Composite

Part:BBa_K1212012

Designed by: Amy Soon   Group: iGEM13_UC_Davis   (2013-09-16)

pBAD+Riboswitch2+TAL8

TAL repressor 1 expression under the control of a pBAD promoter and a theophylline riboswitch inducible by arabinose and theophylline. The data below shows the behavior of this part in the context of a cotransformation between pBAD+RS2+TAL 8 in pSB3K3 (low copy number) and pTet+TBS 2+GFP in pSB1C3 (high copy number). See BBa_K1212016 for further characterization of pTet+TBS 2+GFP.
See our wiki's data page for more information.


UCD_2013_RiboTALE_Tunability.PNG



The image above displays the peak fluorescence of two RiboTALe constructs, one expressing TALe 1 and the other expressing TALe 8, under different induction conditions for arabinose and theophylline. Both RiboTALes exhibit the expected behavior pattern given the induction conditions, but at consistently different levels of fluorescence. We have attributed this to the difference in binding affinities of the two TAL repressors to their respective binding sites.This variable, if well characterized for different TAL repressors, will provide a powerful means to control the tunability of these devices.


It is similarly interesting to note that under conditions of 1% arabinose, but no theophylline, there was clearly some reduction in fluorescence. We concluded that the riboswitch we used in this experiment had some degree of leakiness. We next investigated the possibility of altering riboswitch leakiness as another means to increase the tunability of our RiboTALe devices.



UCD_2013_RiboTALE_Leakiness.PNG



The image above displays the fluorescence results for the two RiboTALe devices tested in this experiment. According to the literature both riboswitches have similar reported fold activation ratios[1,2]. But it is clear that the two RiboTALe devices, differing only in the riboswitch controlling the translation of the TAL repressor, exhibit consistently different behavior. The data show that at 1% arabinose (the inducer for the RiboTALe transcript), but in the absence of theophylline, the RiboTALe under control of riboswitch Clone E is active. Under identical induction conditions, the RiboTALe under riboswitch Clone 8.1* exhibits no repression activity. The fluorescence measured was in fact higher than the baseline for reasons not understood. From the data we conclude that Clone E is leakier and yet stronger than Clone 8.1*, generating a 3.68 fold reduction in fluorescence as opposed to a 2.42 fold reduction. These data indicate that differences riboswitch leakiness and strength do impact RiboTALe system behavior, and can be engineered into RiboTALe designs as sources of tunability.



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 180
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
    Illegal PstI site found at 180
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
    Illegal BamHI site found at 203
    Illegal BamHI site found at 2597
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 180
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 180
    Illegal NgoMIV site found at 221
    Illegal NgoMIV site found at 2423
    Illegal AgeI site found at 2604
  • 1000
    COMPATIBLE WITH RFC[1000]


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