Project

Part:BBa_K404129

Designed by: Freiburg Bioware 2010   Group: iGEM10_Freiburg_Bioware   (2010-10-11)

[AAV2]-left-ITR_phTERT_betaglobin_mCherry_hGH_[AAV2]-right-ITR


[AAV2-left-ITR_phTERT_betaglobin_mCherry_hGH_[AAV2]-right-ITR ]
Freiburg10 Vectorplasmid composite 11.png
BioBrick Nr. BBa_K404129
RFC standard RFC 10
Requirement pSB1C3
Source pAAV_MCS: provided by Stratagene

BBa_J06504

Submitted by FreiGEM 2010


Freiburg10 Vectorplasmid composite 11.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.

Furthermore, by providing the tumor-specific promoter with the “Virus Vonstruction Kit”, the iGEM Freiburg_Bioware team 2010 ensures another layer of specificity and safety to the recombinant viral vector system. Telomerase activation is a critical step in human tumorigenesis and about 90% of human tumors show telomerase activity. In most somatic cells, the phTERT promoter is inactive. This prevents expression of the hTERT protein subunit and renders the healthy tissue telomerase negative.

 


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 2383


[edit]
Categories
//chassis/eukaryote/human
//viral_vectors
//viral_vectors/aav
//viral_vectors/aav/vector_plasmid
Parameters
None