To meet the criteria of the Gold Medal, we submitted BBa_K1632020, BBa_K1632022
and BBa_K1632023.
To meet the criteria of the Silver Medal, we submitted BBa_K1632007, BBa_K1632008, BBa_K1632012 and BBa_K1632013.
To meet the criteria of the Bronze Medal, we submitted BBa_K1632002 and BBa_K1632003.

Favorite Tokyo Tech 2015 iGEM Team Parts

Tokyo Tech 2015 iGEM Team Parts

1. 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_K395160 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 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.5-1-1-1). From our experiment, CmRssrA work better than CmR without ssrA tag for our project.

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

2. Best New Basic Part and Best New Composite part: BBa_K1632010, BBa_K1632012

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 in the ON-to-OFF direction and in the OFF-to-ON direction (Fig.5-1-2-1.).

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.5-1-2-2.). The expression of FimB is controlled by arabinose in PBAD/araC_fimB(wild-type) (BBa_K1632012).

Fig.5-1-2-1. Design of fim switch (wild-type)

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

3. Best Part Collection: BBa_K1632004, BBa_K1632005, BBa_K1632007, BBa_K1632008, BBa_K1632011, BBa_K1632013

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 ON and the fim switch default OFF and experimented them. These fim switch is derived from a wild type. The fim switch(wild-type) has a sigma 70 promotor which functions constitutively. We submitted two parts, one in the default ON (BBa_K1632004) and the other in the default OFF (BBa_K1632005)(Fig.5-1-3-1). The fim switch 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 by adding the Fim recombinase. From our results of experiment, they work ideally (Fig.5-1-3-2 and Fig.5-1-3-3).

Fig.5-1-3-1. The design of fim switch (wild-type)

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

Fig.5-1-3-3. The result of our experiment used BBa_K1632007,BBa_K1632008 and BBa_K1632013 with flow cytometers

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 to OFF direction. The expression of this Fim recombinase is controlled by arabinose in PBAD/araC_fimE (wild-type)(BBa_K1632013). From our experimental results (Fig.5-1-3-4.), they work ideally.

Fig.5-1-3-4. The result of our experiment used BBa_K1632007,BBa_K1632008 and BBa_K1632013 with flow cytometers

4. Part Collection: BBa_K1632000, BBa_K1632001, BBa_K1632002, BBa_K1632003, BBa_K1632006

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 fim switch (wild-type) and the fim switch (Tokyo_Tech) is that we replaced the sigma 70 promotor to the J23119 promotor" (BBa_J23119) and two restriction enzyme cut sites are added in each side of the promotor.(Fig.5-1-4-1). Due to this addition of the restriction enzyme cut sites, we were able to replace the J23119 promotor (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 promotor (BBa_J23119) and inserted Plac promotor (BBa_R0010) (Fig.5-1-4-2) .

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

Fig.5-1-4-2. Replace the promotor of fim switch (Tokyo_Tech)

5. Submitted parts : BBa_K1632018, BBa_K1632019

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