Difference between revisions of "Part:BBa K1632010"
Line 4: Line 4:
 
[[Image:Tokyo_Tech_fimB_summary1.png |thumb|center|900px|<b>Fig. 1. </b>]]<br>
 
[[Image:Tokyo_Tech_fimB_summary1.png |thumb|center|900px|<b>Fig. 1. </b>]]<br>
  
−
With the expression of FimB, the <i>fim</i> switch(example (<partinfo>BBa_K1632000</partinfo>), (<partinfo>BBa_K1632004</partinfo>) etc...) inverts from the ON state to the OFF state and from the OFF state to the ON state. <br>
+
<span style="margin-left: 10px;">The <i>fim</i> switch is inverted by FimB.The FimB protein inverts the <i>fim</i> switch in the ON-to-OFF direction.<br>
−
We constructed PBAD/''araC''_fimB(wild-type) (<partinfo>BBa_K1632012</partinfo>) that produces FimB (wild-type). We also prepared two other new plasmids, <partinfo>BBa_K1632007</partinfo> and <partinfo>BBa_K1632008</partinfo>. In BBa_K1632007 and BBa_K1632008, either [ON] or [OFF] ''fim'' switch (wild-type) is placed upstream of GFP coding sequence. <br>
+
−
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.<br>
+
  
−
[[Image:Tokyo_Tech_FimB_assay.png |thumb|center|900px|<b>Fig. 1. </b>Plasmids used in our assay]]<br>
+
<span style="margin-left: 10px;">In order to assay the function of our FimB, we added a GFP coding sequence on the downstream of the <i>fim</i> switch.The <i>fim</i> switch[default ON](wild-type)_gfp (BBa_K1632007) emitts fluorescence when expressed, while the <i>fim</i> switch [default OFF](wild-type)_gfp(BBa_K1632008) does not emit florescence when expressed. We also added 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 <i>fim</i> 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.
−
 
+
−
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>
+
−
 
+
−
[[Image:Tokyo_Tech_FimB_assay_Results.png |thumb|center|900px|<b>Fig. 2. </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>
+
  
 +
[[Image:Tokyo_Tech_FimB_assay_Results.png |thumb|center|700px|<b>Fig. 2. </b>The histogram of the samples measured by flow cytometer]]<br>
  
 +
<span style="margin-left: 10px;">From the experimental results, our fimB inverted the <i>fim</i> switch[default ON](wild-type) from ON-to-OFF and the <i>fim</i> switch[defult OFF](wild-type) from OFF-to-ON, depending on the concentration of arabinose.<br><br>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 05:58, 18 September 2015

fimB (wild-type)

Fig. 1.

The fim switch is inverted by FimB.The FimB protein inverts the fim switch in the ON-to-OFF direction.

In order to assay the function of our FimB, we added a GFP coding sequence on the downstream of the fim switch.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.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. The histogram of the samples measured by flow cytometer

From the experimental results, our fimB inverted the fim switch[default ON](wild-type) from ON-to-OFF and the fim switch[defult OFF](wild-type) from OFF-to-ON, depending on the concentration of arabinose.

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]