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From 2010.igem.org

Version 1 : ==Testing individual parts== ----- To realize the genetic double click system, we made two plasmids. First one is ''Pulse generator'', which generate the pulse of T7 RNAP in response to the 1st and 2nd input. The second one is ''pulse -> GFP generator'', which generates the output (GFP expression) on condition that T7 RNAP activates the GFP upstream promoter and also CI protein does not repress that promoter. The subparts of those plasmids are shown below.
[[Image:Chiba_icon_1.jpg]]
[[Image:Chiba_icon_2.jpg]]
Regarding with those two plasmids, we tested the function of each parts in the plasmids.
==1. Pulse generator== For Pulse generator, the parts shown below were checked.Also, we checked if there were no crosstalk between CI/CI434 and CI promoter/CI434 promoter. ===T7/CI-OR1 hybrid promoter=== *[http://partsregistry.org/wiki/index.php?title=Part:BBa_K396000 BBa_K396000] T7/CI-OR1 hybrid promoter was designed to be activated by T7 RNA Polymerase and repressed by lambda CI protein. In sequence design, lambda CI operator site 1 (OR1, Part of BBa_R1051) was directly attached to the downstream of T7 promoter sequence (BBa_I719005). This characterization was implemented to validate the promoter function. In results, the activation and repression was actually observed. ====Experiment&Results==== [[Image:CI-1.jpg|frame|center|Fig. How to check the parts]] [[Image:CI-2.jpg|frame|center|Fig. Data: Under the situation of CI non-expressed , T7 RNAP causes GFP expression by activating T7/CI hybrid promoter (Fig.1). On the other hand, under the situation of CI expressed, T7 RNAP does not cause GFP expression (Fig.2). The reason of this is considered as following. Under the situation of CI expressed, even though T7 RNAP tries to activate T7/CI promoter, the promoter Is repressed by CI because the ability of CI repression is superior to the one of T7 activation for this promoter. So it is concluded that T7/CI hybrid promoter is to be activated by T7 RNA Polymerase and repressed by lambda CI protein, following the rules of CI repression > T7 activation.]] *Dubendorf, J.W. & Studier, F.W. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. Journal of Molecular Biology 219, 45-59 (1991). ==2. Pulse to GFP generator== For GFP generator, the parts shown below were checked. ===Lux/CI434 hybrid promoter=== We got the idea that cI and cI434 repressor don’t crosstalk[1], so we construct Pt7/cI and pLux/cI434 hybrid promoter. ====Rationale==== This hybrid promoter is designed to activated by LuxR when it is bound to AHL and repressed by the phage 434 cI repressor. The operator site sequence come from the biobrick part (BBa_R0052) and we set OR2(ttacaatgtatcttg) between -35 and -10, OR1(ttacaaactttcttg) at downstream of -10. There are only 16 bp between the -10 and -35 boxes we have confirmed that lux promoter works naturally while repression is under observation. ====Ref==== #http://web.mit.edu/synbio/x/htdocs/BB_Parts/BBa_R0050/Part.htm #http://partsregistry.org/Part:BBa_I12040 #Basu S, Mehreja R, Thiberge S, Chen MT and Weiss R (2004). Spatiotemporal control of gene expression with pulse-generating networks. Proc Natl Acad Sci USA 101: 6355-6360.