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From 2010.igem.org

Testing individual parts

To realize the genetic double click system, we made two plasmids. First one is “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. Second one is “Pulse generator”, which generate the pulse of T7 RNAP in response to the 1st and 2nd input. The subparts of those plasmids are shown in Fig.1.

Regarding with those two plasmids, we tested the function of each parts in the plasmids.

For GFP generator, the parts shown below were checked.

1. Constitutive promoter
2. LuxR
3. T7/CI-OR1 hybrid promoter

For Pulse generator, the parts shown below were checked.

1. Lux/CI434 hybrid promoter
2. Pulse generator

Also, we checked if there were no crosstalk between CI/CI434 and CI promoter/CI434 promoter.

In detail

Hybrid promoter Design

We got the idea that cI and cI434 repressor don’t crosstalk[1], so we construct Pt7/cI and pLux/cI434 hybrid promoter.

About the design

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1. 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) 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.

References and links

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[1] http://web.mit.edu/synbio/x/htdocs/BB_Parts/BBa_R0050/Part.htm
[2] http://partsregistry.org/Part:BBa_I12040
[3] 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.