On the previous assay we confirmed crosstalk, which 3OC12HSL-LasR complex activates not only las promoter but also lux promoter too (Gray KM et al., 1994). We came up with idea that crosstalk can be suppressed if lux/tet hybrid promoter is inhibited by protein TetR only when 3OC12HSL-LasR complex is around. Our purpose is to check whether lux/tet hybrid promoter would be repressed or not when 3OC12HSL-LasR complex and protein TetR existed. We tried to compare the frequency of crosstalk in the presence or absence of the TetR inhibitor aTc. aTc weakens the bonding of TetR and TetO.
We made a simple crosstalk circumvention system and named it "Crosstalk Circumvention Switch" (Fig. 2). To construct the circuit in above (Fig. 1), we ligated Pcon-RBS-lasR-TT (BBa_K553003) and Plux/tet-RBS-GFP-TT (BBa_K934025) as a reporter plasmid.
We used Pcon-RBS-luxR-TT-Ptrc-RBS-tetR-TT as the regulator plasmid.
This part was designed for a crosstalk circumvention system for two signal molecules (our wiki). Our crosstalk circumvention system has a network topology which is composed of two repressor proteins, one repressor and one hybrid promoter (Fig. 2). Along with the topology, one can just choose any combination of sets of repressor proteins and promoters. This system can be used for any genetic circuit that has potential crosstalk. This part is useful to confirm the circumvention because the lux/tet hybrid promoter can be regulated common regulatory protein, TetR and LuxR. In addition, this part also contains LasR protein which has potential cross talk to luxR-binding sequence.
We also screened the concentration of 3OC6HSL and aTc, and examined activation of the crosstalk circumvention system circuit. Then we determined the transfer function of 3OC6HSL-LuxR complex by fitting Hill’s equation and introduced into our modeling
Fig. 4 shows activation of the crosstalk circumvention system circuit. It shows transition of the fluorescence intensity of GFP. It indicates that the concentration of C6 saturates between 10 mM and 30 mM.
For more information, see our work in Tokyo_Tech 2013 wiki.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 383
Illegal AgeI site found at 580
- 1000Illegal BsaI.rc site found at 927
Illegal BsaI.rc site found at 1675