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

 
(52 intermediate revisions by 2 users not shown)
Line 227: Line 227:
 
top: 32px;
 
top: 32px;
 
bottom: 0px;
 
bottom: 0px;
−
left: 50%;
+
left: 51%;
 
right: 15%;
 
right: 15%;
 
list-style:none;
 
list-style:none;
Line 308: Line 308:
 
}
 
}
  
−
#width_small {
+
.width_small {
 
width: 100px; /*--改主要選單寬度--*/
 
width: 100px; /*--改主要選單寬度--*/
−
}
 
−
 
−
#width_large {
 
−
width: 140px; /*--改主要選單寬度--*/
 
 
}
 
}
  
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padding-top: 5px;
 
padding-top: 5px;
 
padding-bottom: 5px;
 
padding-bottom: 5px;
 +
line-height: 30px;
 
}
 
}
  
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height: 94%;
 
height: 94%;
 
}
 
}
 +
  
  
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border-top-color: #079235;
 
border-top-color: #079235;
 
border-top-width: 3px;
 
border-top-width: 3px;
 +
}
 +
 +
.Container_Top1{
 +
height: 30px;
 +
width: 100%;
 
}
 
}
  
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text-align: center;
 
text-align: center;
 
line-height: 20px;
 
line-height: 20px;
 +
}
 +
 +
 +
/*-------------------------------------------*/
 +
/*---------------Article Picture-------------*/
 +
/*-------------------------------------------*/
 +
 +
.Container_Article_Picture {
 +
height: 280px;
 +
width: 100%;
 +
padding-top: 25px;
 +
padding-bottom: 25px;
 +
}
 +
 +
.Article_Picture {
 +
width: 480px;
 +
height: auto;
 +
left: 50%;
 +
right: auto;
 +
background-color: #FFF;
 +
margin: 0 0 0 -240px;
 +
position: absolute;
 +
}
 +
 +
.Article_PictureText {
 +
width: 430px;
 +
height: auto;
 +
padding-left: 25px;
 +
padding-right: 25px;
 +
border-top-width: 2px;
 +
border-top-style: dashed;
 +
border-top-color: #fe5838;
 +
}
 +
 +
.Text_Picture {
 +
font-size: 18px;
 +
font-family: Calibri;
 +
text-align: justify;
 +
line-height: 26px;
 +
color: #08923a;
 
}
 
}
  
Line 593: Line 636:
  
 
.Text_underline {
 
.Text_underline {
 +
font-size: 20px;
 +
font-family: Calibri;
 +
padding-top: 20px;
 +
padding-right: 150px;
 +
padding-bottom: 0px;
 +
padding-left: 150px;
 +
line-height: 26px;
 
text-decoration: underline;
 
text-decoration: underline;
 
}
 
}
Line 700: Line 750:
 
<div class="Slidemenu">
 
<div class="Slidemenu">
 
<ul>
 
<ul>
−
<li><div id=width_small><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan">Home</a></div>
+
<li><div class=width_small><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan">Home</a></div>
 
</li>
 
</li>
−
+
 
−
<li><div id=width_small><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Team">Team</a></div>
+
<li><div class=width_small span style="cursor:default"><a>Team</a></div>
−
</li>
+
−
+
−
<li><div id=width_small span style="cursor:default"><a>Project</a></div>
+
 
<ul class="subs">
 
<ul class="subs">
−
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Project/Abstract">Abstract</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Team">Team</a></li>
−
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Project/Overview">Overview</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Attributions">Attributions</a></li>
−
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Project/Parts">Parts</a></li>
+
−
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Project/Results">Results</a></li>
+
 
</ul>
 
</ul>
 
</li>
 
</li>
 
 
−
<li><div id=width_small span style="cursor:default"><a>Modeling</a></div>
+
<li><div class=width_small span style="cursor:default"><a>Project</a></div>
 
<ul class="subs">
 
<ul class="subs">
−
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Modeling/Abstract">Abstract</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Description">Description</a></li>
−
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Modeling/Simulation">Simulation</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Design">Design</a></li>
−
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Modeling/Results">Results</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Results">Results</a></li>
 +
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Modeling">Modeling</a></li>
 +
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Experiments">Protocols</a></li>
 
</ul>
 
</ul>
 
</li>
 
</li>
 
 
−
<li><div id=width_small span style="cursor:default"><div id=Position_Now><a>Notebook</a></div></div>
+
<li><div class=width_small span style="cursor:default"><a>Parts</a></div>
 
<ul class="subs">
 
<ul class="subs">
−
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Notebook">Note</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Parts">Team Parts</a></li>
−
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Protocols">Protocols</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>
 
</ul>
 
</ul>
 +
</li>
 +
 +
<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>
 
          
 
          
−
<li><div id=width_small><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Safety">Safety</a></div>
+
<li><div class=width_small><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Safety">Safety</a></div>
 
</li>
 
</li>
 
 
−
<li><div id=width_large><a href="#">Human Practice</a></div>
+
<li><div class=width_small span style="cursor:default"><a>Society</a></div>
 
<ul class="subs">
 
<ul class="subs">
−
<li><a href="#">Sub Item</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Practices">Human Practices</a></li>
−
<li><a href="#">Sub Item</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Collaborations">Collaborations</a></li>
−
<li><a href="#">Sub Item</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Entrepreneurship">Entrepreneurship</a></li>
 
</ul>
 
</ul>
 
</li>
 
</li>
Line 753: Line 804:
 
<li><a href="#First1">July</a></li>
 
<li><a href="#First1">July</a></li>
 
<li><a href="#First2">August</a></li>
 
<li><a href="#First2">August</a></li>
 +
<li><a href="#First3">September</a></li>
 
</ul>
 
</ul>
 
</li>
 
</li>
Line 787: Line 839:
 
<div class="ContentBox">
 
<div class="ContentBox">
 
<div class="ContentHolder">
 
<div class="ContentHolder">
−
<div class="Text1" id="First1">July</div>
+
<div class="Container_Top1"></div>
  
−
<div class="Container2"><div class="Text2">7/4</div></div>
+
<div class="Container2" id="First1"><div class="Text2">7/4</div></div>
 
<div class="Text3">
 
<div class="Text3">
 
<Green>Manufacture <i>CP<Super>+</Super></i> plate</Green>
 
<Green>Manufacture <i>CP<Super>+</Super></i> plate</Green>
Line 800: Line 852:
 
</div>
 
</div>
 
<div class="Text3">
 
<div class="Text3">
−
<Green>Apoptosis part:</Green>
+
<Green>Suicide Part:</Green>
 
</div>
 
</div>
 
<div class="Text3_Sub">
 
<div class="Text3_Sub">
Line 827: Line 879:
 
</div>
 
</div>
 
<div class="Text3">
 
<div class="Text3">
−
<Green>Apoptosis part:</Green>
+
<Green>Suicide Part:</Green>
 
</div>
 
</div>
 
<div class="Text3_Sub">
 
<div class="Text3_Sub">
Line 845: Line 897:
 
</div>
 
</div>
 
<div class="Text3">
 
<div class="Text3">
−
<Green>Apoptosis part:</Green>
+
<Green>Suicide Part:</Green>
 
</div>
 
</div>
 
<div class="Text3_Sub">
 
<div class="Text3_Sub">
Line 878: Line 930:
 
<div class="Container2"><div class="Text2">7/9</div></div>
 
<div class="Container2"><div class="Text2">7/9</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<Green>Apoptosis part:</Green>
+
<Green>Suicide Part:</Green>
 
</div>
 
</div>
 
<div class="Text3_Sub">
 
<div class="Text3_Sub">
Line 927: Line 979:
 
</div>
 
</div>
 
<div class="Text3">
 
<div class="Text3">
−
<Green>Apoptosis part:</Green>
+
<Green>Suicide Part:</Green>
 
</div>
 
</div>
 
<div class="Text3_Sub">
 
<div class="Text3_Sub">
Line 948: Line 1,000:
 
<div class="Container2"><div class="Text2">7/11</div></div>
 
<div class="Container2"><div class="Text2">7/11</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<Green>Apoptosis part:</Green>
+
<Green>Suicide Part:</Green>
 
</div>
 
</div>
 
<div class="Text3_Sub">
 
<div class="Text3_Sub">
Line 969: Line 1,021:
 
<div class="Container2"><div class="Text2">7/12</div></div>
 
<div class="Container2"><div class="Text2">7/12</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
<Green>Apoptosis part:</Green>
+
<Green>Suicide Part:</Green>
 
</div>
 
</div>
 
<div class="Text3_Sub">
 
<div class="Text3_Sub">
Line 1,068: Line 1,120:
 
[terminator] ----- Result: Succeeded
 
[terminator] ----- Result: Succeeded
 
</div>
 
</div>
 +
 +
 +
<div class="Container2" id="First2"><div class="Text2">8/2</div></div>
 +
<div class="Text3">
 +
<Green>Suicide Part:</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</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">
 +
<Blue>Passaged L.c.393</Blue>
 +
</div>
 +
<div class="Text3">
 +
<Blue>Manufacture MRS plate</Blue>
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Redissoluted DNA of total synthesis
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</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">
 +
<Blue>Passaged L.c.393</Blue>
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</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">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
signalpeptide-PYY] ----- Failed (Smeared band was too blurred)
 +
</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">
 +
<Blue>Manufacture CP<sup>+</sup> plate</Blue>
 +
</div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</div>
 +
<div class="Text_underline">
 +
Result:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[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 class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Clean up:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[signalpeptide-PYY]+ [pSB1A3]
 +
</div>
 +
<div class="Text3_SubSub">
 +
[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">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Clean up:
 +
</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">
 +
[RFP+Terminator]----- Failed (Smeared band was too blurred)
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Cl+Terminator] ----- Failed (Smeared band was too blurred)
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY], [Y2R]
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/12</div></div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Redissoluted DNA of total synthesis
 +
</div>
 +
<div class="Text3_Sub">
 +
Restriction enzyme:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Lac-RBS] cleave by EcoRI and PstI
 +
</div>
 +
<div class="Text3_SubSub">
 +
[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">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Gel electrophoresis:
 +
</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">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</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_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 class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[LcSPddhol]+ [pSB1C3]
 +
</div>
 +
<div class="Text3_Sub">
 +
Clean up:
 +
</div>
 +
<div class="Text3_SubSub">
 +
[TAT-PYY]
 +
</div>
 +
 +
 +
<div class="Container2"><div class="Text2">8/14</div></div>
 +
<div class="Text3">
 +
<Blue>DNA sequencing:</Blue>
 +
</div>
 +
<div class="Text3_Sub">
 +
[SP-PYY], [PYY], [Y2R], [Lac-RBS], [NucA-Terminator], [NIsI], [LcSPddhol]
 +
</div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Ligation:
 +
</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">
 +
<Blue>Passaged L.c.393</Blue>
 +
</div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</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">
 +
[RFP-terminator] ----- Succeeded
 +
</div>
 +
<div class="Text3_SubSub">
 +
[Cl-terminator] ----- Succeeded
 +
</div>
 +
<div class="Text3">
 +
<Green>PYY part</Green>
 +
</div>
 +
<div class="Text3_Sub">
 +
Extract the plasmid:
 +
</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">
 +
<Blue>Passaged L.c.393</Blue>
 +
</div>
 +
<div class="Text3">
 +
<Blue>Manufacture LB broth</Blue>
 +
</div>
 +
<div class="Text3">
 +
<Blue>Manufacture MRS broth</Blue>
 +
</div>
 +
<div class="Text3">
 +
<Green>Suicide part</Green>
 +
</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 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>
  
  

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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