Difference between revisions of "Part:BBa K1632010"

Revision as of 20:58, 18 September 2015

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 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. 2. 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) in the [ON] state to [OFF] state direction and the fim switch[defult OFF](wild-type) in the [OFF] state to [ON] state, dependent on the concentration of arabinose.

Fig. 3. 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) in the [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 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.


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


Sequence and Features