Part:BBa_K1632013
PBAD/araC_rbs_fimE(wild-type)
The fim switch is inverted by FimE.The FimE protein inverts the fim switch predominantly in [ON] state to [OFF] state.
In order to assay the function of our FimE, we added a GFP coding sequence on the downstream of the fim switch(wild-type).The fim switch[default ON](wild-type)_gfp (BBa_K1632007) emitts fluorescence when expressed, while the fim switch[default OFF](wild-type)_gfp(BBa_K1632008) does not emit florescence when expressed. We also added PBAD/araC on the upstream of fimE.PBAD/araC_fimE (<partsinfo>BBa_K1632013<partsinfo>) can induce the expression of FimE(wild-type) in the presence of arabinose. We co-transformed a fim switch(wild-type)_gfp and a PBAD/araC_fim recombinase in the E. coli DH5alpha strain.
We tried to confirm that fim switch(wild-type) is predominantly inverted in the presence of FimE(wild-type) by using GFP as a reporter, under 4 different concentrations of arabinose. In the medium with 0 M arabinose, we supplemented the medium with 1.0 % glucose in order to repress the leakage in the PBAD/araC promoter. Fig. 2 shows the histograms of the samples measured by the flow cytometer. In the results of the reporter cell (1), when the induction of FimE(wild-type) expression increases, the fluorescence intensity decreases. From this fact, we confirmed that the fim switch(wild-type) is inverted from [ON] state to [OFF] state by FimE(wild-type). From the result of the reporter cell (2), even when the expression amount of FimE(wild-type) increases, the expression amount of GFP in the reporter cell (2) does not change. From this fact, we confirmed that the fim switch(wild-type) is inverted only from [ON] state to [OFF] state by FimE(wild-type). From the results of the two reporter cells (1) and (2), we successfully confirmed that FimE(wild-type) inverts the fim switch(wild-type) only from [ON] state to [OFF] state.
After measurement of flow cytometer, we minipreped the cell culture and got plasmid mixture which contain pSB6A1 and pSB3K3 in each sample.The state of fim switch(wild-type) either [ON] state or [OFF] state in colonies is evaluated from fluorescence. Thus, colonies which contain fim switch[default ON](wild-type) expressed GFP. On the other hand, colonies which contain fim switch[default OFF](wild-type) do not express GFP. We counted out the all colonies and those with fluorescence. In the results of the reporter cell (1), when inducing the expression of FimE(wild-type), the percentage of [ON] state decreased dramatically. On the other hand, from the results of the reporter cell (2), when inducing the expression of FimE(wild-type), the percentage of [ON] state remained being small. From the results of the two reporter cells (1) and (2), we successfully confirmed that FimE(wild-type) inverts the fim switch(wild-type) predominantly from [ON] state to [OFF] state. (Fig.3). This result was consistent with the histograms (Fig.2.).
Also, we incubated the colonies with fluorescence and those without fluorescence. We minipreped cell culture. Sequence complementarity in the specific region of the switch shows intended inversion of the switch from [ON] state to [OFF] state in all sample (Fig.4.).
More information
For more information, see http://2015.igem.org/Team:Tokyo_Tech/Project Our work in Tokyo_Tech 2015 wiki, http://2015.igem.org/Team:Tokyo_Tech/Experiment/ssrA_tag_degradation_assay About ssrA-tag, http://2015.igem.org/Team:Tokyo_Tech/Experiment/Overview_of_fim_inversion_system About ''fim'' inversion system
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
Illegal AgeI site found at 1260 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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