Difference between revisions of "Part:BBa K1632012"

Revision as of 00:54, 19 September 2015

PBAD/araC_rbs_fimB(wild-type)

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

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

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. 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.

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.).

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

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