Part:BBa_K1632012

PBAD/araC_rbs_fimB(wild-type)

Fig. 1. FimB allows Decision making coli to select option at random, inverting a promoter in a fim switch.

To enable prisoner coli to have their own option selected randomly, we designed Decision making coli which expresses FimB protein (Fig.1). Even only corporation-mode plasmid was used for transformation of Decision making coli, two types of plasmids, ones in cooperation and defection mode, co-exist in the single cell due to inversion of the fim switch. We are then able to extract mixture of the both plasmid. When we use the plasmid mixture for transformation of prisoner coli, either one of the mixture will be introduced into a single cell. We thus cannot know the prisoner coli’s option selection at that time. To confirm the activity by Decision making coli, we firstly used Fim switch GFP plasmids: GFP [ON] and GFP [OFF] (Fig.2).

Fig. 2. 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.3)

Fig. 3. 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. 3 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.4). This result was consistent with the histograms (Fig. 4)

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.).

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

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