Difference between revisions of "Part:BBa K404119"

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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K404119 short</partinfo>
 
<partinfo>BBa_K404119 short</partinfo>
 
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<br><br><br>
 
[[Image:Freiburg10_Vectorplasmid composite 1.png|thumb|center|480px]]<br>
 
[[Image:Freiburg10_Vectorplasmid composite 1.png|thumb|center|480px]]<br>
  
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</head>
 
</head>
 
<body lang="EN-US">
 
<body lang="EN-US">
<div class="WordSection1">
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<div class="WordSection1"><br>
<h1><span style="color: windowtext;">[AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR</span></h1>
+
<p class="MsoNormal">&nbsp;<big><big><big><big><big><big><big><big><big><big><big><big><big><span
<p class="MsoNormal">&nbsp;</p>
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<p class="MsoNormal"><big><big><big><big><big><big><big><big><big><big><big><big><big><span
+
 
  style="font-size: 10pt; line-height: 115%;">Producing
 
  style="font-size: 10pt; line-height: 115%;">Producing
 
recombinant virus particles for therapeutical applications is, besides
 
recombinant virus particles for therapeutical applications is, besides
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Comparing mVenus expression of the reference plasmid and the
 
Comparing mVenus expression of the reference plasmid and the
 
pSB1C3_mVenus plasmid revealed
 
pSB1C3_mVenus plasmid revealed
that biological functionality of the reassembled plasmid was preserved.</small>
+
that biological functionality of the reassembled plasmid was preserved.</small></p>
</p>
+
<p class="MsoNormal"></p>
 +
<p class="MsoNormal"><small></small> </p>
 
<p class="MsoNormal"><span
 
<p class="MsoNormal"><span
 
  style="color: rgb(0, 176, 80);"></span></p>
 
  style="color: rgb(0, 176, 80);"></span></p>
 
<table class="MsoTableGrid"
 
<table class="MsoTableGrid"
  style="border: medium none ; border-collapse: collapse; width: 657px; height: 1044px;"
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  style="border: medium none ; border-collapse: collapse; width: 644px; height: 935px;"
 
  border="1" cellpadding="0" cellspacing="0">
 
  border="1" cellpadding="0" cellspacing="0">
 
   <tbody>
 
   <tbody>
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  style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
 
  style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
 
  valign="top" width="641">
 
  valign="top" width="641">
       <p class="MsoCaption"><a name="_Ref275784576">Figure</a>
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       <p class="MsoCaption"><small><a
1: Flow cytometry analysis of fluorescent protein expression in
+
name="_Ref275784576">Figure</a> 1: Flow cytometry
transduced HT1080 cells. <span style="font-weight: normal;">For
+
analysis of fluorescent protein expression in transduced HT1080 cells. <span
viral particle production, AAV-293 cells were transfected with the
+
style="font-weight: normal;">For viral particle production,
reassembled vector plasmid (BBa_K404119) or the reference plasmid,
+
AAV-293 cells were transfected with the reassembled vector plasmid
respectively.</span> A<span style="font-weight: normal;">:
+
(BBa_K404119) or the reference plasmid, respectively.</span> A</small><span
Gating non transduced cells (control); subcellular debris and cellular
+
style="font-weight: normal;"><small>: Gating non
aggreates can be distinguished from single cells by size, estimated
+
transduced cells (control); subcellular debris and cellular aggreates
forward scatter (FS Lin) and granularity, estimated side scatter (SS
+
can be distinguished from single cells by size, estimated forward
Lin) B: Non-transduced cells plotted against cells expressing mVenus
+
scatter (FS Lin) and granularity, estimated side scatter (SS Lin) B:
 +
Non-transduced cells plotted against cells expressing mVenus
 
(Analytical gate was set such that 1% or fewer of negative control
 
(Analytical gate was set such that 1% or fewer of negative control
 
cells fell within the positive region (R5) C: Gating transduced cells
 
cells fell within the positive region (R5) C: Gating transduced cells
(R2 </span><span
+
(R2 </small></span><small><span
 
  style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙
 
  style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙
 
       </span><span style="font-weight: normal;">R14)
 
       </span><span style="font-weight: normal;">R14)
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  style="font-weight: normal;">: Transduced cells plotted
 
  style="font-weight: normal;">: Transduced cells plotted
 
against cells expressing mVenus. R10 comprises transduced cells
 
against cells expressing mVenus. R10 comprises transduced cells
detected by mVenus fluorescence.</span> J<span
+
detected by mVenus fluorescence.</span> J</small><span
  style="font-weight: normal;">: Overlay of non-transduced
+
  style="font-weight: normal;"><small>: Overlay of
(red) and transduced (green) cells plotted against mVenus expression. </span></p>
+
non-transduced (red) and transduced (green) cells plotted against
 +
mVenus expression.</small> </span></p>
 
       </td>
 
       </td>
 
       <td></td>
 
       <td></td>
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<p class="MsoNormal">&nbsp;</p>
 
<p class="MsoNormal">&nbsp;</p>
 
<table class="MsoTableGrid"
 
<table class="MsoTableGrid"
  style="border: medium none ; border-collapse: collapse; width: 659px; height: 474px;"
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  style="border: medium none ; border-collapse: collapse; width: 640px; height: 413px;"
 
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   <tbody>
 
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  style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
 
  style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
 
  valign="top" width="641">
 
  valign="top" width="641">
       <p class="MsoNormal"
+
       <p class="MsoCaption"><img alt=""
style="margin-bottom: 0.0001pt; line-height: normal;"><img
+
  src="%5BImage:Freiburg10%20Quantification%20of%20mVenus%20fluorescence%20by%20flow%20cytometry%20analysis.png%5D">&nbsp;<br>
alt=""
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       <img style="width: 481px; height: 381px;" alt=""
  src="%5BImage:Freiburg10%20Quantification%20of%20mVenus%20fluorescence%20by%20flow%20cytometry%20analysis.png%5D">&nbsp;</p>
+
id="Diagramm 90"
       <p class="MsoNormal"
+
  src="https://static.igem.org/mediawiki/2010/8/8e/Freiburg10_Quantification_of_mVenus_fluorescence_by_flow_cytometry_analysis.png"><br>
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal; page-break-after: avoid;"
+
       <small><a name="_Ref275784852">Figure</a>
align="center"><img style="width: 481px; height: 381px;"
+
2: Quantification of mVenus fluorescence by flow cytometry analysis. </small><span
alt="" id="Diagramm 90"
+
  style="font-weight: normal;"><small>The data source is
  src="https://static.igem.org/mediawiki/2010/8/8e/Freiburg10_Quantification_of_mVenus_fluorescence_by_flow_cytometry_analysis.png"></p>
+
identical to Figure 13. Compared to the reference vector plasmid,
       <p class="MsoCaption"><a name="_Ref275784852">Figure</a>
+
mVenus expression in HT1080 cells is enhanced in case of the assembled
2: Quantification of mVenus fluorescence by flow cytometry analysis. <span
+
plasmid (pSB1C3_mVenus)</small> </span></p>
  style="font-weight: normal;">The data source is identical to
+
Figure 13. Compared to the reference vector plasmid, mVenus expression
+
in HT1080 cells is enhanced in case of the assembled plasmid
+
(pSB1C3_mVenus) </span></p>
+
 
       </td>
 
       </td>
 
     </tr>
 
     </tr>
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</body>
 
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</html>
 
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Revision as of 15:02, 27 October 2010

[AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR


Freiburg10 Vectorplasmid composite 1.png


 Producing recombinant virus particles for therapeutical applications is, besides specific cell targeting, purification and quantification assays of AAV-2, one intention of the Virus Construction Kit provided by the iGEM team Freiburg_Bioware 2010. For obtaining a modular toolkit, the complex biological system of the Adeno-associated virus serotype 2 was examined by an exhaustive literature search. Subsequently, the essential components for AAV-2 particle production were extracted and redesigned to match the iGEM standard.

The provided tripartite system is independent of a superinfection  of Adeno- or herpes simplex viruses since the genes encoding the required helper-proteins are co-transfected. Inside the eukaryotic host cell, the DNA sequence containing the inverted terminal repeats (ITRs) is extracted and later encapsidated into the preformed capsids after production of single-stranded DNA. Consequently, this plasmid is known as the vector plasmid (pGOI). Promoter, beta-globin intron and the hGH terminator signal are flanked by the ITRs (ITRs, BBa_K404100 and BBa_K404101) and regulate transgene expression. The vector plasmid containing the desired gene of interest is cotransfected with the RepCap plasmid (BBa_K404001, BBa_K404002 or BBa_K404003) and the pHelper plasmid. To obtain the fully assembled vector plasmid, several assembly steps have to be performed. 

After assembly of plasmids containing all required elements, vector plasmid functionality was confirmed in cell culture. AAV-293 cells stably expressing the E1A and E1B proteins were transfected with three plasmids (pHelper, pRC, pGOI). Virus particles were harvested 72 hours post transfection and the tumor cell line HT1080 was transduced with the recombinant viral vectors encapsidating the gene of interest mVenus (BBa_I757008).

In the beginning, the iGEM team Freiburg_Bioware 2010 used a commercial vector plasmid, pAAV-MCS (Stratagene), to determine whether virus particle production by AAV-293 cells could be achieved. iGEM RFC 25 restriction enzyme sites were introduced and the fluorescent protein mVenus was subcloned into this plasmid. Subsequently, this plasmid was used as a reference and compared to the assembled vector plasmid pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (abbreviated pSB1C3_mVenus, BBa_K404119). Fluorescence expression data obtained by flow cytometry analysis are shown in Figure 11 and Figure 12. Comparing mVenus expression of the reference plasmid and the pSB1C3_mVenus plasmid revealed that biological functionality of the reassembled plasmid was preserved.

pSB1C3_mVenus (BBa_K404119)

Figure 1: Flow cytometry analysis of fluorescent protein expression in transduced HT1080 cells. For viral particle production, AAV-293 cells were transfected with the reassembled vector plasmid (BBa_K404119) or the reference plasmid, respectively. A: Gating non transduced cells (control); subcellular debris and cellular aggreates can be distinguished from single cells by size, estimated forward scatter (FS Lin) and granularity, estimated side scatter (SS Lin) B: Non-transduced cells plotted against cells expressing mVenus (Analytical gate was set such that 1% or fewer of negative control cells fell within the positive region (R5) C: Gating transduced cells (R2 R14) (plasmids used for transfection: pGOI: pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus: BBa_K404119), pHelper, pRC. D: Transduced cells plotted against cells expressing mVenus. R10 comprises transduced cells detected by mVenus fluorescence. E: Overlay of non-transduced (red) and transduced (green). F: Gating non-transduced cells (control). G: Non-transduced cells plotted against cells expressing mVenus (R5). H: Gating transduced cells (R14 R2) (plasmids used for transfection: pGOI: pAAV_mVenus, pHelper). I: Transduced cells plotted against cells expressing mVenus. R10 comprises transduced cells detected by mVenus fluorescence. J: Overlay of non-transduced (red) and transduced (green) cells plotted against mVenus expression.

 

 

 

Figure 2: Quantification of mVenus fluorescence by flow cytometry analysis. The data source is identical to Figure 13. Compared to the reference vector plasmid, mVenus expression in HT1080 cells is enhanced in case of the assembled plasmid (pSB1C3_mVenus)

 

 

 



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1319
    Illegal AgeI site found at 2039
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2441