Team:Tokyo Tech/Parts

From 2014.igem.org

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                   <td><h2>Best New Basic Part<span class="head">:<a href="http://parts.igem.org/Part:BBa_K1529301"> BBa_K1529301</a>, <a href="http://parts.igem.org/Part:BBa_K1529311">BBa_K1529311</a>, <a href="http://parts.igem.org/Part:BBa_K1529321">BBa_K1529321</a></span></h2></td>
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                   <td><h2>1. Best New Basic Part<span class="head">:<a href="http://parts.igem.org/Part:BBa_K1529301"> BBa_K1529301</a>, <a href="http://parts.igem.org/Part:BBa_K1529311">BBa_K1529311</a>, <a href="http://parts.igem.org/Part:BBa_K1529321">BBa_K1529321</a></span></h2></td>
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                   <td><h2>Best New Composite Part<span class="head">: <a href="http://parts.igem.org/Part:BBa_K1529302">BBa_K1529302</a>, <a href="http://parts.igem.org/Part:BBa_K1529797">BBa_K1529797</a></span></h2></td>
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                   <td><h2>2. Best New Composite Part<span class="head">: <a href="http://parts.igem.org/Part:BBa_K1529302">BBa_K1529302</a>, <a href="http://parts.igem.org/Part:BBa_K1529797">BBa_K1529797</a></span></h2></td>
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                   <td><h2>Best  Part Collection: <span class="head"><a href="http://parts.igem.org/Part:BBa_K1529302">BBa_K1529302</a>, <a href="http://parts.igem.org/Part:BBa_K1529797">BBa_K1529797</a><a href="http://parts.igem.org/Part:BBa_K1139201" rel="nofollow"></a></span> </h2></td>
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                   <td><h2>3. Best  Part Collection: <span class="head"><a href="http://parts.igem.org/Part:BBa_K1529302">BBa_K1529302</a>, <a href="http://parts.igem.org/Part:BBa_K1529797">BBa_K1529797</a><a href="http://parts.igem.org/Part:BBa_K1139201" rel="nofollow"></a></span> </h2></td>
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Revision as of 14:13, 17 October 2014

Tokyo_Tech

Parts

Each part give us a whole new experience

 

Favorite Tokyo Tech 2014 iGEM Team Parts

 
Name
Type
Description
Design
Length (bp)
  BBa_K1529301 Composite Prhl(RL)-GFP Kohdai Hibi 990
  BBa_K1529302 Composite Prhl(RL)-CmR-LasI Kohdai Hibi 1435
  BBa_K1529797 Composite Plux-CmR-RhlI Shoko Suzuki 1435

 

Tokyo Tech 2014 iGEM Team Parts

 
Name
Type
Description
Design
Length (bp)
  BBa_K1529311 Composite Prhl(LR)-GFP Kohdai Hibi 990
  BBa_K1529321 Composite Prhl(RR)-GFP Kohdai Hibi 990

 

1. Best New Basic Part: BBa_K1529301, BBa_K1529311, BBa_K1529321

• Prhl(RL)-GFP (BBa_K1529301)

Prhl(RL) is a promoter that is activated by C4HSL-RhlR complex. 
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. 5-1-1-1.)

Fig. 5-1-1-1. Fluorescence intensity detected by flow cytometer
 

2. Best New Composite Part: BBa_K1529302, BBa_K1529797

• 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.

To confirm CmR production, we added chloramphenicol into the medium containing Prhl(RL)-CmR-LasI cell and measured optical density for about 8 h to estimate the concentration of the cell. (Fig. 5-1-2-1.)

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. 5-1-2-1. C4HSL-Dependent CmR Expression Assay
 

Prhl(RL)-CmR-LasI (BBa_K1529797)

We constructed this part by combining BBa_K395162, BBa_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.

To confirm C4HSL production, we measured the expression of GFP in reporter cells by flow cytometer. (Fig. 5-1-2-2.)
Fig. 5-1-2-2. 3OC12HSL-Dependent C4HSL Production Assay
 

3. Best Part Collection: BBa_K1529302, BBa_K1529797

 
 
 
 
 

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