Difference between revisions of "Part:BBa K1632000"

Latest revision as of 03:23, 19 September 2015

fim switch[deault ON](Tokyo_Tech/J23119)

Fig. 1. fim switch(Tokyo_Tech/J23119) design (Up:[ON] state Down:[OFF] state)

The fim switch is the promoter containing repeated DNA sequence which is inverted by the Fim recombinase. Therefore, we can control the expression of the gene downstream of the fim switch by adding the Fim recombinase.
We designed this fim switch which has a J23119 promoter (BBa_J23119). Also between the promoter and the inverting site, there are two restriction enzyme cut sites are added in each side of the promoter (Fig. 1.). So the promoter can easily be interchanged. For example,BBa_K1632005, we replaced J23119 promoter with Lac promoter(BBa_R0010)(Fig. 2.). Except for insertion of restriction enzyme sites, basically, the design of fim switch(Tokyo_Tech) is similar with fim switch(wild-type).

Fig. 2. Fim switch(Tokyo_Tech/R0010) design

From our results of our assay, the inversion of fim switch(Tokyo_Tech/J23119) by FimB(wild-type) (BBa_K1632010)/FimE(wild-type) (BBa_K1632011) was not confirmed correctly. The FimB protein(wild-type) inverts fim switch from [ON] state to [OFF] state and from [OFF] state to [ON] state correctly(Fig. 3.). However the FimE protein(wild-type) didn't invert fim switch predominantly from [ON] state to [OFF] state. In the assay, the FimE protein(wild-type) inverts fim switch from [ON] state to [OFF] state and from [OFF] state to [ON] state(Fig. 4.). In other words, the FimE protein works as the FimB protein.

Fig. 3. The histograms of the samples with FimB measured by flow cytometer

Fig. 4. The histograms of the samples with FimE measured by flow cytometer

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
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 345
Illegal NheI site found at 368
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 374
Illegal BamHI site found at 333
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 __NOTOC__
 <partinfo>BBa_K1632000 short</partinfo>
+[[Image:Tokyo Tech fim switch Tokyo Tech design.png|thumb|center|400px|Fig. 1.  ''fim'' switch(Tokyo_Tech/J23119) design (Up:[ON] state  Down:[OFF] state)]]<br>
 <span style="margin-left: 10px;">The <i>fim</i> switch is the promoter containing repeated DNA sequence which is inverted by the Fim recombinase. Therefore, we can control the expression of the gene downstream of the <i>fim</i> switch by adding the Fim recombinase.<br>
-<span style="margin-left: 10px;">We designed this ''fim'' switch which has a J23119 promoter (<partinfo>BBa_J23119</partinfo>) . Also between the promoter and the inverting site, there are two restriction enzyme sites in each front (SalIand BamHI) and back (BglII and MluI)(Fig. 1. Tokyo Tech ''fim'' switch (Tokyo_Tech/J23119) design). So the promoter can easily be interchanged. Except for insertion of restriction enzyme sites, basically, the design of TokyoTech fim switch is similar with Wild Type fim switch.
+<span style="margin-left: 10px;">We designed this ''fim'' switch which has a J23119 promoter (<partinfo>BBa_J23119</partinfo>). Also between the promoter and the inverting site, there are two restriction enzyme cut sites are added in each side of the promoter (Fig. 1.).  So the promoter can easily be interchanged.  For example,<partinfo>BBa_K1632005</partinfo>, we replaced J23119 promoter with Lac promoter(<partinfo>BBa_R0010</partinfo>)(Fig. 2.).   Except for insertion of restriction enzyme sites, basically, the design of <i>fim</i> switch(Tokyo_Tech) is similar with <i>fim</i> switch(wild-type).
+[[Image:Tokyo Tech fim switch Tokyo Tech Plac design.png|thumb|center|600px|Fig. 2. ''Fim'' switch(Tokyo_Tech/R0010) design]]<br>
+<span style="margin-left: 10px;">From our results of our assay, the inversion of ''fim'' switch(Tokyo_Tech/J23119) by FimB(wild-type) (<partinfo>BBa_K1632010</partinfo>)/FimE(wild-type) (<partinfo>BBa_K1632011</partinfo>) was not confirmed correctly. The FimB protein(wild-type) inverts ''fim'' switch from  [ON] state to [OFF] state and from [OFF] state to [ON] state correctly(Fig. 3.). However the FimE protein(wild-type) didn't invert ''fim'' switch predominantly from [ON] state to [OFF] state. In the assay, the FimE protein(wild-type) inverts ''fim'' switch from [ON] state to [OFF] state and from [OFF] state to [ON] state(Fig. 4.). In other words, the FimE protein works as the FimB protein.
+[[Image:Tokyo_Tech_fim_switch_TT_FimB_assay_Results.png |thumb|center|700px|<b>Fig. 3. </b>The histograms of the samples with FimB measured by flow cytometer]]<br>
+[[Image:Tokyo_Tech_fim_switch_TT_FimE_assay_Results.png |thumb|center|700px|<b>Fig. 4. </b>The histograms of the samples with FimE measured by flow cytometer]]<br>
+===More information===
-[[Image:Tokyo Tech fim switch Tokyo Tech design.png|thumb|center|600px|Fig. 1. Tokyo Tech ''fim'' switch (Tokyo_Tech/J23119) design    (Up:on state  Down:off state)]]<br>
+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]]
-<span style="margin-left: 10px;">We constructed ''fim'' switch [default ON](Tokyo_Tech/J23119)_rbs_gfp(<partinfo>BBa_K1632002</partinfo>) to characterize the function of this part, by inserting this promoter upstream of a GFP coding sequence. <br>
-<span style="margin-left: 10px;">From our results of our assay, the inversion of ''fim'' switch (Tokyo_Tech/J23119) by FimB/FimE was not confirmed correctly. The FimB protein inverts ''fim'' switch in the ON-to-OFF and the OFF-to-ON direction correctly. However the FimE protein didn’t inverts ''fim'' switch predominantly in the ON-to-OFF direction. In the assay, the FimE protein inverts ''fim'' switch in the ON-to-OFF and the OFF-to-ON direction. In other words, the FimE protein works as the FimB protein.
 <!-- Add more about the biology of this part here