Difference between revisions of "Part:BBa K404121"

 
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K404121 short</partinfo>
 
<partinfo>BBa_K404121 short</partinfo>
 +
<br/><br><br>
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{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
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! colspan="2" style="background:#66bbff;"|[https://parts.igem.org/Part:BBa_K404121 [AAV2]-left-ITR_pCMV_betaglobin_mCherry_hGH_[AAV2]-right-ITR ]
 +
|-
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! colspan="2"|[[Image:Freiburg10_Vectorplasmid composite 3.png|300px]]
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|-
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|'''BioBrick Nr.'''
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|[https://parts.igem.org/Part:BBa_K404121 BBa_K404121]
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|-
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|'''RFC standard'''
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|[https://parts.igem.org/Help:Assembly_standard_10 RFC 10]
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|-
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|'''Requirement'''
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|pSB1C3<br>
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|-
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|'''Source'''
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|pAAV_MCS: provided by Stratagene<br/>
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mCherry:[https://parts.igem.org/Part:BBa_J06504 BBa_J06504]
 +
|-
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|'''Submitted by'''
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|[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]
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|}
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<br>
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 +
[[Image:Freiburg10_Vectorplasmid composite 3.png|left|thumb|400px]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
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<div class="WordSection1">
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<p class="MsoNormal" style="text-align: justify;"><span
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style="font-size: 10pt; line-height: 115%;">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.</span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
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style="font-size: 10pt; line-height: 115%;">The provided
 +
tripartite system is independent of a
 +
superinfection&nbsp; 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, <i>beta-globin</i> 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.&nbsp; </span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="font-size: 10pt; line-height: 115%;">The composite
 +
part </span><span style="font-size: 10pt; line-height: 115%;">[AAV2]-left-ITR_pCMV_betaglobin_mCherry_hGH_[AAV2]-right-ITR
 +
provided by the iGEM team Freiburg_Bioware comprises all <i>cis</i>-elements
 +
required for efficient transgene expression in mammalian cells
 +
(polyadenylation
 +
terminator: </span><span
 +
style="font-size: 10pt; line-height: 115%;">BBa_K404108 </span><span
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style="font-size: 10pt; line-height: 115%;">and
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transcription enhancer element: </span><span
 +
style="font-size: 10pt; line-height: 115%;">BBa_K404107)</span><span
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style="font-size: 10pt; line-height: 115%;"> and
 +
encapsidation into virus
 +
particles (</span><span
 +
style="font-size: 10pt; line-height: 115%;">ITRs:
 +
BBa_K404100 and BBa_K404101). The mCherry coding sequence as gene of
 +
interest enables
 +
facile detection of transduced cells using flow cytometry or
 +
fluorescence
 +
microscopy. While mCherry is expressed in the cytosol, mVenus can be detected in nuclei due to the VP1 integrated NLS. </span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="font-size: 10pt; line-height: 115%;"><img alt=""
 +
src="https://static.igem.org/mediawiki/2010/2/22/Freiburg10_TimeLapse_AAV-293_mVenus%2BGOI_mCherry_390px.gif"></span></p>
 +
<p style="float: left; margin: 0px 0px 40px 0px; font-size: 10px;">Movie 1:HT1080: mVenus (nuclei) and mCherry (cytosol) expression</p>
 +
</div>
 +
</body>
 +
</html>
 +
  
−
x
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 12:49, 31 October 2010

[AAV2]-left-ITR_pCMV_betaglobin_mCherry_hGH_[AAV2]-right-ITR


[AAV2-left-ITR_pCMV_betaglobin_mCherry_hGH_[AAV2]-right-ITR ]
Freiburg10 Vectorplasmid composite 3.png
BioBrick Nr. BBa_K404121
RFC standard RFC 10
Requirement pSB1C3
Source pAAV_MCS: provided by Stratagene

mCherry:BBa_J06504

Submitted by [http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]


Freiburg10 Vectorplasmid composite 3.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. 

The composite part [AAV2]-left-ITR_pCMV_betaglobin_mCherry_hGH_[AAV2]-right-ITR provided by the iGEM team Freiburg_Bioware comprises all cis-elements required for efficient transgene expression in mammalian cells (polyadenylation terminator: BBa_K404108 and transcription enhancer element: BBa_K404107) and encapsidation into virus particles (ITRs: BBa_K404100 and BBa_K404101). The mCherry coding sequence as gene of interest enables facile detection of transduced cells using flow cytometry or fluorescence microscopy. While mCherry is expressed in the cytosol, mVenus can be detected in nuclei due to the VP1 integrated NLS.

Movie 1:HT1080: mVenus (nuclei) and mCherry (cytosol) expression


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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2580