Part:BBa_K1212016
pTet + TBS2 + RBS + GFP + T
See our [http://2013.igem.org/Team:UC_Davis/Data wiki's data page] for more information.
pTet promoter upstream of TALe binding site 2 (corresponding to TAL 8 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212007 (BBa_K121207)], with B0034 and Golden Gate compatible GFP.
The graph below shows GFP fluorescence normalized by OD 600 readings over the course of a bit less than 15 hours with pTet induced by 100 ng/mL aTc and not induced. There is little to no fluorescence when no inducer is added and a 27 fold increase in fluorescence is seen when induced with 100 ng/mL aTc. However, this is not as large of a fold increase as what we saw for our other pTet+TBS1+GFP part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212016 (BBa_K1212016)], and it appears to have an oddly delayed reaction to the induction. This experiment was done in E.coli K-12 strain MG1655Z1, which has been engineered to overexpress TetR.
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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212012 BBa_K1212012] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212015 BBa_K1212015] for further characterization of our RiboTALes.
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.
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.
TBS sequence obtained from
J. F. Meckler, M. S. Bhakta, M. S. Kim, R. Ovadia, C. H. Habrian, A. Zykovich, et al., "Quantitative analysis of TALE-DNA interactions suggests polarity effects," Nucleic Acids Res, vol. 41, pp. 4118-28, Apr 2013.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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