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(wild-type) 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

1. fim switch (wild-type): BBa_K1632004, BBa_K1632005

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.5-2-1-1). 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.5-2-1-2 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

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. 5-2-2-1), 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

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.5-2-3-1). 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.5-2-3-2) .

Fig.5-2-3-1. Design of fim switch (Tokyo_Tech)

Fig.5-2-3-2. Exchange the promoter of fim switch (Tokyo_Tech)