Difference between revisions of "Part:BBa K404159"

Revision as of 02:11, 28 October 2010

pCMV_CFP_Middle-Linker_[AAV2]-VP23 (ViralBrick-587KO-Empty)

This part is used for cotranfection with parts containing VP1up (BBa_K404164-BBa_K404166)

Cyan fluorescent protein (CFP) in RFC25 standard

Nearly all cyan fluorescent proteins are generated on the basis of GFP-like proteins from jellyfish Aequorea macrodactyla. mCyan is an enhanced version of the cyan flourescent protein. The cyan spectral region is from 470 up to 500 nm. mCFP has a higher brightness level, acid insensivity and greater photostability. Like GFP, CFP can be used as a reporter gene. So it can be used in vitro for cell culture experiments. In our viral vector system, CFP is used as gene of interest in the vector plasmid. In this content, it is able to detect the efficiency of the constructed viral particles. Sequence and Features BBa_K404319 SequenceAndFeatures

CMV

CMV promoter is derived from human Cytomegalovirus, which belongs to Herpesvirus group. All family members share the ability to remain in latent stage in the human body. CMV is located upstream of immediate-early gene. However, CMV promoter is an example of widely used promoters and is present in mammalian expression vectors. The advantage of CMV is the high-level constitutive expression in mostly all human tissues [Fitzsimons et al., 2002].

Middle Linker ( Gly-Gly-Ser-Gly)x2

This part is a linker, it can be used to connect two parts and add additional space between them. That can be necessary to avoid interactions between these parts.

Capsid

The AAV capsid consists of 60 capsid protein subunits. The three cap proteins VP1, VP2, and VP3 are encoded in an overlapping reading frame. Arranged in a stoichiometric ratio of 1:1:10, they form an icosahedral symmetry. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Alternative splicing and translation initiation of VP2 at a nonconventional ACG initiation codon promote the expression of VP1, VP2 and VP3. The VP proteins share a common C terminus and stop codon, but begin with a different start codon. The N termini of VP1 and VP2 play important roles in infection and contain motifs that are highly homologous to a phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+).

ViralBrick 587-KO empty

(BBa_K4004210) The primary receptor of AAV-2 is the heparan sulfate proteoglycan (HSPG) receptor (Perabo et al. 2006). Its binding motif consists of five amino-acids located on the capsid surface: R484/R487, K532, R585/587. (Trepel et al. 2009). The positively charged arginine residues interact with the HSPGs' negatively charged acid residues. Opie et al. have shown that two point mutations (R585A and R588A) are sufficient to eliminate the heparin binding affinity in AAV2. (Opie et al. 2003). This ViralBrick has been created to introduce this knockout into other constructs. The biobricks with containing this knockout are annotated with „HSPG-ko“.

Sequence and Features

References

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