Part:BBa_K1632013

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.


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. Thus, colonies which contain fim switch[default ON] expressed GFP. On the other hand, colonies which contain fim switch[default OFF] do not express GFP. We also counted out the all colonies and those with fluorescence. In the results of the reporter cell (1), when inducing the expression of FimB(wild-type), the percentage of [ON] state decreased. Furthermore, from the results of the reporter cell (2), when inducing the expression of FimB(wild-type), 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 the [OFF] state and from [OFF] state to [ON] state. (Table. 3-4-3-2). This result was consistent with the histograms (Fig. 2)
Also, we incubated the colonies with fluorescence and those 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.).

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

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

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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  Illegal AgeI site found at 1260
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961