Difference between revisions of "Part:BBa K404159"

Latest revision as of 16:31, 31 October 2010

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

pCMV_CFP_Middle-Linker_(AAV2)-VP23(ViralBrick-587KO-Empty)
BioBrick Nr.	BBa_K404159
RFC standard	RFC 10
Requirement	pSB1C3
Source
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

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

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

(BBa_K243005)

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

(BBa_K404006)

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-terminus of VP1 plays important role in infection and contains a motif highly homologous to a phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+). VP2 contains basic regions, too.

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

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 665
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 3261
Illegal SapI site found at 2172

References

Mellon. 2002. Epidermal growth factor receptor and bladder cancer.Postgraduate medical journal78, no. 924 (October): 584-9. doi:10.1136/pmj.78.924.584. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1742539&tool=pmcentrez&rendertype=abstract.
Friedman, Mikaela, Anna Orlova, Eva Johansson, Tove L J Eriksson, Ingmarie Höidén-Guthenberg, Vladimir Tolmachev, Fredrik Y Nilsson, and Stefan Ståhl. 2008. Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule. Journal of molecular biology376, no. 5: 1388-402. doi:10.1016/j.jmb.2007.12.060. http://www.ncbi.nlm.nih.gov/pubmed/18207161.
Göstring, Lovisa, Ming Tsuey Chew, Anna Orlova, Ingmarie Höidén-guthenberg, Anders Wennborg, Jörgen Carlsson, and Fredrik Y Frejd. 2010. Quantification of internalization of EGFR-binding Affibody molecules: Methodological aspects. International Journal of Oncology 36, no. 4 (March): 757-763. doi:10.3892/ijo_00000551. http://www.spandidos-publications.com/ijo/36/4/757.
Hirsch,Fred R, Marileila Varella-Garcia, Paul a Bunn, Michael V Di Maria, Robert Veve, Roy M Bremmes, Anna E Barón, Chan Zeng, and Wilbur a Franklin. 2003. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 21, no. 20 (October): 3798-807. doi:10.1200/JCO.2003.11.069. http://www.ncbi.nlm.nih.gov/pubmed/12953099.
Nord, K, E Gunneriusson, J Ringdahl, S Ståhl, M Uhlén, and P A Nygren. 1997. Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain. Nature biotechnology 15, no. 8 (August): 772-7. doi:10.1038/nbt0897-772. http://www.ncbi.nlm.nih.gov/pubmed/9255793.
Orlova, Anna, Vladimir Tolmachev, Rikard Pehrson, Malin Lindborg, Thuy Tran, Mattias Sandström, Fredrik Y Nilsson, Anders Wennborg, Lars Abrahmsén, and Joachim Feldwisch. 2007. Synthetic affibody molecules: a novel class of affinity ligands for molecular imaging of HER2-expressing malignant tumors. Cancer research 67, no. 5 (March): 2178-86. doi:10.1158/0008-5472.CAN-06-2887. http://www.ncbi.nlm.nih.gov/pubmed/17332348.
Walker, R a, and S J Dearing. 1999. Expression of epidermal growth factor receptor mRNA and protein in primary breast carcinomas. Breast cancer research and treatment53, no. 2 (January): 167-76. http://www.ncbi.nlm.nih.gov/pubmed/10326794.
Wikman, M, a-C Steffen, E Gunneriusson, V Tolmachev, G P Adams, J Carlsson, and S Ståhl. 2004. Selection and characterization of HER2/neu-binding affibody ligands. Protein engineering, design & selection : PEDS 17, no. 5 (May): 455-62. doi:10.1093/protein/gzh053. http://www.ncbi.nlm.nih.gov/pubmed/15208403.

@@ Line 23: / Line 23: @@
 <h2>Cyan fluorescent protein (CFP) in RFC25 standard </h2>
-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 <br>
+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 <br>
@@ Line 32: / Line 32: @@
 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.<br>
 <h2>Capsid</h2> (BBa_K404006)<br>
-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)(+).
+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-terminus of VP1 plays important role in infection and contains a motif highly homologous to a phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+). VP2 contains basic regions, too.
 <html><center><img src="https://static.igem.org/mediawiki/parts/a/a7/Freiburg10_Cap_proteins_VP1_2%263.png" width="600" height="auto"/></center></html><br>
 <h2>ViralBrick 587-KO empty</h2>