Part:BBa_K1632012

PBAD/araC_rbs_fimB(wild-type)

Fig. 1. New plasmids we constructed to confirm the function of BBa_K1632012 plasmid for Decision making coli.

The fim switch is inverted by FimB. The FimB protein inverts the fim switch from [ON] state to [OFF] state and from [OFF] state to [ON] state with approximately equal probability.

In order to assay the function of our FimB(wild-type), we added a GFP coding sequence on the downstream of the fim switch. The fim switch[default ON](wild-type)_gfp (BBa_K1632007) emits fluorescence when expressed, while the fim switch[default OFF](wild-type)_gfp(BBa_K1632008) does not emit florescence when expressed. We also inserted PBAD/araC on the upstream of fimB. PBAD/araC_fimB (BBa_K1632012) can induce the expression of FimB in the presence of arabinose. We co-transformed a fim switch_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)

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

From the experimental results, our FimB inverted the fim switch[default ON](wild-type) from [ON] state to [OFF] state and the fim switch[defult OFF](wild-type) from [OFF] state to [ON] state, depending on the concentration of arabinose.

Fig. 3. The histogram of reporter cell (2)

When the concentration of FimB(wild-type) increased by increasing concentration of arabinose, we confirmed that the fluorescence intensity decreased in both [ON] to [OFF] process and [OFF] to [ON] process.

The result of the reporter cell (2) shows that when the concentration of arabinose is increased to 0〜20 microM, the fluorescence intensity increases. This shows the function of FimB (wild-type) inverting the fim switch(wild-type) from [OFF] state to [ON] state. However, when the arabinose concentration is excess (5mM), the fluorescence intensity decreases (Fig. 3). According to [1], this is caused by the excess increase of the inversion rate of the fim switch. When the inversion rate is too high, there is not enough time for transcription initiation. Consequently, the GFP expression decreases.


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 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)
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.).

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
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

References

[1]Hung M. et al. (2014) Modulating the frequency and bias of stochastic switching to control phenotypic variation. Nat Commun 5:4574. doi:10.1038/ncomms5574
[2]Timothy S. Ham et al. (2006) A Tightly Regulated Inducible Expression System Utilizing the fim Inversion Recombination Switch. Biotechnol Bioeng 94(1):1-4