Parts
Favorite Tokyo Tech 2015 iGEM Team Parts
| Name | Type | Description | Design | Length(bp) | Experiment |
|---|
| BBa_K1632007 | Composite | fim switch[default ON](wild-type)_gfp | Riku Shinohara | 1128 | Work |
| BBa_K1632012 | Composite | PBAD/araC_fimB(wild-type) | Riku Shinohara | 1839 | Work |
| BBa_K1632020 | Composite | rbs_CmRssrA | Jun Kawamura | 712 | Work |
Tokyo Tech 2015 iGEM Team 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_K1632002 | Composite | fim switch[default ON](Tokyo_Tech/J23119)_gfp | Riku Shinohara | 1178 | Work |
| BBa_K1632003 | Composite | fim switch[default OFF](Tokyo_Tech/J23119)_gfp | Riku Shinohara | 1178 | 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/B0010) | Riku Shinohara | 597 | Work |
| BBa_K1632008 | Composite | fim switch[default OFF](wild-type)_gfp | Riku Shinohara | 1128 | Work |
| BBa_K1632010 | Coding | fimB(wild-type) | Riku Shinohara | 603 | Work |
| BBa_K1632011 | Coding | fimE(wild-type) | Riku Shinohara | 597 | Work |
| BBa_K1632013 | Composite | Pbad/araC_fimE(wild-type) | Riku Shinohara | 1835 | Work |
| BBa_K1632018 | Composite | J23100_lasR_TT_Plux_fimE(wild-type) | Jun Kawamura | 1609 | |
| BBa_K1632019 | Composite | J23100_rhlR_TT_Plux_fimE(wild-type) | Jun Kawamura | 1615 | |
| BBa_K1632022 | Composite | J23100_lasR_TT_Plux_CmRssrA | Jun Kawamura | 1704 | Work |
| BBa_K1632023 | Composite | J23100_rhlR_TT_Plux_CmRssrA | Jun Kawamura | 1710 | Work |
1. Improved Part: BBa_K1632020, BBa_K1632022, BBa_K1632023
At the first stage of our wet experiment, we used “rbs_CmR” (BBa_K395610 by iGEM 2010 team Tokyo_Tech). However, the result showed a leaky expression of CmR. We inserted an ssrA degradation tag to the C-terminal of CmR. In the our experiment using the J23100_lasR_TT_Plux_CmRssrA (BBa_K1632022) and J23100_rhlR_TT_Plux_CmRssrA (BBa_K1632023), we could not observe cell growth for cells that owned the ssrA-tagged plasmid, in the absence of AHL (Fig.5-1-1-1). From our experiment, CmRssrA work better than CmR without ssrA tag for our project.
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Fig.5-1-1-1. The cell’s growth with Cm |
2. Best New Basic Part and Best New Composite part: BBa_K1632010, BBa_K1632012
BBa_K1632012 meet the criteria of the Silver Medal
FimB (BBa_K1632010) is a Fim recombinase. This is derived from the wild type MG1655. FimB invert the fim switch from the ON state to the OFF state and from the OFF state to the ON state (Fig.5-1-2-1.).
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.5-1-2-2.). The expression of FimB is controlled by arabinose in BBa_K1632012.
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Fig.5-1-2-1. Design of fim switch (wild-type) |
3. Best Part Collection: BBa_K1632004, BBa_K1632005, BBa_K1632007, BBa_K1632008, BBa_K1632011, BBa_K1632013
We are the first team in iGEM to successfully construct both the fim switch default state ON and the fim switch default state OFF and assay them. These fim switch is derived from a wild type and the gene sequence is the same as that of a wild type E.coli. The fim switch is inverted by the Fim recombinase. Therefore, we can regulate the expression of the gene downstream of the fim switch by adding the Fim recombinase. From the flow cytometers assay, they work ideally.
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Fig.5-1-3-1. The design of fim switch (wild-type) |
BBa_K1632013 meet the criteria of the Silver Medal
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) from the ON state to the OFF state. The expression of this Fim recombinase is controlled by arabinose in BBa_K1632013. From our experimental results (Fig.5-1-3-2.), they work ideally.
4. Part Collection: BBa_K1632000, BBa_K1632001, BBa_K1632002, BBa_K1632003, BBa_K1632006
We designed another fim switch with a standardized interchangeable promotor, 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 promotor" (BBa_J23119). We also inserted two restriction enzyme sites in both the front (SalI and BamHI) and the back (BglII and MluI) of the promotor. By inserting the restriction enzymes, our fim switch (Tokyo_Tech) turned into a fim switch with a standardized interchangeable promotor (Fig.5-1-4-1). There is an example. BBa_K1632006 is made by removing the J23119 promotor (BBa_J23119) and inserted Plac promotor (BBa_B0010) (Fig.5-1-4-2) .
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Fig.5-1-4-1. Design of Fim Switch (Tokyo_Tech) |
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Fig.5-1-4-1. Design of Fim Switch (Tokyo_Tech) |
5. Submitted parts : BBa_K1632018, BBa_K1632019
FimE is a Fim recombinase. This Fim recombinase is derived from the wild type MG1655. FimE invert the fim switch from the ON state to the OFF state. The expression of these Fim recombinases are controlled by AHL in BBa_K1632018 and BBa_K1632019.
