Part:BBa_K2787027
RNA attenuator pT181 with GFP under pT181 target sequence
pT181 is a RNA switch that can repress the expression of target gene by binding its Antisense RNA to its sense target DNA placed upstream of the coding sequenced of target gene. Here the part carries a pT181 Antisense under constitutive promoter J23100 and a GFP reporter whose coding sequence is placed seamlessly after pT181 sense target, also under J23100. This part is used for testing the repressive efficiency of pT181 efficiency by contrast to the results from part BBa_K2787028.
Usage and Biology
A schematic representation of the pT181 attenuator in action
A schematic representation of the experimental group plasmid. This has the basic pT181 attenuator Antisense under control of a constitutive promoter, as well as a GFP gene downstream of the pT181 attenuator sense target under the control of a constitutive promoter.
A schematic representation of the positive control plasmid with the GFP gene downstream of the pT181 attenuator sense target under the control of a constitutive promoter, without a pT181 attenuator Antisense on it.
400x image of positive control under fluorescence microscope.
400x image of experimental group under fluorescence microscope
Characterization of pT181 attenuator in DH5-α E.coli cells. OD600 monitored over time for cell lines incorporating the pT181 attenuator in the absence or presence of the pT181 antisense. The result shows that the pT181 antisense is not harmful to the E.coli, which provides convenience for test for fluorescence as we do not need to normalize the OD600.
Fluorescence monitored over time for cell lines incorporating the pT181 system with pT181 antisense. It shows that the GFP can be expressed in the pT181-attenuator, and the expression level increases gradually.
Fluorescence monitored over time for cell lines incorporating the pT181 system without pT181 antisense. It matches the curve of how GFP’s expression increases without being repressed, which establishes foundation for measure the repression effect of pT181-attenuator.
The combination of the two figures above. We could see the sharp difference in the fluorescence between the two curves. This proves our pT181 could repress the expression of GFP as expected, which means our part C is able to produce repression effect as anticipated. This shows that the controller in our Three-Node Feedback Loop is constructed successfully.
Characterization of pT181 attenuator in DH5-α E.coli cells. endpoint fluorescence (18 hours) for cell lines in the absence or presence of Pt181. The data shows that our Pt181 attenuator could repress the target gene for 98%.
Characterization of dual control pT181 system in DH5α strain with pSB1C3 as a vector. Fluorescence over time for cell growth incorporating dual control pT181 or Kyoto pT181 system. From the figure, we can see the fluorescence of the dual control pT181 is always lower than the Kyoto pT181, which means it shows better repression effect.
Characterization of pT181 attenuator in DH5-α E.coli cells. Endpoint fluorescence (18 hours) for cell lines under the control of dual control or Kyoto Pt181. The data shows that our Pt181 attenuator could repress the target gene for 15% than the Kyoto pT181. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 268
Illegal NheI site found at 291 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1141
| None |