Team:Tokyo Tech/Parts

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Parts

　　

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

　　　　　　

Name	Type	Description	Design	Length(bp)	Experiment
BBa_K1632007	Composite	fim switch[default ON](wild-type)_gfp	Riku Shinohara	1128	Work
BBa_K1632012	Composite	PBAD/araC_fimB(wild-type)	Riku Shinohara	1839	Work
BBa_K1632020	Composite	rbs_CmRssrA	Jun Kawamura	712	Work

Tokyo Tech 2015 iGEM Team Parts

Name	Type	Description	Design	Length(bp)	Experiment
BBa_K1632000	Regulatory	fim switch[default ON](Tokyo_Tech/J23119)	Riku Shinohara	382	Work
BBa_K1632001	Regulatory	fim switch[default OFF](Tokyo_Tech/J23119)	Riku Shinohara	382	Work
BBa_K1632002	Composite	fim switch[default ON](Tokyo_Tech/J23119)_gfp	Riku Shinohara	1178	Work
BBa_K1632003	Composite	fim switch[default OFF](Tokyo_Tech/J23119)_gfp	Riku Shinohara	1178	Work
BBa_K1632004	Regulatory	fim switch[default ON](wild-type)	Riku Shinohara	382	Work
BBa_K1632005	Regulatory	fim switch[default OFF](wild-type)	Riku Shinohara	382	Work
BBa_K1632006	Regulatory	fim switch[default ON](Tokyo_Tech/R0010)	Riku Shinohara	597	Work
BBa_K1632008	Composite	fim switch[default OFF](wild-type)_gfp	Riku Shinohara	1128	Work
BBa_K1632010	Coding	fimB(wild-type)	Riku Shinohara	603	Work
BBa_K1632011	Coding	fimE(wild-type)	Riku Shinohara	597	Work
BBa_K1632013	Composite	Pbad/araC_fimE(wild-type)	Riku Shinohara	1835	Work
BBa_K1632018	Composite	J23100_lasR_TT_Plux_fimE(wild-type)	Jun Kawamura	1609
BBa_K1632019	Composite	J23100_rhlR_TT_Plux_fimE(wild-type)	Jun Kawamura	1615
BBa_K1632022	Composite	J23100_lasR_TT_Plux_CmRssrA	Jun Kawamura	1704	Work
BBa_K1632023	Composite	J23100_rhlR_TT_Plux_CmRssrA	Jun Kawamura	1710	Work

1. Improved Part: BBa_K1632020, BBa_K1632022, BBa_K1632023

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

• BBa_K1632020
  rbs_CmRssrA
• BBa_K1632022
  J23100_lasR_TT_Plux_CmRssrA
• BBa_K1632023
  J23100_rhlR_TT_Plux_CmRssrA

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 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

• BBa_K1632010
  fimB(wild-type)
• BBa_K1632012
  Pbad/araC_fimB(wild-type)

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 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

• BBa_K1632004
  fim switch[default ON](wild-type)
• BBa_K1632005
  fim switch[default OFF](wild-type)
• BBa_K1632007
  fim switch[default ON](wild-type)_gfp
• BBa_K1632008
  fim switch[default OFF](wild-type)_gfp

We are the first team in iGEM to successfully construct both the fim switch default ON state and the fim switch default OFF state and experiment 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 state (BBa_K1632004) and the other in the default OFF state (BBa_K1632005). 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.5-1-3-1. The design of fim switch (wild-type)

BBa_K1632013 meet the criteria of the Silver Medal

• BBa_K1632011
  FimE(wild-type)
• BBa_K1632013
  PBAD/araC_fimE (wild-type)

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 BBa_K1632013. From our experimental results (Fig.5-1-3-2.), they work ideally.

Fig.5-1-3-2. 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

• BBa_K1632000
  fim switch[default ON](Tokyo_Tech/J23119)
• BBa_K1632001
  fim switch[default OFF](Tokyo_Tech/J23119)
• BBa_K1632002
  fim switch[default ON](Tokyo_Tech/J23119)_gfp
• BBa_K1632003
  fim switch[default OFF](Tokyo_Tech/J23119)_gfp
• BBa_K1632006
  fim switch[default ON](Tokyo_Tech/R0010)

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 promoter to the J23119 promotor" (BBa_J23119) and two restriction enzyme cut sites are added in each side of the promoter. Due to this addition of the restriction enzyme cut sites, we were able to replace the J23119 promoter in the fim swtich (Tokyo_Tech). There is an example. 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

• BBa_K1632018
  J23100_lasR_TT_Plux_fimE (wild-type)
• BBa_K1632019
  J23100_rhlR_TT_Plux_fimE (wild-type)

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.