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

 
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Line 636: 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 743: Line 750:
 
<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>
  
Line 768: Line 775:
 
<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 798: 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 1,212: Line 1,219:
 
Gel electrophoresis:
 
Gel electrophoresis:
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,236: Line 1,243:
 
Gel electrophoresis:
 
Gel electrophoresis:
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,248: Line 1,255:
 
Clean up:
 
Clean up:
 
</div>
 
</div>
−
<div class="Text3_Sub">
+
<div class="Text3_SubSub">
 
[RFP+Terminator]
 
[RFP+Terminator]
 
</div>
 
</div>
Line 1,287: Line 1,294:
 
Gel electrophoresis
 
Gel electrophoresis
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,353: Line 1,360:
 
Gel electrophoresis:
 
Gel electrophoresis:
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,398: Line 1,405:
 
Gel electrophoresis:
 
Gel electrophoresis:
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,443: Line 1,450:
 
Gel electrophoresis:
 
Gel electrophoresis:
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,455: Line 1,462:
 
[NIsI] ----- Succeeded
 
[NIsI] ----- Succeeded
 
</div>
 
</div>
−
<div class="Text3_Sub">
+
<div class="Text3_SubSub">
 
[LcSPddhol] ----- Succeeded
 
[LcSPddhol] ----- Succeeded
 
</div>
 
</div>
Line 1,482: Line 1,489:
 
Gel electrophoresis:
 
Gel electrophoresis:
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,512: Line 1,519:
 
Gel electrophoresis:
 
Gel electrophoresis:
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,590: Line 1,597:
 
Gel electrophoresis:
 
Gel electrophoresis:
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,623: Line 1,630:
 
Gel electrophoresis:
 
Gel electrophoresis:
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,683: Line 1,690:
 
Chemiluminescent detection
 
Chemiluminescent detection
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,728: Line 1,735:
 
Gel electrophoresis:
 
Gel electrophoresis:
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,749: Line 1,756:
 
Chemiluminescent detection
 
Chemiluminescent detection
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,827: Line 1,834:
 
Chemiluminescent detection
 
Chemiluminescent detection
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,857: Line 1,864:
 
Gel electrophoresis:
 
Gel electrophoresis:
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,891: Line 1,898:
 
</div>
 
</div>
 
<div class="Text3">
 
<div class="Text3">
−
<Green>Suicide part</Green>
+
<Green>Suicide part</Green>
 
</div>
 
</div>
 
<div class="Text3_Sub">
 
<div class="Text3_Sub">
Line 1,916: Line 1,923:
 
  <div class="Text3_SubSub">
 
  <div class="Text3_SubSub">
 
SDS-page
 
SDS-page
−
< /div>
+
</div>
 
<div class="Text3_SubSub">
 
<div class="Text3_SubSub">
 
Transfer
 
Transfer
Line 1,947: Line 1,954:
 
Gel electrophoresis:
 
Gel electrophoresis:
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 1,980: Line 1,987:
 
Chemiluminescent detection
 
Chemiluminescent detection
 
</div>     
 
</div>     
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
 
<div class="Text3_SubSub">
 
<div class="Text3_SubSub">
 
[SP-PYY] ----- Failed (no band displayed)
 
[SP-PYY] ----- Failed (no band displayed)
−
</div>
+
</div>
−
<div class="Text3_SubSub">
+
<div class="Text3_SubSub">
 
[PYY] ----- Failed (no band displayed)
 
[PYY] ----- Failed (no band displayed)
 
</div>
 
</div>
 
<div class="Text3_SubSub">
 
<div class="Text3_SubSub">
 
[TAT-PYY] ----- Failed (no band displayed)
 
[TAT-PYY] ----- Failed (no band displayed)
−
</div>
+
</div>
−
<div class="Text3_Sub">
+
<div class="Text3_Sub">
 
Transformation of [SP-PYY], [PYY], [TAT-PYY], [RFP] again
 
Transformation of [SP-PYY], [PYY], [TAT-PYY], [RFP] again
−
</div>
+
</div>
−
<div class="Text3_Sub">
+
<div class="Text3_Sub">
 
Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP] in BL21  
 
Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP] in BL21  
 
</div>
 
</div>
−
<div class="Text3_Sub">
+
<div class="Text3_Sub">
 
Competent <i>E. coli</i>
 
Competent <i>E. coli</i>
 
</div>
 
</div>
  
  
−
<div class="Container2"><div class="Text2">8/28</div></div>
+
<div class="Container2"><div class="Text2">8/28</div></div>
−
<div class="Text3">
+
<div class="Text3">
 
<Green>Suicide part</Green>
 
<Green>Suicide part</Green>
 
</div>
 
</div>
Line 2,022: Line 2,029:
 
Gel electrophoresis:
 
Gel electrophoresis:
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
<div class="Text_underline">
 
Result:
 
Result:
 
</div>
 
</div>
Line 2,028: Line 2,035:
 
[Cl-T] ----- Failed (band size was wrong)
 
[Cl-T] ----- Failed (band size was wrong)
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
  <div class="Text3_SubSub">
 
[RFP-T] ----- Failed (band size was wrong)
 
[RFP-T] ----- Failed (band size was wrong)
 
</div>
 
</div>
−
<div class="Text3_Sub">
+
<div class="Text3_Sub">
 
Restriction enzyme:
 
Restriction enzyme:
−
</div>
+
</div>
−
<div class="Text3_SubSub">
+
  <div class="Text3_SubSub">
 
[plac-T] cleave by SpeI and PstI
 
[plac-T] cleave by SpeI and PstI
 
</div>
 
</div>
−
<div class="Text3_SubSub">
+
  <div class="Text3_SubSub">
 
[pJ23102] cleave by SpeI and PstI
 
[pJ23102] cleave by SpeI and PstI
 
</div>
 
</div>
Line 2,054: Line 2,061:
 
  <div class="Text3_SubSub">
 
  <div class="Text3_SubSub">
 
SDS-page
 
SDS-page
−
< /div>
+
</div>
−
<div class="Text3_SubSub">
+
<div class="Text3_SubSub">
 
Transfer
 
Transfer
 
</div>     
 
</div>     
Line 2,065: Line 2,072:
 
</div>
 
</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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