We constructed 13 fim related parts, and established a tripartite relationship between the fim switch and FimB/FimE. Although fim related parts have been submitted from past iGEM teams, respectively, who lack detailed analysis or full length of protein-coding sequence, we are the first team in iGEM to successfully assay and clearly confirm the function of the fim system, by using GFP located downstream of fim switch containing promoter sequence. Our FimB parts clearly showed random bidirectional inversion of fim switch, according to colony formation by plasmid mixture inverted by FimB, on top of FACS and sequence analysis. Our fimE parts having full-length coding sequence also showed expected unidirectional inversion of the switch. Also, the collection includes parts that can be used as controls in the assay. We thus claim that our collection can be applied to various future projects in iGEM, and that is an unprecedented achievement in iGEM.

Best New Basic Part of Tokyo Tech 2015 iGEM Team Parts

Best New Composite Part of Tokyo Tech 2015 iGEM Team Parts

Best Part Collection of Tokyo Tech 2015 iGEM Team Parts

1. fim switch(Tokyo_Tech): 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.5-4-1-1). 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 Plac promoter (BBa_R0010) (Fig.5-4-1-2) .

Fig.5-4-1-1. Design of fim switch (Tokyo_Tech)

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](wild-type) and the fim switch [default OFF](wild-type) 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.5-4-2-1). The fim switch (wild-type) 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 (wild-type) by adding the Fim recombinase. From our results of experiment, they work ideally (Fig.5-4-2-2 and Fig.5-4-2-3).

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

Fig. 5-4-2-2. The result of our experiment used BBa_K1632007, BBa_K1632008 and BBa_K1632012 with flow cytometers.

Fig.5-4-2-3. The result of our experiment used BBa_K1632007,BBa_K1632008 and BBa_K1632013 with flow cytometers

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).

Fig. 5-4-3-1. The result of our experiment used BBa_K1632007, BBa_K1632008 and BBa_K1632012 with flow cytometers.

Fig.5-4-3-2. The result of our experiment used BBa_K1632007,BBa_K1632008 and BBa_K1632013 with flow cytometers