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 about chloramphenicol resistance(CmR), 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 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.5-1-3-1). 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 (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. 5-1-3-4), 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 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.5-1-4-1). 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 lac promoter (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 promoter 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).