Control of the expression of anchored genes using micron scale heater

Applied Physics Letters

Volumn 76, Issue 24, 2000, Pages 3638-3640

  Shivashankar, G V ^a,b   Liu, S ^a,b   Libchaber, A ^a,b  

^a NEC LABORATORIES AMERICA   (United States)

^b ROCKEFELLER UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

EID: 0001745829     PISSN: 00036951     EISSN: None     Source Type: Journal    
DOI: 10.1063/1.126732     Document Type: Article

References (8)

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2. M. V. Olson, Proc. Natl. Acad. Sci. USA 90, 4338 (1993).
3. P. O. Brown and D. Botstein, Nature Genetics. 21, 33 (1999).
4. L. Jermutus, L. A. Ryabova, and A. Pluckthun, Curr. Op. Biotechnology. 9, 534 (1998).
5. 5. The volume of the sample chamber is 25 μl [G. V. Shivashankar and A. Libchaber, Appl. Phys. Lett. 73, 417 (1998)].
6. A Wheat Germ Coupled T7 Transcription and Translation kit (Promega, Madison, WI) is used for in vitro transcription and translation of the luciferase gene and the spacer sequence. In addition to the reaction components, 0.8 mM of luciferin is added to the reaction to detect luminescence.
7. T. Wilson and J. B. Hastings, Annu. Rev. Cell Dev. Biol. 14, 197 (1998).
8. In the standard gene construct the mRNA and the protein are released from the bound DNA. To attach the encoded protein to its DNA we introduce changes at the transcription and translation stages. To prevent transcription termination, we attach avidin to the biotinylated 3′ end of the DNA and the polymerase moves towards that end. The translation termination is prevented by deletion of the stop codon from the initial DNA. Without stop codon, the ribosome does not release the protein. If we prevent transcription and translation termination, the mRNA stays linked to DNA via RNA polymerase, and the protein is bound to mRNA through the ribosome. The testing DNA has three regions; the control region, the luciferase gene sequence (1.65 kb), and a spacer. The control region has a T7 promoter sequence for transcription. In one type of construct the stop codon of the luciferase sequence is deleted by PCR. We use 717 base pairs for the spacer. DNA fragments are first made by PCR. These fragments are ligated together and further amplified by PCR. For immobilization on avidin coated beads, the 3′ end primer (at the end of transcription) is biotinylated.