Project

Part:BBa_K404126

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

[AAV2]-left-ITR_pCMV_betaglobin_CD_hGH_[AAV2]-right-ITR


[AAV2-left-ITR_pCMV_betaglobin_CD_hGH_[AAV2]-right-ITR ]
Freiburg10 Vectorplasmid composite 8.png
BioBrick Nr. BBa_K404126
RFC standard RFC 10
Requirement pSB1C3
Source pAAV_MCS: provided by Stratagene
Submitted by FreiGEM 2010


Freiburg10 Vectorplasmid composite 8.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 gene of interest (GOI) cytosine deaminase ) was inserted into the transgene expression cassette and its biological activity was test in cell culture. In E. coli, Cytosine Deaminase (CD) (EC 3.5.4.1) is encoded by codA. The protein plays a crucial role in nucleotide synthesis since it catalyzes the deamination of cytosine to uracil (Danielsen et al. 1992).
In eukaryotic cells, expression of this protein can be used to convert the non-toxic prodrug 5-fluorocytosine to the toxic compound 5-fluorouracil. The presence of this nucleotide analogue in the target cell blocks the synthesis of thymidine by inhibition of the essential enzyme thymidylate synthase, therefore leading to cell death.

 

Furthermore, another advantage of 5-FUra is its solubility and ability to freely diffuse into adjacent cells [Domin et al., 1993; Huber et al., 1994] therefore enhancing the bystander effect.

References:

Danielsen, S. et al., 1992. Characterization of the Escherichia coli codBA operon encoding cytosine permease and cytosine deaminase. Molecular microbiology, 6(10), pp.1335-44. Available at: http://www.ncbi.nlm.nih.gov/pubmed/1640834.

 




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 2606
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3008


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