Part:BBa_K1632008

fim switch[default OFF](wild-type)_rbs_gfp

Fig. 1. Overview of fim inversion system

Fim switch(wild-type) is derived from wild-type sequence. Fim switch(wild-type) have sigma 70 promoter which is constitutively promoter. The promoter in the fim switch transcripts to the right. From the direction of transcription, the state is defined as [ON] state. On the other hand, fim switch[default OFF](wild-type) (BBa_K1632005) transcripts to the left.

Fim switch is inverted by two recombinase, FimB and FimE.The FimB protein inverts fim switch from [ON] state to [OFF] state and [OFF] state to [ON] state with approximately equal efficiencies. On the other hand, the FimE protein inverts fim switch predominantly from [ON] state to [OFF] state.

We constructed fim switch[default ON](wild-type)_rbs_gfp(BBa_K1632007) to characterize the function of this part, by inserting fim switch[default ON](wild-type)(BBa_K1632004) upstream of a GFP coding sequence.

In the fimB dependent fim switch state assay, we transformed fim switch[default ON](wild-type)_rbs_gfp and PBAD/araC_fimB(BBa_K1632012)in the E.coli DH5alpha strain. We measured the fluorescence intensity of the cells induced by arabinose.

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

We tried to confirm that fim switch is bidirectically inverted in the presence of FimB(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 0.5 % 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 FimB(wild-type) expression increases, the fluorescence intensity decreases. From this fact, we confirmed that the fim switch is inverted from [ON] state to [OFF] state by FimB(wild-type). From the result of the reporter cell (2), when the expression amount of FimB(wild-type) increases, the expression amount of GFP in the reporter cell (2) increases. From this fact, we confirmed that the fim switch is inverted from [OFF] state to [ON] state by FimB(wild-type). From the results of the two reporter cells (1) and (2), we successfully confirmed that FimB(wild-type) inverts the fim switch from [ON] state to [OFF] state and from [OFF] state to [ON] state.

Fig. 4. Determination of percemtage of [ON] state and colony formation using plasmid mixture extracted cell expressing FimB.

To confirm our results that our FimB(wild-type) inverted the fim switch(wild-type) further, after scattering the samples on a plate, we counted the number of colonies which were expressing GFP and the colonies which were not expressing GFP(Fig.4). The state of fim switch either [ON] or [OFF] in colonies is evaluated from fluorescence. In brief, colonies which contain fim switch[default ON] expresse GFP while colonies which contain fim switch[default OFF] do not express GFP. We counted out the all colonies and colonies which contain fim switch[default ON]. In the results of the reporter cell (1), when the expression of FimB(wild-type) was induced by arabinose, the percentage of [ON] state decreased. Furthermore, from the results of the reporter cell (2), when the expression of FimB(wild-type) was induced, the percentage of [ON] state increased. From the results of the two reporter cells (1) and (2), we successfully confirmed that the fimB protein inverts the fim switch(wild-type) from [ON] state to [OFF] state and from [OFF] state to [ON] state. (Fig.3). This result was consistent with the histograms (Fig. 2)

Fig. 5. DNA sequencing results of fim switch(wild-type)

Also, we incubated the colonies with fluorescence and the colonies without fluorescence. We minipreped cell cultures. Sequence complementarity of the each sample in the specific region of the switch shows intended inversion of the switch from [ON] state to [OFF] state in all samples (Fig. 5.).

Similary,in the fimE dependent fim switch state assay, we transformed fim switch[default ON](wild-type)_rbs_gfp and PBAD/araC_fimE(wild-type)(BBa_K1632013) in the E.coli DH5alfpha strain.We measured the fluorescence intensity of the cells induced by arabinose.

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

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1052