Team:Tokyo Tech/Parts

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Tokyo_Tech

Parts

Each part give us a whole new experience

 

Favorite Tokyo Tech 2014 iGEM Team Parts

 
Name
Type
Description
Design
Length (bp)
  K1529301 Composite Prhl(RL)-GFP Kohdai Hibi 801
  K1529265 Composite Plux-CmR-RhlI Kohdai Hibi 1435
  K1529263 Composite Plux-CmR-LasI Kohdai Hibi 1435

 

Tokyo Tech 2013 iGEM Team Parts

 
Name
Type
Description
Design
Length (bp)
  K1529311 Composite Prhl(RL)-GFP Kohdai Hibi 801
  K1529321 Composite Prhl(a)-GFP Kohdai Hibi 801
  K1529264 Composite Plux-CmR-LuxI Kohdai Hibi 1411
  K1529302 Composite Prhl(RL)_CmR_LasI Kohdai Hibi 1435
  K1529020 Composite Ptet-ydeD-tolC Kohdai Hibi 1633

 

Best new Basic part: BBa_K1529300, BBa_K1529310, BBa_K1529320

• Prhl(RL) : BBa_K1529300
• Prhl(LR) : BBa_K1529310
• Prhl(RR) : BBa_K1529320

Prhl(RL) Promoter has the biggest ratio of leakage and expression (when C4HSL is induced) among the three above.

   

Best new Composite part: BBa_K1529301, BBa_K1529302, BBa_K1529797

Prhl(RL)_GFP (BBa_K1529301)

Prhl(RL) is a promoter that is activated by C4HSL. We improved Prhl Promoter(BBa_R0071)  by changing LuxR binding site of Plux Promoter(BBa_R0062) to RhlR binding site. To characterize the function of the Prhl(RL) Promoter (BBa_K1529300), we constructed this part,  Prhl(RL)_GFP (BBa_K1529301) , by inserting a Prhl(RL) promoter into the upstream region of GFP coding sequence. By using reporter cells containing Prhl(RL)_GFP, we measured the fluorescence intensity of the cells induced by C4HSL. We confirmed that our new Prhl(RL) promoter was actually activated by C4HSL through an induction assay (Fig. 5-1-1).

Fig. 5-1-1.  Prhl(RL) has no leakage and higher expression
   

Prhl(RL)_CmR_LasI (BBa_K1529302)

We constructed this part by combining BBa_K1529300, BBa_K395160, BBa_B0034 and BBa_C0078.
(CmR is Chloramphenicol resistance.)

Prhl(RL) Promoter has no leakage and higher expression than Prhl Promoter (BBa_R0071). CmR and LasI are inserted into the downstream of the promoter. This is the first Biobrick part that succeeded in C4HSL dependent CmR and 3OC12HSL production. Using this part with the plasmid that is constitutively expressing RhlR, we succeeded in confirming RhlR activates the expression of CmR and LasI in the presence of C4HSL.

Moreover, by co-culturing the cells containing this part with the cells containing 3OC12HSL dependent C4HSL producer part (BBa_K1529797), we succeeded in constructing a positive feedback system.

Fig. 5-1-2. 
   

Plux_CmR_RhlI (BBa_K1529797)

We constructed this part by combining BBa_K395162BBa_B0034 and BBa_C0070.
(CmR is Chloramphenicol resistance.)

This is the first Biobrick part that succeeded in 3OC12HSL dependent CmR and C4HSL production. In our experiment, we used 3OC12HSL instead of 3OC6HSL as an inducer in order to circumvent crosstalk. Using this part with the plasmid that is constitutively expressing luxR, we succeeded in confirming LuxR activates the expression of CmR and RhlI in the presence of 3OC12HSL.

Fig. 5-1-3. 
   

Best Part Collection: BBa_K1529302, BBa_K1529797, BBa_K1529263

Plux_CmR_LasI (BBa_K1529263)

We constructed this part by combining BBa_K395162BBa_B0034 and BBa_C0078.
(CmR is Chloramphenicol resistance.)

To characterize Plux-CmR-LasI (BBa_K1529263), we introduced Plux-CmR-LasI on pSB3K3 with Ptet-RhlR-Ptet-GFP on pSB6A1 to E.coli as “C4HSL dependent CmR and 3OC12HSL producer cell”. In this E.coli, constitutively expressed RhlR activates the expression of CmR and LasI in the presence of C4HSL.

 

Fig. 5-1-4. 
NameTypeDescriptionDesignerLength
   BBa_K1529000ReporterYdeDRiku Shinohara4
  BBa_K1529001Compositerbs_ydeDRiku Shinohara24
  BBa_K1529002CompositePtet_rbs_ydedRiku Shinohara86
  BBa_K1529003CompositePlux_rbs_ydeDRiku Shinohara87
   BBa_K1529010ReporteryfiKRiku Shinohara10
  BBa_K1529011Compositerbs_yfiKRiku Shinohara30
  BBa_K1529012CompositePtet_rbs_yfiKRiku Shinohara92
  BBa_K1529013CompositePlux_rbs_yfiKRiku Shinohara93
  BBa_K1529020CompositePtet_rbs_ydeD_rbs_tolCRiku Shinohara1633
  BBa_K1529021CompositePlux_rbs_ydeD_rbs_tolCRiku Shinohara1634
  BBa_K1529030CompositePlux-M256I CysENaoto Takahashi902
  BBa_K1529080CompositearaC_Pbad_M256ICysE_Ptet_luxR_TTKohdai Hibi3056
   BBa_K1529100Compositerbs_ppkRiku Shinohara2070
   BBa_K1529101CompositePtet_rbs_ppk1_terRiku Shinohara162
   BBa_K1529102CompositePtet_rbs_ppk1_terRiku Shinohara157
  BBa_K1529103CompositePlac_rbs_ppx_rbs_ppk1Shoko Suzuki2355
  BBa_K1529104CompositePcIλ_rbs_ackA_rbs_ppk1Shoko Suzuki3356
  BBa_K1529105CompositePt7_rbs_ppk1_rbs_ppxShoko Suzuki2167
   BBa_K1529106CompositePt7_rbs_ppk1_rbs_AckAShoko Suzuki3319
   BBa_K1529110CodingppxRiku Shinohara1542
  BBa_K1529111Compositerbs_ppxRiku Shinohara69
  BBa_K1529112CompositePtet_rbs_ppx_terRiku Shinohara131
  BBa_K1529113CompositePc1λ_rbs_ppx_terRiku Shinohara277
  BBa_K1529120CompositephoURiku Shinohara726
  BBa_K1529121Compositerbs_phoURiku Shinohara24
  BBa_K1529122CompositePtet_rbs_phoURiku Shinohara86
  BBa_K1529123CompositePlac_rbs_phoURiku Shinohara232
  BBa_K1529130Compositerbs_ackARiku Shinohara1221
  BBa_K1529131CompositePc1λ_rbs_ackARiku Shinohara1278
  BBa_K1529150CompositePT7_obcAShoko Suzuki1827
  BBa_K1529151CompositePT7_obcBShoko Suzuki763
   BBa_K1529199Composite -- No description -- Riku Shinohara 
  BBa_K1529200CompositePtet_tolCKohdai Hibi1601
  BBa_K1529201Compositerbs_ydeD_rbs_tolCKohdai Hibi1571
  BBa_K1529202CompositePlux_tolCKohdai Hibi1602
  BBa_K1529230CompositePtet_M256ICysEKohdai Hibi901
  BBa_K1529235CompositePlux_M256ICysE_Ptet_luxR_TTKohdai Hibi1908
  BBa_K1529240Compositerbs_aiiAKohdai Hibi832
  BBa_K1529241CompositePlac_cIλ_aiiAKohdai Hibi1841
  BBa_K1529242CompositePcIλ_lacI_luxIKohdai Hibi1897
  BBa_K1529243CompositePtet_luxIKohdai Hibi723
  BBa_K1529250CompositePlac_rbsKohdai Hibi220
  BBa_K1529251Compositerbs_RhlIKohdai Hibi685
  BBa_K1529252CompositePtet_RhlIKohdai Hibi747
  BBa_K1529263CompositePlux_CmR_LasIKohdai Hibi1435
  BBa_K1529264CompositePlux_CmR_luxIKohdai Hibi1411
  BBa_K1529265CompositePlux_CmR_RhlIKohdai Hibi1435
  BBa_K1529267CompositePtet_CmRKohdai Hibi741
  BBa_K1529268CompositePtet_CmR_luxIKohdai Hibi1410
  BBa_K1529270CompositePtet_RhlRKohdai Hibi867
  BBa_K1529271CompositePtet_RhlR_luxIKohdai Hibi1536
  BBa_K1529290CompositePlux/lac_luxIKohdai Hibi743
  BBa_K1529291CompositePlux/lac_aiiAKohdai Hibi914
   BBa_K1529292CompositePlux_luxIRiku Shinohara724
  BBa_K1529293CompositePlux_aiiAKohdai Hibi895
  BBa_K1529300RegulatoryPrhl(RL)Kohdai Hibi55
  BBa_K1529301CompositePrhl(RL)_GFPKohdai Hibi990
  BBa_K1529302CompositePrhl(RL)_CmR_LasIKohdai Hibi1435
  BBa_K1529310RegulatoryPrhl(LR)Kohdai Hibi55
  BBa_K1529311CompositePrhl(LR)_GFPKohdai Hibi990
   BBa_K1529320RegulatoryPrhl(RR)Kohdai Hibi55
   BBa_K1529321CompositePrhl(RR)_GFPKohdai Hibi990
  BBa_K1529430CompositePcIλ_lacIShoko Suzuki1141
  BBa_K1529431CompositePlac_cIλShoko Suzuki981
  BBa_K1529432CompositePlux_cIλShoko Suzuki836
  BBa_K1529567CodingpartsShoko Suzuki51
  BBa_K1529568CompositesssShoko Suzuki967
  BBa_K1529777CompositePlux_CmR_luxIRiku Shinohara1411
  BBa_K1529787ReporterabeGyomon Ryu 
  BBa_K1529789CodingabeGyomon Ryu 
  BBa_K1529797CompositePlux_CmR_RhlIShoko Suzuki1435
   BBa_K1529923TerminatorlksjKohdai Hibi18
  BBa_K1529948RBSabeGyomon Ryu24
  BBa_K1529950Terminator1Xinan Wang 
  BBa_K1529951Composite1Daisuke Kiga967
  BBa_K1529952Composite1Xinan Wang967
  BBa_K1529953GeneratorK1139200 B0030 E0040 B0010 B0012Gyomon Ryu967
  BBa_K1529955Composite1Ayaka Murase967
  BBa_K1529958Composite1Naoto Takahashi967
   BBa_K1529959Regulatory1Xinan Wang 
  BBa_K1529972Compositeoxalic acidMatsumura Keina967
  BBa_K1529973CodingzenMatsumura Keina 
   BBa_K1529978RegulatoryabeDaisuke Kiga55
   BBa_K1529987Regulatory1Naoto Takahashi92
  BBa_K1529996Composite1Kino Yuta132
  BBa_K1529997RBSaaKino Yuta12
  BBa_K1529998Terminator1Ayaka Murase20
 

<groupparts>iGEM014 Tokyo_Tech</groupparts>