFP] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-Cl] ----- Succeeded
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
Sonication of Competent <i>E. coli</i>
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/19</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[NucA]
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[NucA] ----- Succeeded
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
SDS-page
 +
</div>
 +
<div class="Text3_SubSub">
 +
Transfer
 +
</div>   
 +
<div class="Text3_SubSub">
 +
Blocking
 +
</div>
 +
<div class="Text3_SubSub">
 +
Primary antibody
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/20</div></div>
 +
<div class="Text3">
 +
<Blue>Passaged Caco2 cell</Blue>
 +
</div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel Purification:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-Cl-T], [pCl-NucA-T]
 +
</div>
 +
<div class="Text3_Sub">
 +
Ligation:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-Cl-T], [pCl-NucA-T]
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [plac-Cl-T], [pCl-NucA-T]
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
Secondary antibody
 +
</div>   
 +
<div class="Text3_SubSub">
 +
Chemiluminescent detection
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
SDS-page
 +
</div>
 +
<div class="Text3_SubSub">
 +
Transfer
 +
</div>   
 +
<div class="Text3_SubSub">
 +
Blocking
 +
</div>
 +
<div class="Text3_SubSub">
 +
Primary antibody
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/21</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Culture [[plac-Cl-T], [pCl-NucA-T] in LB broth
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-Cl-T], [pCl-NucA-T]
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-Cl-T] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pCl-NucA-T] ----- Failed (band was too blurred)
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
Secondary antibody
 +
</div>   
 +
<div class="Text3_SubSub">
 +
Chemiluminescent detection
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Failed (band was too blurred)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [SP-PYY], [PYY], [TAT-PYY], [RFP] again. (use [RFP] as negative control)
 +
</div>
 +
<div class="Text3_Sub">
 +
Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP] in BL21
 +
</div>
 +
<div class="Text3_Sub">
 +
Competent <i>E. coli</i> and Rosetta Competent <i>E. coli</i>
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/22</div></div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY] , [RFP]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
Sonication of Competent <i>E. coli</i>
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/23</div></div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY] , [RFP]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
SDS-page
 +
</div>
 +
<div class="Text3_SubSub">
 +
Transfer
 +
</div>   
 +
<div class="Text3_SubSub">
 +
Blocking
 +
</div>
 +
<div class="Text3_SubSub">
 +
Primary antibody
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/24</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[NucA] cleave by SpeI and PstI
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
Secondary antibody
 +
</div>   
 +
<div class="Text3_SubSub">
 +
Chemiluminescent detection
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Failed (band was not specificity)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Failed (band was not specificity)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Failed (band was not specificity)
 +
</div>
 +
<div class="Text3_Sub">
 +
Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP]in mini
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/25</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-C1-T-backbone] cleave by EcoRI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-C1-T] ----- Failed
 +
</div>
 +
<div class="Text3_Sub">
 +
Redissoluted [pJ23102]
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [pCl], [pCll], [RBS], [pJ23102]
 +
</div>
 +
<div class="Text3_Sub">
 +
Culture [pCl], [pCll], [RBS] in LB broth
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP] in midi
 +
</div>
 +
<div class="Text3_Sub">
 +
Centrifugal and took the supernatant
 +
</div>   
 +
<div class="Text3_Sub">
 +
Bradford protein assay
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/26</div></div>
 +
<div class="Text3">
 +
<Blue>Passaged Caco2 cell</Blue>
 +
</div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Ligation:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-Cl-T]
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [plac-Cl-T]
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac], [pCl], [pCll], [RBS]
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
SDS-page
 +
</div>
 +
<div class="Text3_SubSub">
 +
Transfer
 +
</div>   
 +
<div class="Text3_SubSub">
 +
Blocking
 +
</div>
 +
<div class="Text3_SubSub">
 +
Primary antibody
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/27</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [RBS-Cl-T], [RBS-RFP-T]
 +
</div>
 +
<div class="Text3_Sub">
 +
Culture [plac-Cl-T],[RBS-Cl-T],[RBS-RFP-T],[pJ23102] in LB broth
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-Cl-T], [pJ23102]
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-Cl-T] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pJ23102] ----- Succeeded
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pCl-T] cleave by SpeI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel Purification:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pCll]
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
Secondary antibody
 +
</div>
 +
<div class="Text3_SubSub">
 +
Chemiluminescent detection
 +
</div>   
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [SP-PYY], [PYY], [TAT-PYY], [RFP] again
 +
</div>
 +
<div class="Text3_Sub">
 +
Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP] in BL21
 +
</div>
 +
<div class="Text3_Sub">
 +
Competent <i>E. coli</i>
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/28</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Ligation:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pCIt]+[NucA]
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Cl-T], [RFP-T]
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Cl-T] ----- Failed (band size was wrong)
 +
</div>
 +
  <div class="Text3_SubSub">
 +
[RFP-T] ----- Failed (band size was wrong)
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
  <div class="Text3_SubSub">
 +
[plac-T] cleave by SpeI and PstI
 +
</div>
 +
  <div class="Text3_SubSub">
 +
[pJ23102] cleave by SpeI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel Purification:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pCI-T]
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
SDS-page
 +
</div>
 +
<div class="Text3_SubSub">
 +
Transfer
 +
</div>   
 +
<div class="Text3_SubSub">
 +
Blocking
 +
</div>
 +
<div class="Text3_SubSub">
 +
Primary antibody
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/29</div></div>
 +
<div class="Text3">
 +
<Blue>Passaged Caco2 cell</Blue>
 +
</div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RBS-Cl-T], [RBS-RFP-T]
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
Secondary antibody
 +
</div>
 +
<div class="Text3_SubSub">
 +
Chemiluminescent detection
 +
</div>   
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_Sub">
 +
Histag column([SP-PYY], [PYY], [TAT-PYY] , [RFP])
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
SDS-page
 +
</div>
 +
<div class="Text3_SubSub">
 +
Transfer
 +
</div>   
 +
<div class="Text3_SubSub">
 +
Blocking
 +
</div>
 +
<div class="Text3_SubSub">
 +
Primary antibody
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/30</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RBS], [pCl]
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RBS] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pCl] ----- Failed
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
Secondary antibody
 +
</div>
 +
<div class="Text3_SubSub">
 +
Chemiluminescent detection
 +
</div>   
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Failed (no band displayed)
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/31</div></div>
 +
<div class="Text3">
 +
<Blue>Passaged hek293 cell</Blue>
 +
</div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel Purification:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RBS], [CI-T]
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
SDS-page
 +
</div>
 +
<div class="Text3_SubSub">
 +
Transfer
 +
</div>   
 +
<div class="Text3_SubSub">
 +
Blocking
 +
</div>
 +
<div class="Text3_SubSub">
 +
Primary antibody
 +
</div>
 +
 +
 +
<div class="Container2" id="First3"><div class="Text2">9/1</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Ligation:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RBS] + [CI-T]
 +
</div>
 +
<div class="Text3_Sub">
 +
Culture [RBS- CI-T] in LB broth
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
 +
</div>
 +
<div class="Text3_SubSub">
 +
Secondary antibody
 +
</div>
 +
<div class="Text3_SubSub">
 +
Chemiluminescent detection
 +
</div>   
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Failed (no band displayed)
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
 +
</div>
 +
<div class="Text3">
 +
<Green>Selection part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Culture [nisI] in MRS broth
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">9/2</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RBS- CI-T]
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RBS- CI-T] ----- Succeeded
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] cleave by EcoRI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] cleave by EcoRI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] cleave by EcoRI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] cleave by EcoRI
 +
</div>   
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] ----- Succeeded
 +
</div>
 +
<div class="Text3">
 +
<Green>Selection part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Nisin Immunity Test
 +
</div>
 +
<div class="Text3_Sub">
 +
Culture [nisI] in LB broth
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">9/3</div></div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Failed (small band didn’t display)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Failed (small band didn’t display)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Failed(small band didn’t display)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] ----- Succeeded
 +
</div>
 +
<div class="Text3_Sub">
 +
Again -->
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>   
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] ----- Succeeded
 +
</div>
 +
<div class="Text3_Sub">
 +
16℃ Ligation 4hr:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY]+ [pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY]+ [pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY]+ [pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3">
 +
<Green>Selection part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[nisI-pSB1C3]
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[nisI-pSB1C3] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[nisI-pSB1C3] ----- Succeeded
 +
</div>
 +
<div class="Text3_Sub">
 +
Nisin Immunity Test
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">9/4</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Ligation:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pJ23102] + [RBS-CI-T]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac] + [RBS-CI-T]
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [pJ23102-RBS-CI-T], [plac-RBS-CI-T]
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>   
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] ----- Succeeded
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel Purification:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
16℃ Ligation 4hr:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY]+ [pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY]+ [pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY]+ [pETDuet-1] ----- Failed
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">9/5</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Culture[pJ23102-RBS-CI-T], [plac-RBS-CI-T] in LB broth
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>   
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] ----- Succeeded
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel Purification:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
16℃ Ligation 4hr:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY]+ [pETDuet-1]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY]+ [pETDuet-1]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY]+ [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Measure the resistance of Caco2 cell
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">9/6</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[placE-RBS-CI-T], [pJ23102-RBS-CI-T], [placE]
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[placE-RBS-CI-T] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pJ23102-RBS-CI-T] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[placE] ----- Succeeded
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1],[PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY-pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY-pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>   
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1]-----Failed(Ligation failed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY-pETDuet-1] ----- Failed(Ligation failed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY-pETDuet-1]-----Failed(Ligation failed)
 +
</div>
 +
<div class="Text3_Sub">
 +
Measure the resistance of Caco2 cell
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">9/7</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[placE] cleave by SpeI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[RBS-CI-T] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [pCLP7]
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pCLP7] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[nisi] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY-pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY-pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>   
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Failed(small band didn’t display)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Failed(small band didn’t display)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Failed(small band didn’t display)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1]-----Failed(small band didn’t display)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1]-----Failed(Ligation failed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY-pETDuet-1] ----- Failed(Ligation failed)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY-pETDuet-1]-----Failed(Ligation failed)
 +
</div>
 +
<div class="Text3_Sub">
 +
Measure the resistance of Caco2 cell
 +
</div>
 +
<div class="Text3">
 +
<Green>Selection part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Construction of the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[nisI- pCLP7]
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[nisI- pCLP7] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[nisI- pCLP7] ----- Succeeded
 +
</div>
 +
<div class="Text3_Sub">
 +
Clean up:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pCLP7]
 +
</div>
 +
<div class="Text3_Sub">
 +
Ligation:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[nisI] + [pCLP7]
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [nisI- pCLP7]
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">9/8</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pCLP7] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-RBS-RFP-T] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Ligation:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pCLP7] + [plac-RBS-RFP-T]
 +
</div>
 +
<div class="Text3_Sub">
 +
Manufacture competent cell with [pJ23102-RBS-CI-T]
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>   
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1]-----Succeeded
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel Purification:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
4℃ Ligation overnight:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY]+ [pETDuet-1]
 +
</div>   
 +
<div class="Text3_SubSub">
 +
[PYY]+ [pETDuet-1]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY]+ [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Measure the resistance of Caco2 cell
 +
</div>
 +
<div class="Text3_Sub">
 +
Transfect hek293 cell
 +
</div>
 +
<div class="Text3">
 +
<Green>Selection part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Culture [nisI- pCLP7] in LB broth
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">9/9</div></div>
 +
<div class="Text3">
 +
<Blue>Passaged hek293 cell</Blue>
 +
</div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pCLP7], [place-RBS-CI-T]
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pCLP7] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[place-RBS-CI-T]-----Failed(concentration to low)
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [plac-RBS-RFP-T- pCIp]
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [plac-RBS-CI-T-NucA] in competent cell with [pJ23102-RBS-CI-T]
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>   
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1]-----Succeeded
 +
</div>
 +
<div class="Text3_Sub">
 +
Measure the resistance of Caco2 cell
 +
</div>
 +
<div class="Text3">
 +
<Green>Selection part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[nisI- pCLP7]
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[nisI- pCLP7] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[nisI- pCLP7] ----- Failed
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">9/10</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-RBS-RFP-T- pCLP7]
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac-RBS-RFP-T- pCLP7] ----- Failed
 +
</div>
 +
<div class="Text3_Sub">
 +
Manufacture L.casei competent cell
 +
</div>
 +
<div class="Text3_Sub">
 +
Electroporation of [pCIp] in L.casei competent cell
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel Purification:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
4℃ Ligation overnight:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY]+ [pETDuet-1]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY]+ [pETDuet-1]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY]+ [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1],[PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
 +
</div>   
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet1] cleave by EcoRI and SpeI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY-pETDuet-1] cleave by EcoRI and SpeI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY-pETDuet1] cleave by EcoRI and SpeI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] ----- Succeeded
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel Purification:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Measure the resistance of Caco2 cell
 +
</div>
 +
<div class="Text3">
 +
<Green>Selection part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[nisI- pCLP7]
 +
</div>
 +
<div class="Text3_Sub">
 +
Nisin Immunity Test
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">9/11</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac+RBS-RFP-T- pCLP7]
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[plac+RBS-RFP-T- pCLP7] ----- Failed
 +
</div>
 +
<div class="Text3_Sub">
 +
Test electroporation of <i>L.casei</i>
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
4℃ Ligation overnight:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY]+ [pETDuet-1]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY]+ [pETDuet-1]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY]+ [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1] in XL1blue
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>   
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet1] cleave by XbaI and KpnI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY-pETDuet-1] cleave by XbaI and KpnI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] cleave by EcoRI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] cleave by EcoRI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] cleave by EcoRI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] cleave by EcoRI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] ----- Succeeded
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] cleave by XbaI and PstI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[pETDuet-1] ----- Succeeded
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel Purification:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
4℃ Ligation overnight:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY]+ [pETDuet-1]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY]+ [pETDuet-1]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY]+ [pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Measure the resistance of Caco2 cell
 +
</div>
 +
<div class="Text3">
 +
<Green>Selection part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Nisin Immunity Test
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">9/12</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Test absorbance(O.D. 600nm) of E.coli in Suicide circuit
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>   
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet1] cleave by XbaI and KpnI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY-pETDuet-1] cleave by XbaI and KpnI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_Sub">
 +
Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1] in XL1blue
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet1] cleave by XbaI and KpnI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY-pETDuet-1] cleave by XbaI and KpnI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY-pETDuet-1] ----- Failed
 +
</div>
 +
<div class="Text3">
 +
<Green>Selection part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Nisin Immunity Test
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">9/13</div></div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>   
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[SP-PYY-pETDuet1] cleave by XbaI and KpnI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[PYY-pETDuet-1] cleave by XbaI and KpnI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
 +
</div>
 +
<div class="Text3">
 +
<Green>Selection part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Nisin Immunity Test
 +
</div>
  
  
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Maintained by the iGEM team NTU-LIHPAO-Taiwan&nbsp;&nbsp;&nbsp;&nbsp;©2015 NTU-LIHPAO-Taiwan
 
Maintained by the iGEM team NTU-LIHPAO-Taiwan&nbsp;&nbsp;&nbsp;&nbsp;©2015 NTU-LIHPAO-Taiwan
 
</div>
 
</div>
 +
 +
 +
 
</div>
 
</div>
 
</div>
 
</div>
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Latest revision as of 13:22, 18 September 2015

NTU-LIHPAO-Taiwan

7/4
Manufacture CP+ plate
7/6
Learn how to transform
Suicide Part:
Transformation of:
[RBS] (BBa_B0034), [C2] (BBa_C0053)
7/7
Learn how to manufacture competent cell
Manufacture competent cell
PYY part:
Transformation of:
[promoter] (BBa_J23100), [signal peptide] (BBa_K554002), [terminator] (BBa_B0015)
Suicide Part:
Transformation of:
[pC1] (BBa_R0051), [pC2] (BBa_R0053), [endolysin] (BBa_K112806), [C1] (BBa_C0051)
7/8
Learn how to extract the plasmid
Learn how to conduct the gel extraction and electrophoresis
Suicide Part:
Extract the plasmid:
[RBS] (BBa_B0034), [C2] (BBa_C0053)
Restriction enzyme:
[RBS] cleave by EcoRI, [C2] cleave by EcoRI and PstI
Gel electrophoresis:
[RBS] ----- Result: Failed (Smeared band was too blurred)
[C2] ----- Result: Succeeded
Transformation of:
[pC1] (BBa_R0051), [pC2] (BBa_R0053), [endolysin] (BBa_K112806) again
7/9
Suicide Part:
Extract the plasmid:
[RBS] (BBa_B0034), [C1] (BBa_C0051), [endolysin] (BBa_K112806)
Gel electrophoresis:
[RBS] ----- Result: Failed (Smeared band was too blurred)
[C1] ----- Result: Succeeded
[endolysin] ----- Result: Failed (Smeared band was too blurred)
PYY part:
Extract the plasmid:
[signal peptide] (BBa_K554002), [terminator] (BBa_B0015)
Gel electrophoresis:
[signal peptide] ----- Result: Succeeded
[terminator] ----- Result: Succeeded
7/10
Manufacture CP+ plate
Manufacture Amp+ plate
Suicide Part:
Gel electrophoresis:
[RBS] (BBa_B0034) ----- Result: Failed (Smeared band was too blurred)
[C2] (BBa_C0053) ----- Result: Succeeded
Gel extraction:
[C2]
7/11
Suicide Part:
Extract the plasmid:
[RBS] (BBa_B0034), [C1] (BBa_C0051), [endolysin] (BBa_K112806)
PYY part:
Transformation of:
[promoter] (BBa_J23101)
7/12
Suicide Part:
Extract the plasmid:
[pC1] (BBa_R0051)
7/13
PYY part:
Extract the plasmid:
[promoter] (BBa_J23101)
Gel electrophoresis (two white colonies & two red colonies):
[promoter] ------ Result: Failed (white colonies didn’t have band, red colonies had band but not the correct size, we speculated that was pollution)
7/20
Discuss about total synthesis
7/13
PYY part:
Transformation of:
[terminator] (BBa_B0015)
7/24
Discuss our project with professor
PYY part:
Extract the plasmid:
[terminator] (BBa_B0015)
Gel electrophoresis:
[terminator] ----- Result: Failed
7/25
Discuss our problems with Instructor
7/26
PYY part:
Cultured [terminator] again
7/27
PYY part:
Extract the plasmid:
[terminator] (BBa_B0015)
Gel electrophoresis:
[terminator] ----- Result: Succeeded
8/2
Suicide Part:
Extract the plasmid:
[RFP](BBa_E1010)
Restriction enzyme:
[RFP] cleave by EcoRI and SpeI
[Cl] cleave by XbaI and PstI
[pSB1A3] cleave by EcoRI and PstI
8/5
Passaged L.c.393
Manufacture MRS plate
PYY part
Redissoluted DNA of total synthesis
Restriction enzyme:
[signalpeptide -PYY] cleave by EcoRI and PstI
Ligation:
[signalpeptide-PYY]+ [pSB1C3]
Transformation
8/6
Passaged L.c.393
PYY part
Restriction enzyme:
[PYY] cleave by EcoRI and PstI
[TAT-PYY] cleave by EcoRI and PstI
[Y2R] cleave by KpnI and XhoI
Ligation:
[PYY]+ [pSB1C3]
[TAT-PYY]+ [pSB1C3]
[Y2R]+[pcDNA]
Transformation
8/7
PYY part
Gel electrophoresis:
Result:
signalpeptide-PYY] ----- Failed (Smeared band was too blurred)
[PYY] ----- Failed (Smeared band was too blurred)
Transform again
8/10
Manufacture CP+ plate
Suicide part
Gel electrophoresis:
Result:
[RFP+Terminator] ----- Failed (Confirmed it was pollution)
[Cl+Terminator] ----- Failed
Clean up:
[RFP+Terminator]
PYY part
Clean up:
[signalpeptide-PYY]+ [pSB1A3]
[PYY]+ [pSB1A3]
[TAT-PYY]+ [pSB1A3]
[Y2R]+[pcDNA]
Transformation
8/11
Suicide part
Clean up:
[Cl+Terminator]
Gel electrophoresis
Result:
[RFP+Terminator]----- Failed (Smeared band was too blurred)
[Cl+Terminator] ----- Failed (Smeared band was too blurred)
PYY part
Extract the plasmid:
[TAT-PYY], [Y2R]
8/12
Suicide part
Redissoluted DNA of total synthesis
Restriction enzyme:
[Lac-RBS] cleave by EcoRI and PstI
[NucA-Terminator] cleave by EcoRI and PstI
[NIsI] cleave by EcoRI and PstI
[LcSPddhol] cleave by EcoRI and PstI
Ligation:
[Lac-RBS]+ [pSB1C3]
[NucA-Terminator]+ [pSB1C3]
[NIsI]+ [pSB1C3]
[LcSPddhol]+ [pSB1C3]
Transformation
PYY part
Gel electrophoresis:
Result:
[TAT-PYY]----- Failed(Confirmed it was pollution)
[Cll] ----- Succeeded
8/13
Suicide part
Extract the plasmid:
[Lac-RBS]+ [pSB1C3]
[NucA-Terminator]+ [pSB1C3]
[NIsI]+ [pSB1C3]
Clean up:
[Cl], [terminator], [RFP]
Ligation:
[Cl]+ [terminator]
[RFP]+ [terminator]
Gel electrophoresis:
Result:
[Cl]+ [terminator] ----- Succeeded
[RFP]+ [terminator] ----- Succeeded
PYY part
Extract the plasmid:
[LcSPddhol]+ [pSB1C3]
Clean up:
[TAT-PYY]
8/14
DNA sequencing:
[SP-PYY], [PYY], [Y2R], [Lac-RBS], [NucA-Terminator], [NIsI], [LcSPddhol]
Suicide part
Ligation:
[Cl]+ [terminator]
Gel electrophoresis:
Result:
[Lac-RBS] ----- Succeeded
[NucA-Terminator] ----- Succeeded
[NIsI] ----- Succeeded
[LcSPddhol] ----- Succeeded
Extract the plasmid:
[RFP-terminator]
8/15
Passaged L.c.393
Suicide part
Extract the plasmid:
[Cl-terminator]
Gel electrophoresis:
Result:
[RFP-terminator] ----- Succeeded
[Cl-terminator] ----- Succeeded
PYY part
Extract the plasmid:
[TAT-PYY]
Transformation of [TAT-PYY]
Restriction enzyme:
[TAT-PYY] cleave by EcoRI and PstI
Gel electrophoresis:
Result:
[TAT-PYY] ----- Succeeded
8/16
Passaged L.c.393
Manufacture LB broth
Manufacture MRS broth
Suicide part
Restriction enzyme:
[Cl-Terminator] cleave by EcoRI and PstI
[RFP-Terminator] cleave by EcoRI and PstI
PYY part
Transformation of [SP-PYY], [PYY], [TAT-PYY]
8/17
Suicide part
Ligation:
[plac-RFP-T], [plac-Cl-T]
Transformation of [plac-RFP-T], [plac-Cl-T]
PYY part
Culture [SP-PYY], [PYY], [TAT-PYY] in BL21 Competent
E. coli and Rosetta Competent E. coli
8/18
Suicide part
Culture [plac-RFP], [plac-Cl], [NucA] in LB broth
Extract the plasmid:
[plac-RFP], [plac-Cl]
Gel electrophoresis:
Result:
[plac-RFP] ----- Succeeded
[plac-Cl] ----- Succeeded
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY]):
Sonication of Competent E. coli
8/19
Suicide part
Extract the plasmid:
[NucA]
Gel electrophoresis:
Result:
[NucA] ----- Succeeded
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY]):
SDS-page
Transfer
Blocking
Primary antibody
8/20
Passaged Caco2 cell
Suicide part
Gel Purification:
[plac-Cl-T], [pCl-NucA-T]
Ligation:
[plac-Cl-T], [pCl-NucA-T]
Transformation of [plac-Cl-T], [pCl-NucA-T]
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY]):
Secondary antibody
Chemiluminescent detection
Result:
[SP-PYY] ----- Failed (no band displayed)
[PYY] ----- Failed (no band displayed)
[TAT-PYY] ----- Failed (no band displayed)
Western blot ([SP-PYY], [PYY], [TAT-PYY]):
SDS-page
Transfer
Blocking
Primary antibody
8/21
Suicide part
Culture [[plac-Cl-T], [pCl-NucA-T] in LB broth
Extract the plasmid:
[plac-Cl-T], [pCl-NucA-T]
Gel electrophoresis:
Result:
[plac-Cl-T] ----- Succeeded
[pCl-NucA-T] ----- Failed (band was too blurred)
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY]):
Secondary antibody
Chemiluminescent detection
Result:
[SP-PYY] ----- Failed (band was too blurred)
[PYY] ----- Failed (no band displayed)
[TAT-PYY] ----- Failed (no band displayed)
Transformation of [SP-PYY], [PYY], [TAT-PYY], [RFP] again. (use [RFP] as negative control)
Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP] in BL21
Competent E. coli and Rosetta Competent E. coli
8/22
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY] , [RFP]):
Sonication of Competent E. coli
8/23
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY] , [RFP]):
SDS-page
Transfer
Blocking
Primary antibody
8/24
Suicide part
Restriction enzyme:
[NucA] cleave by SpeI and PstI
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY]):
Secondary antibody
Chemiluminescent detection
Result:
[SP-PYY] ----- Failed (band was not specificity)
[PYY] ----- Failed (band was not specificity)
[TAT-PYY] ----- Failed (band was not specificity)
Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP]in mini
8/25
Suicide part
Restriction enzyme:
[plac-C1-T-backbone] cleave by EcoRI
Gel electrophoresis:
Result:
[plac-C1-T] ----- Failed
Redissoluted [pJ23102]
Transformation of [pCl], [pCll], [RBS], [pJ23102]
Culture [pCl], [pCll], [RBS] in LB broth
PYY part
Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP] in midi
Centrifugal and took the supernatant
Bradford protein assay
8/26
Passaged Caco2 cell
Suicide part
Ligation:
[plac-Cl-T]
Transformation of [plac-Cl-T]
Extract the plasmid:
[plac], [pCl], [pCll], [RBS]
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
SDS-page
Transfer
Blocking
Primary antibody
8/27
Suicide part
Transformation of [RBS-Cl-T], [RBS-RFP-T]
Culture [plac-Cl-T],[RBS-Cl-T],[RBS-RFP-T],[pJ23102] in LB broth
Extract the plasmid:
[plac-Cl-T], [pJ23102]
Gel electrophoresis:
Result:
[plac-Cl-T] ----- Succeeded
[pJ23102] ----- Succeeded
Restriction enzyme:
[pCl-T] cleave by SpeI and PstI
Gel Purification:
[pCll]
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
Secondary antibody
Chemiluminescent detection
Result:
[SP-PYY] ----- Failed (no band displayed)
[PYY] ----- Failed (no band displayed)
[TAT-PYY] ----- Failed (no band displayed)
Transformation of [SP-PYY], [PYY], [TAT-PYY], [RFP] again
Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP] in BL21
Competent E. coli
8/28
Suicide part
Ligation:
[pCIt]+[NucA]
Extract the plasmid:
[Cl-T], [RFP-T]
Gel electrophoresis:
Result:
[Cl-T] ----- Failed (band size was wrong)
[RFP-T] ----- Failed (band size was wrong)
Restriction enzyme:
[plac-T] cleave by SpeI and PstI
[pJ23102] cleave by SpeI and PstI
Gel Purification:
[pCI-T]
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
SDS-page
Transfer
Blocking
Primary antibody
8/29
Passaged Caco2 cell
Suicide part
Extract the plasmid:
[RBS-Cl-T], [RBS-RFP-T]
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
Secondary antibody
Chemiluminescent detection
Result:
[SP-PYY] ----- Failed (no band displayed)
[PYY] ----- Failed (no band displayed)
[TAT-PYY] ----- Failed (no band displayed)
Histag column([SP-PYY], [PYY], [TAT-PYY] , [RFP])
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
SDS-page
Transfer
Blocking
Primary antibody
8/30
Suicide part
Extract the plasmid:
[RBS], [pCl]
Gel electrophoresis:
Result:
[RBS] ----- Succeeded
[pCl] ----- Failed
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
Secondary antibody
Chemiluminescent detection
Result:
[SP-PYY] ----- Failed (no band displayed)
[PYY] ----- Failed (no band displayed)
[TAT-PYY] ----- Failed (no band displayed)
8/31
Passaged hek293 cell
Suicide part
Gel Purification:
[RBS], [CI-T]
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
SDS-page
Transfer
Blocking
Primary antibody
9/1
Suicide part
Ligation:
[RBS] + [CI-T]
Culture [RBS- CI-T] in LB broth
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
Secondary antibody
Chemiluminescent detection
Result:
[SP-PYY] ----- Failed (no band displayed)
[PYY] ----- Failed (no band displayed)
[TAT-PYY] ----- Failed (no band displayed)
Transformation of [SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
Selection part
Culture [nisI] in MRS broth
9/2
Suicide part
Extract the plasmid:
[RBS- CI-T]
Gel electrophoresis:
Result:
[RBS- CI-T] ----- Succeeded
PYY part
Extract the plasmid:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
Restriction enzyme:
[SP-PYY] cleave by EcoRI
[PYY] cleave by EcoRI
[TAT-PYY] cleave by EcoRI
[pETDuet-1] cleave by EcoRI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1] ----- Succeeded
Selection part
Nisin Immunity Test
Culture [nisI] in LB broth
9/3
PYY part
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Failed (small band didn’t display)
[PYY] ----- Failed (small band didn’t display)
[TAT-PYY] ----- Failed(small band didn’t display)
[pETDuet-1] ----- Succeeded
Again -->
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1] ----- Succeeded
16℃ Ligation 4hr:
[SP-PYY]+ [pETDuet-1] ----- Failed
[PYY]+ [pETDuet-1] ----- Failed
[TAT-PYY]+ [pETDuet-1] ----- Failed
Selection part
Extract the plasmid:
[nisI-pSB1C3]
Restriction enzyme:
[nisI-pSB1C3] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[nisI-pSB1C3] ----- Succeeded
Nisin Immunity Test
9/4
Suicide part
Ligation:
[pJ23102] + [RBS-CI-T]
[plac] + [RBS-CI-T]
Transformation of [pJ23102-RBS-CI-T], [plac-RBS-CI-T]
PYY part
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1] ----- Succeeded
Gel Purification:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
16℃ Ligation 4hr:
[SP-PYY]+ [pETDuet-1] ----- Failed
[PYY]+ [pETDuet-1] ----- Failed
[TAT-PYY]+ [pETDuet-1] ----- Failed
9/5
Suicide part
Culture[pJ23102-RBS-CI-T], [plac-RBS-CI-T] in LB broth
PYY part
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1] ----- Succeeded
Gel Purification:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
16℃ Ligation 4hr:
[SP-PYY]+ [pETDuet-1]
[PYY]+ [pETDuet-1]
[TAT-PYY]+ [pETDuet-1]
Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Transformation of [SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
Measure the resistance of Caco2 cell
9/6
Suicide part
Extract the plasmid:
[placE-RBS-CI-T], [pJ23102-RBS-CI-T], [placE]
Gel electrophoresis:
Result:
[placE-RBS-CI-T] ----- Failed
[pJ23102-RBS-CI-T] ----- Succeeded
[placE] ----- Succeeded
PYY part
Extract the plasmid:
[SP-PYY-pETDuet-1],[PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Restriction enzyme:
[SP-PYY-pETDuet-1] cleave by XbaI and PstI
[PYY-pETDuet-1] cleave by XbaI and PstI
[TAT-PYY-pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY-pETDuet-1] ----- Failed
[PYY-pETDuet-1] ----- Failed
[TAT-PYY-pETDuet-1] ----- Failed
Extract the plasmid:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY-pETDuet-1]-----Failed(Ligation failed)
[PYY-pETDuet-1] ----- Failed(Ligation failed)
[TAT-PYY-pETDuet-1]-----Failed(Ligation failed)
Measure the resistance of Caco2 cell
9/7
Suicide part
Restriction enzyme:
[placE] cleave by SpeI and PstI
[RBS-CI-T] cleave by XbaI and PstI
Transformation of [pCLP7]
Restriction enzyme:
[pCLP7] cleave by XbaI and PstI
[nisi] cleave by XbaI and PstI
PYY part
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Restriction enzyme:
[SP-PYY-pETDuet-1] cleave by XbaI and PstI
[PYY-pETDuet-1] cleave by XbaI and PstI
[TAT-PYY-pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Failed(small band didn’t display)
[PYY] ----- Failed(small band didn’t display)
[TAT-PYY] ----- Failed(small band didn’t display)
[pETDuet-1]-----Failed(small band didn’t display)
[SP-PYY-pETDuet-1]-----Failed(Ligation failed)
[PYY-pETDuet-1] ----- Failed(Ligation failed)
[TAT-PYY-pETDuet-1]-----Failed(Ligation failed)
Measure the resistance of Caco2 cell
Selection part
Construction of the plasmid:
[nisI- pCLP7]
Restriction enzyme:
[nisI- pCLP7] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[nisI- pCLP7] ----- Succeeded
Clean up:
[pCLP7]
Ligation:
[nisI] + [pCLP7]
Transformation of [nisI- pCLP7]
9/8
Suicide part
Restriction enzyme:
[pCLP7] cleave by XbaI and PstI
[plac-RBS-RFP-T] cleave by XbaI and PstI
Ligation:
[pCLP7] + [plac-RBS-RFP-T]
Manufacture competent cell with [pJ23102-RBS-CI-T]
PYY part
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1]-----Succeeded
Gel Purification:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
4℃ Ligation overnight:
[SP-PYY]+ [pETDuet-1]
[PYY]+ [pETDuet-1]
[TAT-PYY]+ [pETDuet-1]
Measure the resistance of Caco2 cell
Transfect hek293 cell
Selection part
Culture [nisI- pCLP7] in LB broth
9/9
Passaged hek293 cell
Suicide part
Extract the plasmid:
[pCLP7], [place-RBS-CI-T]
Gel electrophoresis:
Result:
[pCLP7] ----- Succeeded
[place-RBS-CI-T]-----Failed(concentration to low)
Transformation of [plac-RBS-RFP-T- pCIp]
Transformation of [plac-RBS-CI-T-NucA] in competent cell with [pJ23102-RBS-CI-T]
PYY part
Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1]-----Succeeded
Measure the resistance of Caco2 cell
Selection part
Extract the plasmid:
[nisI- pCLP7]
Restriction enzyme:
[nisI- pCLP7] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[nisI- pCLP7] ----- Failed
9/10
Suicide part
Extract the plasmid:
[plac-RBS-RFP-T- pCLP7]
Gel electrophoresis:
Result:
[plac-RBS-RFP-T- pCLP7] ----- Failed
Manufacture L.casei competent cell
Electroporation of [pCIp] in L.casei competent cell
PYY part
Gel Purification:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
4℃ Ligation overnight:
[SP-PYY]+ [pETDuet-1]
[PYY]+ [pETDuet-1]
[TAT-PYY]+ [pETDuet-1]
Extract the plasmid:
[SP-PYY-pETDuet-1],[PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Restriction enzyme:
[SP-PYY-pETDuet1] cleave by EcoRI and SpeI
[PYY-pETDuet-1] cleave by EcoRI and SpeI
[TAT-PYY-pETDuet1] cleave by EcoRI and SpeI
Gel electrophoresis:
Result:
[SP-PYY-pETDuet-1] ----- Failed
[PYY-pETDuet-1] ----- Failed
[TAT-PYY-pETDuet-1] ----- Failed
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1] ----- Succeeded
Gel Purification:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
Measure the resistance of Caco2 cell
Selection part
Extract the plasmid:
[nisI- pCLP7]
Nisin Immunity Test
9/11
Suicide part
Extract the plasmid:
[plac+RBS-RFP-T- pCLP7]
Gel electrophoresis:
Result:
[plac+RBS-RFP-T- pCLP7] ----- Failed
Test electroporation of L.casei
PYY part
4℃ Ligation overnight:
[SP-PYY]+ [pETDuet-1]
[PYY]+ [pETDuet-1]
[TAT-PYY]+ [pETDuet-1]
Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1] in XL1blue
Extract the plasmid:
[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Restriction enzyme:
[SP-PYY-pETDuet1] cleave by XbaI and KpnI
[PYY-pETDuet-1] cleave by XbaI and KpnI
[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
Gel electrophoresis:
Result:
[SP-PYY-pETDuet-1] ----- Failed
[PYY-pETDuet-1] ----- Failed
[TAT-PYY-pETDuet-1] ----- Failed
Extract the plasmid:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
Restriction enzyme:
[SP-PYY] cleave by EcoRI
[PYY] cleave by EcoRI
[TAT-PYY] cleave by EcoRI
[pETDuet-1] cleave by EcoRI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1] ----- Succeeded
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1] ----- Succeeded
Gel Purification:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
4℃ Ligation overnight:
[SP-PYY]+ [pETDuet-1]
[PYY]+ [pETDuet-1]
[TAT-PYY]+ [pETDuet-1]
Measure the resistance of Caco2 cell
Selection part
Nisin Immunity Test
9/12
Suicide part
Test absorbance(O.D. 600nm) of E.coli in Suicide circuit
PYY part
Extract the plasmid:
[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Restriction enzyme:
[SP-PYY-pETDuet1] cleave by XbaI and KpnI
[PYY-pETDuet-1] cleave by XbaI and KpnI
[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
Gel electrophoresis:
Result:
[SP-PYY-pETDuet-1] ----- Failed
[PYY-pETDuet-1] ----- Failed
[TAT-PYY-pETDuet-1] ----- Failed
Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1] in XL1blue
Extract the plasmid:
[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Restriction enzyme:
[SP-PYY-pETDuet1] cleave by XbaI and KpnI
[PYY-pETDuet-1] cleave by XbaI and KpnI
[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
Gel electrophoresis:
Result:
[SP-PYY-pETDuet-1] ----- Failed
[PYY-pETDuet-1] ----- Failed
[TAT-PYY-pETDuet-1] ----- Failed
Selection part
Nisin Immunity Test
9/13
PYY part
Extract the plasmid:
[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Restriction enzyme:
[SP-PYY-pETDuet1] cleave by XbaI and KpnI
[PYY-pETDuet-1] cleave by XbaI and KpnI
[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
Selection part
Nisin Immunity Test
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