Team:Tokyo Tech/Composite Part

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Composite Part

　　

　　　　　　

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 in the [ON] to [OFF] direction and in the [OFF] to [ON] direction (Fig.5-3-0-1.).

From our experimental results, we confirmed that the FimB protein inverts the fim switch(wild-type) in the [ON] to [OFF] direction and in the [OFF] to [ON] direction with approximately equal probability and works ideally (Fig.5-3-0-2.). The expression of FimB is controlled by arabinose in PBAD/araC_fimB(wild-type) (BBa_K1632012).

Fig.5-3-0-1. Design of fim switch (wild-type)

Fig. 5-3-0-2. The result of our experiment used BBa_K1632007, BBa_K1632008 and BBa_K1632012 with flow cytometers.

　　　　　　

0.Tokyo Tech 2015 iGEM Team: The Others 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 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.5-3-1-1). 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. 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 ON](wild-type) and the fim switch [default OFF](wild-type) 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.5-3-2-1). The fim switch (wild-type) 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 (wild-type) by adding the Fim recombinase. From our results of experiment, they work ideally (Fig.5-3-2-2 and Fig.5-3-2-3).

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

Fig.5-3-2-2. The result of our experiment used BBa_K1632007,BBa_K1632008 and BBa_K1632013 with flow cytometers

3. 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 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.5-3-3-1). 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 Plac promoter (BBa_R0010) (Fig.5-3-3-2) .

Fig.5-3-3-1. Design of fim switch (Tokyo_Tech)

Fig.5-3-3-2. Replace the promoter of fim switch (Tokyo_Tech)

4. 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 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).