Difference between revisions of "Part:BBa K1632013"

Revision as of 19:50, 18 September 2015

PBAD/araC_rbs_fimE(wild-type)

Fig. 1. New plasmids we constructed to confirm the function of BBa_K1632013 plasmid for Decision making coli.

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 (BBa_K1632013) 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 measured the fluorescence intensity of the cells induced by different concentraions of arabinose.

Fig. 2. The histograms of the samples measured by flow cytometer

From the experimental results,our FimE inverted the fim switch[default ON](wild-type) from [ON] state to [OFF] state but did not invert the fim switch[default OFF](wild-type) from the [OFF] state, dependent on the concentration of arabinose.


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.)

Fig. 3. The histograms of the samples measured by flow cytometer

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.).

Fig. 4. The histograms of the samples measured by flow cytometer

For more information, see [http://2015.igem.org/Team:Tokyo_Tech/Project our work in Tokyo_Tech 2015 wiki].

Sequence and Features