Favorite Tokyo Tech 2015 iGEM Team Parts

Tokyo Tech 2015 iGEM Team Parts

1. Part Collection: BBa_K1632000, BBa_K1632001, BBa_K1632002, BBa_K1632003, BBa_K1632007, BBa_K1632018, BBa_K1632019

We designed anotherfim switch with a standardized interchangeable promotor, fim switch (Tokyo-Tech). The only difference between the Wild Type fim switch and the fim switch (Tokyo-Tech) is that we changed the sigma 70 promoter to the J23119 promotor and inserted two restriction enzyme sites in both the front (SalIand 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.5-1-1-1). As an example is BBa_K1632006. Except for the fact we inserted the restriction enzyme sites, the basic design of fim switch (Tokyo Tech) is similar to the Wild Type fim switch.

Fig.5-3-1-1. Design of Fim Switch (Tokyo-Tech)

Fim E is a Fim recombinase. With the expression of FimE, the fim switch only inverts from the ON state to the OFF state. This Fim recombinase is derived from the wild type MG1655. From our results, they work ideally.

The expression of these Fim recombinases are controlled by AHL in BBa_K1632018 and BBa_K1632019. We are the first team in iGEM to construct a part with Fim recominase, which can be expressed with cell-cell communication.

2. New Part Collection: BBa_K1632004, BBa_K1632005, BBa_K1632007, BBa_K1632008, BBa_K1632013, BBa_K1632011

We are the first team in iGEM to successfully construct and assay both the fim switch default state ON and the fim switch default state OFF. These fim switch is derived from a wild type and the gene sequence is the same as that of a wild type E.coli. From the FLA assay, they work ideally. The fim switch is the promoter containing a repeated DNA sequence which is inverted by the Fim recombinase. Therefore, we can control the expression of the gene downstream of the fim switch by adding the Fim recombinase.

Fim E (Wild Type) is Fim recombinases. This Fim recombinase is derived from the wild type MG1655. From our results (Fig. 5-1-2-1.), they work ideally. The expression of this Fim recombinase is controlled by arabinose in BBa_K1632013. With the expression of FimE, the fim switch (wild-type) is only inverted from the ON state to the OFF state.

Fig.5-1-2-1. The result of our assay with flow cytometers

3. Best New Improved Part: BBa_K1632020, BBa_K1632022, BBa_K1632023

At the first stage of our wet lab experiment, we used “rbs_cmR” (BBa_K395160). However, the result showed a leaky expression of CmR (Fig.5-1-3-1). Therefore we suspected that there was a leakage in the promoter. We constructed a new plasmid with an ssrA degradation tag. From the results of 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.5-1-3-2, Fig.5-1-3-3). From our experiment, cmRssrA work better than CmR without ssrA tag.

Fig.5-1-3-1. The cell’s growth with Cm without cmRssrA

Fig.5-1-3-2. The cell’s growth with Cm without cmRssrA

Fig.5-1-3-3. ssrA tag reduces the influence of the leak of CmR

4. Best New Basic Part: BBa_K1632012, BBa_K1632010

FimB (BBa_K1632010) is a Fim recombinase. With the expression of FimB, the fim switch inverts from the ON state to the OFF state and from the OFF state to the ON state (Fig.5-1-4-1.).

This fim recombinase is derived from the wild type MG1655. From our assay 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. The expression of FimB is controlled by arabinose in BBa_K1632012 (Fig.5-1-4-2.).

Fig.5-1-4-1. 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.5-1-4-2. The result of our assay with with flow cytometers.