Parts Collection
We constructed 13 fim related parts, and established a tripartite relationship between the fim switch and FimB/FimE, which is an unprecedented achievement in iGEM. Although fim related parts have been submitted from past iGEM teams, since the results of their experiments lack reproducibility, we are the first team in iGEM to successfully assay and confirm the function of the all three types of fim related parts. Since the assay results matched with the results reported in past reference papers, we confirmed that our fim switch (both default ON and default OFF), FimB, and FimE is functioning. Combining the parts in the collection enables the fim switch from inverting either back and forth, or in one direction with the induction of arabinose. Since the collection includes parts that can be used as controls in the assay, this collection can be applied to various future projects in iGEM.
Best New Basic Part of Tokyo Tech 2015 iGEM Team Parts
| Name | Type | Description | Design | Length(bp) | Experiment |
|---|
| BBa_K1632010 | Coding | fimB(wild-type) | Riku Shinohara | 603 | Work |
Best New Composite Part of Tokyo Tech 2015 iGEM Team Parts
| Name | Type | Description | Design | Length(bp) | Experiment |
|---|
| BBa_K1632012 | Composite | PBAD/araC_fimB(wild-type) | Riku Shinohara | 1839 | Work |
Best Part Collection of 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 OFF](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 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 | Work |
| BBa_K1632007 | Composite | fim switch[default ON](wild-type)_gfp | Riku Shinohara | 1128 | Work |
| BBa_K1632008 | Composite | fim switch[default OFF](wild-type)_gfp | Riku Shinohara | 1128 | Work |
| BBa_K1632011 | Coding | fimE(wild-type) | Riku Shinohara | 597 | Work |
| BBa_K1632013 | Composite | Pbad/araC_fimE(wild-type) | Riku Shinohara | 1835 | Work |
1. fim switch(Tokyo_Tech): BBa_K1632000, BBa_K1632001, BBa_K1632002, BBa_K1632003, BBa_K1632006
We designed the fim switch(Tokyo_Tech), the promoter of the fim switch(wild-type) is replaced with J23119 promoter (BBa_J23119) and two restriction enzyme cut sites are added in each side of the promoter. 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)(Fig.5-4-1-1). For example, we made fim switch[default ON](Tokyo_Tech/R0010)(BBa_K1632006) by replaced J23119 promoter with Lac promoter (BBa_R0010)(Fig.5-4-1-2).
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Fig.5-4-1-1. Design of fim switch (Tokyo_Tech) |
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Fig.5-4-1-2. Replace the promoter of fim switch (Tokyo_Tech) |
2. fim switch (wild-type): BBa_K1632004, BBa_K1632005, BBa_K1632007, BBa_K1632008
We are the first team in iGEM to successfully construct both the fim switch default ON and the fim switch default OFF and experienced them. These fim switch is derived from a wild type and the gene sequence is the same as that of a wild type. 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 (Fig.5-4-2-1). From the results of our experiment with flow cytometers , they work ideally(Fig.5-4-2-2 and Fig.5-4-2-3).
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Fig.5-4-2-1. 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 |
3. Fim recombinases (wild-type): BBa_K1632010, BBa_K1632011, BBa_K1632012, BBa_K1632013
FimB (BBa_K1632010) and FimE are a Fim recombinase. These are derived from the wild type MG1655. FimB invert the fim switch in the ON-to-OFF direction and in the OFF-to-ON direction, FimE invert the fim switch (wild-type) in the ON-to-OFF direction. The expression of these Fim recombinases are controlled by arabinose in both PBAD/araC_fimB(BBa_K1632013) and PBAD/araC_fimE(BBa_K1632012).
From our experimental results, we confirmed that the Fim recombinases inverts the fim switch ideally. (Fig.5-4-3-1. Fig.5-4-3-2).
