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 | Translational_Unit | 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 | 432 | Work |
| BBa_K1632001 | Regulatory | fim switch[default OFF](Tokyo_Tech/J23119) | Riku Shinohara | 432 | 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 ON](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 | |
| 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 about chloramphenicol resistance(CmR), we used “rbs_CmR” (BBa_K395160 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 Pcon_lasR_TT_Plux_CmRssrA (BBa_K1632022) and Pcon_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 in the [ON] to [OFF] direction and in the [OFF] to [ON] direction (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 PBAD/araC_fimB(wild-type) (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 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.5-1-3-1). 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.5-1-3-2 and Fig.5-1-3-3).
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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 (BBa_K1632011) is a Fim recombinase. This is derived from the wild type MG1655. FimE invert the fim switch in the [ON] to [OFF] direction.
From our experimental results, we confirmed that the FimE protein inverts the fim switch(wild -type) predominantly in [ON] state to [OFF] state direction. The expression of FimE is controlled by arabinose in PBAD/araC_fimB(wild-type) (BBa_K1632012). From our experimental results (Fig. 5-1-3-4), 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 promoter, fim switch (Tokyo_Tech). A difference between the fim switch (wild-type) and the fim switch (Tokyo_Tech) is that we replaced the sigma 70 promoter to the J23119 promoter" (BBa_J23119) and two restriction enzyme cut sites are added in each side of the promoter.(Fig.5-1-4-1). Due to this addition of the restriction enzyme cut sites, we were able to replace the J23119 promoter (BBa_J23119) in the fim swtich (Tokyo_Tech). There is an example. fim switch [default ON] (Tokyo_Tech/R0010) (BBa_K1632006) is made by removing the J23119 promoter (BBa_J23119) and inserted lac promoter (BBa_R0010) (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.5-1-4-2. Replace the promoter 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 in the [ON] to [OFF]. The expression of these Fim recombinases are controlled by AHL in Pcon_lasR_TT_Plux_fimE(wild-type)(BBa_K1632018) and Pcon_rhlR_TT_Plux_fimE(wild-type)(BBa_K1632019).
