Difference between revisions of "Part:BBa K1632010"

Revision as of 01:04, 18 September 2015

fimB (wild-type)

With the expression of FimB, the fim switch(example (BBa_K1632000), (BBa_K1632004) etc...) inverts from the ON state to the OFF state and from the OFF state to the ON state.
We constructed PBAD/araC_fimB(wild-type) (BBa_K1632012) that produces FimB (wild-type). We also prepared two other new plasmids, BBa_K1632007 and BBa_K1632008. In BBa_K1632007 and BBa_K1632008, either [ON] or [OFF] fim switch (wild-type) is placed upstream of GFP coding sequence.
Our purpose is to confirm that FimB (wild-type) inverts the fim switch (wild-type) from ON to OFF and from OFF to ON (Fig. 1.). We measured the fluorescence intensity from the GFP expression in the presence of arabinose. From the results, we confirmed that our fim switch (wild-type) is inverted from ON to OFF and OFF to ON. From the results we also confirmed our fim switch (wild-type) is not inverted by the endogenous FimB and FimE and that FimB expression doesn’t affect the GFP expression.

Fig. 1. Plasmids used in our assay

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. 1. Histogram of the samples measured by flow cytometer

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 to OFF 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 to ON 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 to OFF and from OFF to ON.

The results of positive control 1 and negative control 1 confirmed that the endogenous FimB and FimE did not invert our fim switch (wild-tyoe). Also, the result of negative control 2, indicates that the expression of FimB (wild-type) do not have effects on the gfp expression.

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]

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 [[Image:Tokyo_Tech_FimB_assay.png |thumb|center|900px|<b>Fig. 1. </b>Plasmids used in our assay]]<br>
-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 to OFF 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 to ON 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 to OFF and from OFF to ON.<br>
+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.<br>
-The results of positive control 1 and negative control 1 confirmed that the endogenous FimB and FimE did not invert our ''fim'' switch (wild-tyoe). Also, the result of negative control 2, indicates that the expression of FimB (wild-type) do not have effects on the gfp expression. The reason the fluorescence intensity of the positive control 2 is increasing in proportion to the arabinose concentration is described in 4. Discussion section. <br>
 [[Image:Tokyo_Tech_FimB_assay_Results.png |thumb|center|900px|<b>Fig. 1. </b>Histogram of the samples measured by flow cytometer]]<br>
+ 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 to OFF 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 to ON 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 to OFF and from OFF to ON.<br>
+The results of positive control 1 and negative control 1 confirmed that the endogenous FimB and FimE did not invert our ''fim'' switch (wild-tyoe). Also, the result of negative control 2, indicates that the expression of FimB (wild-type) do not have effects on the gfp expression. <br>