Part:BBa_K1632010

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. 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 in the [ON] state to [OFF] state direction.

In order to assay the function of our FimB, 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 fimB(wild-type).PBAD/araC_fimB(wild-type) (BBa_K1632012) can induce the expression of FimB(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. 3. The histograms of the samples measured by flow cytometer

From the experimental results, our FimB(wild-type) 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, dependent on the concentration of arabinose.

Fig. 4. The histogram of reporter cell (2)

When the concentration of FimB(wild-type) increased by increasing the 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. 4). According to [1], this is caused by the excess increase in the inversion rate of the fim switch(wild-type). When the inversion rate is too high, there is not enough time for transcription initiation. Consequently, the GFP expression decreases.

Fig. 5. 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.This result was consistent with the histograms (Fig. 4)

Fig. 6. 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 in the [ON] state to [OFF] state in all samples (Fig. 6).

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

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