- BBa_K1632010
fimB(wild-type)
Difference between revisions of "Team:Tokyo Tech/Basic Part"
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− | <p class="text">We are the first team in iGEM to successfully construct both the <i>fim</i> switch default ON and the <i>fim</i> switch default OFF and experimented them. These <i>fim</i> switch is derived from a wild type. The <i>fim</i> switch(wild-type) has a sigma 70 | + | <p class="text">We are the first team in iGEM to successfully construct both the <i>fim</i> switch default ON and the <i>fim</i> switch default OFF and experimented them. These <i>fim</i> switch is derived from a wild type. The <i>fim</i> switch(wild-type) has a sigma 70 promoter which functions constitutively. We submitted two parts, one in the default ON (<a href="http://parts.igem.org/Part:BBa_K1632004" target="_brank">BBa_K1632004</a>) and the other in the default OFF (<a href="http://parts.igem.org/Part:BBa_K1632005" target="_brank">BBa_K1632005</a>) (Fig.5-2-1-1). The <i>fim</i> switch is inverted by two recombinases, FimB (<a href="http://parts.igem.org/Part:BBa_K1632010" target="_brank">BBa_K1632010</a>) and FimE (<a href="http://parts.igem.org/Part:BBa_K1632011" target="_brank">BBa_K1632011</a>). Therefore, we can regulate the expression of the gene downstream of the <i>fim</i> switch by adding the Fim recombinase. From our results of experiment, they work ideally(Fig.5-2-1-2 and Fig.5-2-1-3).</p> |
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− | <p class="text">We designed <i>fim</i> switch with a standardized interchangeable | + | <p class="text">We designed <i>fim</i> switch with a standardized interchangeable promoter, <i>fim</i> switch (Tokyo_Tech). A difference between the wild type <i>fim</i> switch and the <i>fim</i> switch (Tokyo_Tech) is that we replaced the sigma 70 promoter to the J23119 promoter" (<a href="http://parts.igem.org/Part:BBa_J23119" target="_brank">BBa_J23119</a>). We also inserted two restriction enzyme sites in both the front (SalI and BamHI) and the back (BglII and MluI) of the promoter. By inserting the restriction enzymes, our <i>fim</i> switch (Tokyo_Tech) turned into a <i>fim</i> switch with a standardized interchangeable promoter (Fig.5-2-3-1). There is an example. <i>fim</i> switch[default ON](Tokyo_Tech/R0010) (<a href="http://parts.igem.org/Part:BBa_K1632006" target="_brank">BBa_K1632006</a>) is made by removing the J23119 promoter (<a href="http://parts.igem.org/Part:BBa_J23119" target="_brank">BBa_J23119</a>) and inserted Plac promoter (<a href="http://parts.igem.org/Part:BBa_R0010" target="_brank">BBa_R0010</a>) (Fig.5-2-3-2) . </p> |
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<table width="940px"><tbody><tr><td align="center"><img src="https://static.igem.org/mediawiki/2015/c/cb/Tokyo_Tech_parts1.png" width="60%"></td></tr><tr><td align="center"><h4 class="fig">Fig.5-2-3-1. Design of <i>fim</i> switch (Tokyo_Tech)</h4></tr></td></tbody></table> | <table width="940px"><tbody><tr><td align="center"><img src="https://static.igem.org/mediawiki/2015/c/cb/Tokyo_Tech_parts1.png" width="60%"></td></tr><tr><td align="center"><h4 class="fig">Fig.5-2-3-1. Design of <i>fim</i> switch (Tokyo_Tech)</h4></tr></td></tbody></table> | ||
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− | <table width="940px"><tbody><tr><td align="center"><img src="https://static.igem.org/mediawiki/2015/b/b7/Tokyo_Tech_parts10.png" width="60%"></td></tr><tr><td align="center"><h4 class="fig">Fig.5-2-3-2. Exchange the | + | <table width="940px"><tbody><tr><td align="center"><img src="https://static.igem.org/mediawiki/2015/b/b7/Tokyo_Tech_parts10.png" width="60%"></td></tr><tr><td align="center"><h4 class="fig">Fig.5-2-3-2. Exchange the promoter of <i>fim</i> switch (Tokyo_Tech)</h4></tr></td></tbody></table> |
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Revision as of 16:35, 18 September 2015
Basic Parts
Best Basic Part: fimB (wild-type) (BBa_K1632010)
FimB (BBa_K1632010) is a Fim recombinase. This is derived from the wild type MG1655. FimB invert the fim switch in the ON-to-OFF direction and in the OFF-to-ON direction (Fig.5-2-0-1.).
From our experimental results, we confirmed that the FimB protein inverts the fim switch in the ON-to-OFF direction and in the OFF-to-ON direction with approximately equal probability and works ideally (Fig.5-2-0-2.). The expression of FimB is controlled by arabinose in PBAD/araC_fimB(wild-type) (BBa_K1632012).
Fig.5-2-0-1. Design of fim switch (wild-type) |
Fig. 5-2-0-2. The result of our experiment used BBa_K1632007, BBa_K1632008 and BBa_K1632012 with flow cytometers. |
0.Tokyo Tech 2015 iGEM Team: The Others Basic Parts
Name | Type | Description | Design | Length(bp) | Experiment |
---|---|---|---|---|---|
BBa_K1632000 | Regulatory | fim switch[default ON](Tokyo_Tech/J23119) | Riku Shinohara | 382 | Work |
BBa_K1632001 | Regulatory | fim switch[default ON](Tokyo_Tech/J23119) | Riku Shinohara | 382 | Work |
BBa_K1632004 | Regulatory | fim switch[default OFF](wild-type) | Riku Shinohara | 382 | Work |
BBa_K1632005 | Regulatory | fim switch[default OFF](wild-type) | Riku Shinohara | 382 | Work |
BBa_K1632006 | Regulatory | fim switch[default ON](Tokyo_Tech/R0010) | Riku Shinohara | 597 | Work |
BBa_K1632010 | Coding | fimB(wild-type) | Riku Shinohara | 603 | Work |
BBa_K1632011 | Coding | fimE(wild-type) | Riku Shinohara | 597 | Work |
1. fim switch (wild-type): BBa_K1632004, BBa_K1632005
- BBa_K1632004
fim switch[default ON](wild-type) - BBa_K1632005
fim switch[default OFF](wild-type)
We are the first team in iGEM to successfully construct both the fim switch default ON and the fim switch default OFF and experimented them. These fim switch is derived from a wild type. The fim switch(wild-type) has a sigma 70 promoter which functions constitutively. We submitted two parts, one in the default ON (BBa_K1632004) and the other in the default OFF (BBa_K1632005) (Fig.5-2-1-1). The fim switch is inverted by two recombinases, FimB (BBa_K1632010) and FimE (BBa_K1632011). Therefore, we can regulate the expression of the gene downstream of the fim switch by adding the Fim recombinase. From our results of experiment, they work ideally(Fig.5-2-1-2 and Fig.5-2-1-3).
Fig.5-2-1-1. fim switch is inverted by two recombinases, FimB and FimE. These proteins have distinct activities. The FimB protein inverts fim switch in the ON-to-OFF and the OFF-to-ON direction with approximately equal probability |
Fig. 5-2-1-2. The result of our assay used BBa_K1632007, BBa_K1632008 and BBa_K1632012 with flow cytometers. |
Fig.5-2-1-3. The result of our assay used BBa_K1632007,BBa_K1632008 and BBa_K1632013 with flow cytometers |
2. fimE (wild-type): BBa_K1632011
- BBa_K1632011
fimE(wild-type)
FimE(wild-type)(BBa_K1632011) is Fim recombinases. This Fim recombinase is derived from the wild type MG1655. FimE invert the fim switch (wild-type) in the ON-to-OFF direction. The expression of this Fim recombinase is controlled by arabinose in PBAD/araC_fimE (BBa_K1632013). From our experimental results (Fig. 5-2-2-1.), they work ideally.
Fig.5-2-2-1. The result of our assay used BBa_K1632007,BBa_K1632008 and BBa_K1632013 with flow cytometers |
3. fim switch (Tokyo_Tech): BBa_K1632000, BBa_K1632001, BBa_K1632006
- BBa_K1632000
fim switch[default ON](Tokyo_Tech/J23119) - BBa_K1632001
fim switch[default OFF](Tokyo_Tech/J23119) - BBa_K1632006
fim switch[default ON](Tokyo_Tech/R0010)
We designed fim switch with a standardized interchangeable promoter, fim switch (Tokyo_Tech). A difference between the wild type fim switch and the fim switch (Tokyo_Tech) is that we replaced the sigma 70 promoter to the J23119 promoter" (BBa_J23119). We also inserted two restriction enzyme sites in both the front (SalI and BamHI) and the back (BglII and MluI) of the promoter. By inserting the restriction enzymes, our fim switch (Tokyo_Tech) turned into a fim switch with a standardized interchangeable promoter (Fig.5-2-3-1). There is an example. fim switch[default ON](Tokyo_Tech/R0010) (BBa_K1632006) is made by removing the J23119 promoter (BBa_J23119) and inserted Plac promoter (BBa_R0010) (Fig.5-2-3-2) .
Fig.5-2-3-1. Design of fim switch (Tokyo_Tech) |
Fig.5-2-3-2. Exchange the promoter of fim switch (Tokyo_Tech) |