Question5

=Text of the Question:= toc You have previously read about the edge detector circuit. Redesign this circuit to utilize the photoswitchable tryptophan repressor. The end result, edge detection, should be the same. Show on your diagram how you expect your circuit to work.

=Definitions of the Terms:=

trp operon
Sequence of DNA containing (in order): trpR, a promotor, operon, trpL (leader, with embedded attenuator), trpE, trpD, trpC, trpB, trpA.

trpR, the promotor, operon, trpL (leader, with embedded attenuator) form a regulator region.

The remainder is the regulated genes.

The contains 2 tryptophan residues which stall the ribosome such that the mRNA being transcribed can from an attenuation structure which ends translation.[5.5]

TrpR (Trp repressor)
The gene product of the trpR gene. It contains a helix-turn-helix motif allowing it to bind to DNA.

LOV
Abbreviation for "light, oxygen, voltage". The LOV domain of a protein photosensor, included in proteins such as the phototropins (light-activated serine/threonine kinases). The LOV domain of A. sativa is known as AsLOV2.[5.3] LOV's typically have a prosthetic FMN (flavin nucleotide) chromophore.

Lov-TAP
LOV and tryptophan-activated protein. It is the end-to-end fusion product of TrpR and AsLOV2.[5.3] When illuminated by by light, it binds cognate DNA protecting it.[5.2]

Presumably, AsLOV2 activates the TrpR and then TrpR would bind to the operator of the trp operon. This allows the fusion product to inhibit trp transcription.

=The Original Circuit (from 5.1):=

=The Circuit Redesigned:=

=Mechanism of the Circuit:=

=Relevant Literature:=

5.1. [|Tabor, J. J., H. M. Salis, et al. (2009). "A Synthetic Genetic Edge Detection Program." Cell 137(7): 1272-1281.] 5.2. [|Strickland, D., K. Moffat, et al. (2008). "Light-activated DNA binding in a designed allosteric protein." Proceedings of the National Academy of Sciences 105(31): 10709-10714.] 5.3. [|Strickland, D., X. Yao, et al. (2010). "Rationally improving LOV domain-based photoswitches." Nat Meth 7(8): 623-626.] 5.4. [|Takayama, Y., M. Nakasako, et al. (2011). "Light-induced movement of the LOV2 domain in an Asp720Asn mutant LOV2-kinase fragment of Arabidopsis phototropin 2." Biochemistry. 50(7): 1174-1183. Epub 2011 Jan 1128.] 5.5. Nelson and Cox. Lehninger Principles of Biochemistry. 2008, 5th edition, WH Freeman. pp. 1128-9