Difference between revisions of "Part:BBa K404200"

 
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<title>BBa_K404003</title>
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<br>
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<h3 style="margin-left: 0cm; text-indent: 0cm;"><a
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name="_Toc275885926"></a><a name="_Toc275817885"><span
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lang="EN-US">Modularization: Adapting
 +
pSB1C3 to loop
 +
insertions – pSB1C3_001</span></a></h3>
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<p class="MsoNormal"
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style="text-indent: 0cm; line-height: 150%;"><span
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lang="EN-US">To
 +
fulfill iGEM requirements, all plasmids need to be submitted in pSB1C3.
 +
Therefore, primers were ordered for amplifying <i>RepVP123</i>
 +
containing all
 +
modifications done so far by PCR and cloning them into pSB1C3. Still,
 +
pSB1C3
 +
contains two restriction sites for SspI and PvuII restriction enzymes
 +
in its
 +
CAT marker. Since these are necessary for cloning ViralBricks in this
 +
vector,
 +
the iGEM Team Freiburg_Bioware 2010 decided in agreement with iGEM
 +
Headquarters
 +
to implement a new standard for the pSB1C3 backbone which was named
 +
pSB1C3_001.
 +
Both restriction sites interfering with ViralBrick insertions were
 +
mutated to
 +
make SspI and PvuII single-cutters (see method development).</span></p>
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<table class="MsoTableGrid"
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style="border: medium none ; margin-left: auto; border-collapse: collapse; text-align: left; margin-right: auto;"
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border="0" cellpadding="0" cellspacing="0">
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  <tbody>
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    <tr>
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      <td
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style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;">
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      <p class="MsoNormal"
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style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
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align="center"><img style="width: 584px; height: 278px;"
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id="Picture 16"
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src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg10_pSB1C3_001_mutations.png"
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alt=""></p>
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      <p class="MsoCaption"><span lang="EN-US">Figure
 +
1 </span><span style="color: windowtext; font-weight: normal;"
 +
lang="EN-US">Comparison
 +
of pSB1C3 (upper row) and pSB1C3_001 (lower row). Deletions of SspI and
 +
PvuII are marked by red boxes.</span></p>
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      </td>
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    </tr>
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  </tbody>
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</table>
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<p class="MsoNormal"><i><span lang="EN-US">RepVP123</span></i><span
 +
lang="EN-US">
 +
containing both <i>rep</i> and <i>cap</i>
 +
synthetic gene fragments including
 +
the re-mutation of KpnI and the downstream p5TATA-less promotor was
 +
cloned into
 +
the newly constructed pSB1C3_001. Testing this newly assembled plasmid
 +
in cell
 +
culture revealed unexpected data: Not only did the newly assembled
 +
plasmid work
 +
(see Figure 2), but in comparison to pAAV containing the same <i>RepVP123</i>
 +
construct, pSB1C3_001 showed an about 3 times higher transduction
 +
efficiency.
 +
Although exact reasons are still unknown, these results are probably
 +
related to
 +
the length reduction of pSB1C3_001 compared to the original pAAV
 +
plasmid
 +
of
 +
approximately 1000 base pairs.</span></p>
 +
<span
 +
style="font-size: 11pt; line-height: 200%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
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lang="EN-US"></span><span lang="EN-US">&nbsp;</span>
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<table
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style="border: medium none ; width: 466px; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
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class="MsoTableGrid" border="0" cellpadding="0"
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cellspacing="0">
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  <tbody>
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    <tr style="height: 209.85pt;">
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      <td
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style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 4661px; height: 209.85pt;">
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      <p class="MsoNormal"
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style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
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align="center"><span
 +
style="font-size: 10pt; line-height: 200%;"><img
 +
style="width: 516px; height: 258px;" alt="" id="Chart 15"
 +
src="https://static.igem.org/mediawiki/2010/2/2b/Freiburg10_pAAV_pSB1C3_001.png"></span></p>
 +
      <p class="MsoCaption"><span lang="EN-US">Figure
 +
2 </span><span style="color: windowtext; font-weight: normal;"
 +
lang="EN-US">AAV-293
 +
cells were transfected with three plasmids pHelper,
 +
pSB1C3_001_[AAV2]-Rep-VP123_p5-TATAless or pAAV_RC_IRCK and
 +
pSB1C3_[AAV2]-left-ITR_pCMV_beta-globin_mVenus_hGH_[AAV2]-right-ITR
 +
providing essential genes and proteins for producing viral particles.
 +
48 hours post transfection, viral particles were harvested by
 +
freeze-thaw lysis and centrifugation followed by HT1080 transduction.
 +
mVenus expression of viral genomes was determined by flow cytometry
 +
analysis
 +
24 hours post infection. </span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">Fluorescence
 +
is measured in surviving cells.</span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">&nbsp;Results
 +
showed functionality of <i>RepVP123</i>
 +
within the pSB1C3_001 vector and additionally increased transduction
 +
efficiency.</span></p>
 +
      </td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
<h4 style="margin-left: 0cm; text-indent: 0cm;"><a
 +
name="_Toc275885927"></a><a name="_Toc275817886"><span
 +
lang="EN-US"></span></a></h4>
 +
</body>
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</html>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 18:27, 27 October 2010

pSB1C3-001

BBa_K404003 BBa_K404003

Modularization: Adapting pSB1C3 to loop insertions – pSB1C3_001

To fulfill iGEM requirements, all plasmids need to be submitted in pSB1C3. Therefore, primers were ordered for amplifying RepVP123 containing all modifications done so far by PCR and cloning them into pSB1C3. Still, pSB1C3 contains two restriction sites for SspI and PvuII restriction enzymes in its CAT marker. Since these are necessary for cloning ViralBricks in this vector, the iGEM Team Freiburg_Bioware 2010 decided in agreement with iGEM Headquarters to implement a new standard for the pSB1C3 backbone which was named pSB1C3_001. Both restriction sites interfering with ViralBrick insertions were mutated to make SspI and PvuII single-cutters (see method development).

Figure 1 Comparison of pSB1C3 (upper row) and pSB1C3_001 (lower row). Deletions of SspI and PvuII are marked by red boxes.

RepVP123 containing both rep and cap synthetic gene fragments including the re-mutation of KpnI and the downstream p5TATA-less promotor was cloned into the newly constructed pSB1C3_001. Testing this newly assembled plasmid in cell culture revealed unexpected data: Not only did the newly assembled plasmid work (see Figure 2), but in comparison to pAAV containing the same RepVP123 construct, pSB1C3_001 showed an about 3 times higher transduction efficiency. Although exact reasons are still unknown, these results are probably related to the length reduction of pSB1C3_001 compared to the original pAAV plasmid of approximately 1000 base pairs.

 

Figure 2 AAV-293 cells were transfected with three plasmids pHelper, pSB1C3_001_[AAV2]-Rep-VP123_p5-TATAless or pAAV_RC_IRCK and pSB1C3_[AAV2]-left-ITR_pCMV_beta-globin_mVenus_hGH_[AAV2]-right-ITR providing essential genes and proteins for producing viral particles. 48 hours post transfection, viral particles were harvested by freeze-thaw lysis and centrifugation followed by HT1080 transduction. mVenus expression of viral genomes was determined by flow cytometry analysis 24 hours post infection. Fluorescence is measured in surviving cells. Results showed functionality of RepVP123 within the pSB1C3_001 vector and additionally increased transduction efficiency.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 2059
    Illegal suffix found in sequence at 10
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2059
    Illegal SpeI site found at 11
    Illegal PstI site found at 25
    Illegal NotI site found at 18
    Illegal NotI site found at 2065
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2059
    Illegal XhoI site found at 1043
    Illegal XhoI site found at 1935
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 2059
    Illegal suffix found in sequence at 11
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 2059
    Illegal suffix found in sequence at 1
  • 1000
    COMPATIBLE WITH RFC[1000]