Team:Tokyo Tech/Composite Part

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Composite Part

　　

　　　　　　

Tokyo Tech 2015 iGEM Team Composite Parts

Name	Type	Description	Design	Length(bp)	Experiment
BBa_K1632002	Regulatory	fim switch[default ON](Tokyo_Tech/J23119)_gfp	Riku Shinohara	1178	Work
BBa_K1632003	Regulatory	fim switch[default OFF](Tokyo_Tech/J23119)_gfp	Riku Shinohara	1178	Work
BBa_K1632007	Composite	fim switch[default ON](wild-type)_gfp	Riku Shinohara	1128	Work
BBa_K1632008	Composite	fim switch[default OFF](wild-type)_gfp	Riku Shinohara	1128	Work
BBa_K1632012	Composite	PBAD/araC_fimB(wild-type)	Riku Shinohara	1839	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_K1632020	Composite	rbs_cmRssrA	Jun Kawamura	712	Work
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. Best New 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 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.5-3-1-1). From our experiment, cmRssrA work better than cmR without ssrA tag for our project.

Fig.5-3-1-1. The cell’s growth with Cm

2. fimB(wild-type) controlled arabinose: BBa_K1632012

BBa_K1632012 meet the criteria of the Silver Medal

• 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 from the ON state to the OFF state and from the OFF state to the ON state (Fig.5-3-2-1.).

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

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

3. fim switch(wild-type) with GFP: BBa_K1632007, BBa_K1632008

BBa_K1632007 and BBa_K1632008 meet the criteria of the Silver Medal

• 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 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.

4. fim switch(Tokyo_Tech) with GFP: BBa_K1632002, BBa_K1632003

BBa_K1632002 and BBa_K1632003 meet the criteria of the Bronze Medal

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

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.5-3-4-1).

Fig.5-3-4-1. Design of Fim Switch (Tokyo_Tech)

5. fimE(wild-type) controlled by AHL: 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.