Team:Tokyo Tech/Parts

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Parts

Each part given us a whole new experience

　　

Favorite Tokyo Tech 2015 iGEM Team Parts

　　　　　　

Name	Type	Description	Design	Length(bp)	Experiment
BBa_K1632007	Composite	fim switch[default ON](wild type)_rbs_gfp	Riku Shinohara	1128	Work
BBa_K1632012	Composite	Pbad/araC_fimB	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)	Riku Shinohara	382	Work
BBa_K1632001	Regulatory	fim switch[default ON](Tokyo-Tech)	Riku Shinohara	382	Work
BBa_K1632002	Regulatory	fim switch[default ON](Tokyo-Tech)_rbs_gfp	Riku Shinohara	1178	Work
BBa_K1632003	Regulatory	fim switch[default OFF](Tokyo-Tech)_rbs_gfp	Riku Shinohara	1178	Work
BBa_K1632004	Regulatory	fim switch[default OFF](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 Lac promoter)	Riku Shinohara	597	Work
BBa_K1632008	Composite	fim switch[default OFF](wild type)_rbs_gfp	Riku Shinohara	1128	Work
BBa_K1632010	Coding	FimB	Riku Shinohara	603	Work
BBa_K1632011	Coding	FimE	Riku Shinohara	597	Work
BBa_K1632013	Composite	Pbad/araC_rbs_fimE(Wild Type)	Riku Shinohara	1835	Work
BBa_K1632018	Composite	J23100_lasR_TT_Plux_fimE	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. Part Collection: BBa_K1632000, BBa_K1632001, BBa_K1632002, BBa_K1632003, BBa_K1632007, BBa_K1632018, BBa_K1632019

• BBa_K1632000
  fim switch[default ON](Tokyo-Tech)
• BBa_K1632001
  fim switch[default OFF](Tokyo-Tech)
• BBa_K1632002
  fim switch[default ON](Tokyo-Tech)_rbs_gfp
• BBa_K1632006
  fim switch[default OFF](Tokyo-Tech Lac promoter)

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)

• BBa_K1632018
  J23100_lasR_TT_Plux_fimE (Wild Type)
• BBa_K1632019
  J23100_rhlR_TT_Plux_fimE (Wild Type)

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

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

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.

• BBa_K1632013
  Pbad/araC_rbs_fimE (wild type)
• BBa_K1632011
  FimE(wild-type)

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

• 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 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

4. Best New Basic Part: BBa_K1632012, BBa_K1632010 BBa_K1632012 Pbad/araC_rbs_fimB BBa_K1632010 fimB 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.5-14-1. 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.