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

BBa_K1632002 and BBa_K1632003 meet the criteria of the Bronze Medal

We designed another fim switch with a standardized interchangeable promotor, fim switch (Tokyo_Tech). A difference between the wild type fim switch and the fim switch (Tokyo_Tech) is that we replaced the sigma 70 promoter to the J23119 promotor" (BBa_J23119). We also inserted two restriction enzyme sites in both the front (SalI and 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). There is an example. BBa_K1632006 is made by removing the J23119 promotor (BBa_J23119) and inserted Plac promotor (BBa_B0010) .

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

FimE is a Fim recombinase. This Fim recombinase is derived from the wild type MG1655. FimE invert the fim switch from the ON state to the OFF state. The expression of these Fim recombinases are controlled by AHL in BBa_K1632018 and BBa_K1632019.

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

BBa_K1632007 and BBa_K1632008 meet the criteria of the Silver Medal

We are the first team in iGEM to successfully construct both the fim switch default state ON and the fim switch default state OFF and assay them. These fim switch is derived from a wild type and the gene sequence is the same as that of a wild type E.coli. 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. From the flow cytometers assay, they work ideally.

BBa_K1632013 meet the criteria of the Silver Medal

FimE(wild-type)(BBa_K1632011) is Fim recombinases. This Fim recombinase is derived from the wild type MG1655. FimE invert the fim switch (wild-type) from the ON state to the OFF state. The expression of this Fim recombinase is controlled by arabinose in BBa_K1632013. From our experimental results (Fig. 5-1-2-1.), they work ideally.

Fig.5-1-2-1. The result of our assay used BBa_K1632007,BBa_K1632008 and BBa_K1632013 with flow cytometers

3. Best New Basic Part: BBa_K1632012, BBa_K1632010

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 from the ON state to the OFF state and from the OFF state to the ON state (Fig.5-1-3-1.).

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.5-1-3-2.). The expression of FimB is controlled by arabinose in BBa_K1632012.

Fig.5-1-3-1. 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.5-1-3-2. The result of our assay used BBa_K1632007, BBa_K1632008 and BBa_K1632012 with flow cytometers.

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

BBa_K1632020, BBa_K1632022 and BBa_K1632023 meet the criteria of the Gold Medal

At the first stage of our wet experiment, we used “rbs_cmR” (BBa_K395610 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 J23100_lasR_TT_Plux_cmRssrA (BBa_K1632022) and J23100_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.5-1-4-2). From our experiment, cmRssrA work better than cmR without ssrA tag for our project.

Fig.5-1-4-1. The cell’s growth with Cm