Difference between revisions of "Part:BBa K1632003"
 
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<partinfo>BBa_K1632003 short</partinfo>
 
<partinfo>BBa_K1632003 short</partinfo>
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[[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;">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>
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<span style="margin-left: 10px;">We designed this ''fim'' switch which has a J23119 promoter. 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. ''fim'' switch (Tokyo_Tech/J23119) design). So the promoter can easily be interchanged. 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).
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<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).
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[[Image:Tokyo Tech fim switch Tokyo Tech Plac design.png|thumb|center|600px|Fig. 2. ''Fim'' switch(Tokyo_Tech/R0010) design]]<br>
  
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[[Image:Tokyo Tech fim switch Tokyo Tech design.png|thumb|center|600px|Fig. 1. ''fim'' switch (Tokyo_Tech/J23119) design (Up:on state Down:off state)]]<br>
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<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.
  
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<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.
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[[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>
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[[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>
  
  
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===More information===
  
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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]]
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 03:24, 19 September 2015

fim switch[default OFF](Tokyo_Tech/J23119)_rbs_gfp


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 98
    Illegal NheI site found at 121
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 92
    Illegal BamHI site found at 133
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1102