Project

Part:BBa_K404117

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

[AAV2]-left-ITR_pCMV_betaglobin

[AAV2-left-ITR_pCMV_betaglobin]
Freiburg10 Vectorplasmid precursors 4.png
BioBrick Nr. BBa_K404117
RFC standard RFC 10
Requirement pSB1C3
Source pAAV_MCS provided by Stratagene
Submitted by FreiGEM 2010



Part: [AAV2]-left-ITR_pCMV_betaglobin

 

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. 

Facilitating the cloning steps the iGEM team Freiburg_Bioware 2010 provides the 5´nucleotide components in respect to the desired gene of interest, which are the left inverted terminal repeat (BBa_K404100) followed by the CMV promoter (BBa_K404102) and a putative enhancer-element (BBa_K404107). Compared to the parent construct (BBa_K404114), the presence of the beta-globin intron may enhance the expression of the downstream transgene. In context of the viral particle assembly, the presence of this genetic element may lower the packaging efficiency since the latter is affected by the size of the gene of interest encapsidated into the virus particle. Consequently, fewer functional particles are produced by the eukaryotic host cell.

 


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
    COMPATIBLE WITH RFC[1000]


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