Part:BBa_K2787029
RNA attenuator pT181 with GFP under pT181 target sequence by Kyoto 2013 iGEM Team
Measurement of repression efficiency of pT181 sense target submitted by Kyoto 2013 iGEM team(BBa_K1126003) under the expression kit(BBa_K2787027) we designed to test the functionality of our improved version of pT181 sense target(BBa_K2787030). This part is identical to BBa_K2787027 except for the pT181 sense target is changed to BBa_K1126003 and a RBS(BBa_K1893033).
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%.
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 1247
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