Part:BBa_K189000

Targeted BioBrick

The Targeted Biobrick is a specially constructed for cell-specific attachment and subsequent internalization. It consists of a TAT peptide for attachment (can be modified by other peptide for different specificity), the selected segment in fiber shaft of adenovirus and RGD sequence. Different modules or domains are linked by linker sequences.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 501
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 256
  Illegal AgeI site found at 94
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Experiment1	Characteristic1	Value1
Transfection Specificity	HeLa	Low
	MRC-5	High
Response Time	1.5h
Compatibility	Plasmid	pEGFP-N2,pET-28a
	Protein for fusion	C of bacteriophage lambda, GFP
Stability	Genetic Stability	To be measured
	Fluorescence Maintainance	To be measured
Demand	Induction	Not needed
	Cell Density	80%

¹Measured by Feng Tian and Feng Xu 2008-2009

Safety Issues

Targeted BioBrick is composed of three functional domains: RGD domain, fiber protein from adenovirus genome and targeted peptide selected from phage display. Fiber protein is the L5 gene product of adenovirus, while the proteins that are responsible for immuno-response are early proteins such as E2A. RGD domain is common in cell-cell junctions and unlikely to be involved in immuno-response. The cell-specific peptide might be potentially unsafe, especially randomly generated peptides. So cellular and animal experiments are recommended for Targeted BioBrick characterization before clinical uses.

Acknowledgement

We are grateful to Guoqiang Chen, Zhao Wang, Junmin Pan, Wei Wu, Li Yu, Yinghua Chen and Liping Xie of School of Life Sciences, Tsinghua University for assistance in the design, construction and characterization of this part.

References

[1] Glen RN, and Phoebe LS. Role of αv integrins in adenovirus cell entry and gene delivery. Microbiology and Molecular Biology reviews. 1999, 63, 725-734.

[2] Brammar WJ, and Hadfield C. A programme for the construction of a lambda phage. J. Embryol. exp. Morph. 1984, 83, Supplement, 75-88.

[3] Harrison echols, and Helios Murialdo. Genetic map of bacteriophage lambda. Microbiological Reviews. 1978, 42, 577-591.

[4] Christine N. Zanghi, Ramil Sapinoro, Birgit Bradel-Tretheway, and Stephen Dewhurst. A tractable method for simultaneous modifications to the head and tail of bacteriophage lambda and its application to enhancing phage-mediated gene delivery. Nucleic Acids Res. 2007, 35, 8.

[5] http://www.biosea.net/shownews.asp?id=79

[6] Sambrook J, Maniatis T, Fritsch E F. Molecular Cloning: a Laboratory Manual. cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 3rd ed., 2001.

[7] Dale Kaiser, and Terrie Masuda. In Vitro Assembly of Bacteriophage Lambda Heads. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 260-264.

[8] Sachdev S Sidhu, and Shohei Koide. Phage display for engineering and analyzing protein interaction interfaces. Current Opinion in Structural Biology. 2007, 17, 481–487.

[9] Jennifer Xavier, Shashi Singh, David A. Dean, N. Madhusudhana Rao, and Vijaya Gopal. Designed multi-domain protein as a carrier of nucleic acids into cells. Journal of Controlled Release. 2009, 133, 154–160.

[10] C Kalka, and Iris Baumgartner. Gene and stem cell therapy in peripheral arterial occlusive disease. Vascular Medicine. 2008, 13, 157 - 172.