Team:NYMU-Taipei/Project/Inhibition/Inhibition circuit

Circuit

• [http://parts.igem.org/Part:BBa_K1104901 BBa_K1104901]: AhpCp2D1+ RBS+ LuxI+ RBS+ LuxR+ RBS+ TetR+ Term
• [http://parts.igem.org/Part:BBa_K1104902 BBa_K1104902]: pTet + RBS+ cI + Term
• [http://parts.igem.org/Part:BBa_K1104903 BBa_K1104903]: pLux/cI + RBS + Defensin1 + RBS + Abaecin + RBS + LuxR + RBS + LuxI + Term

Circuit Regulation Video

　　In a healthy bee, since there is no N. ceranae, no ROS will be produced. Consequently the sensor will be off and the Tet protein will not be produced. The Tet regulated promoter will be on with no Tet protein expressed, so the cI protein will be expressed and binds to the pLux/cI hybrid promoter, casing the hybrid promoter to turn off. When the hybrid promoter is off, the killing protein will not be produced.
　　While in a sick bee, N. ceranae invaded and ROS will be produced. The sensor will be on due to the ROS produced causing the Tet protein be produced and repress the Tet regulated promoter. When the promoter is off, cI protein will not be produced. And the cI protein that remain on the hybrid promoter will eventually be degraded. Also we can notice that at the downstream of the sensor, there is LacI and LacR gene. LacI protein is an enzyme that can produce AHL. AHL will bind with LacR to form a combination. This combination can bind to the hybrid promoter and activate it. With no repressor and the appearance of activator, the hybrid promoter will be on and our killing protein will be produced. Also, we have designed a positive feedback in order to enhance the production of the killing protein.
　　Ａfter N. ceranae is eliminated, since there is no N.ceranae, no ROS will be produced. The sensor will be off, so the Tet protein will not be produced and those remain on the promoter will be degraded. As a result, Tet regulated promoter will be on and cI protein will again be produced to repress the hybrid promoter. Therefore, our killing protein will not be produced. This is the initial status of our circuit, which means our circuit can again be turn on at the next time N. ceranae invaded and kill it through the process that I have just mentioned.

Circuit Regulation Testing

1.For testing pTet promoter (R0040)：

(1)pTet(R0040)+ E0840....(control)

(2)J23102+ RBS+ tetR+ Term+ pTet+ E0840

(3)J23102+ RBS+ lacI+ Term+ pTet+ E0840

2. For testing pLux/cI hybrid promoter (R0065)：

(1)pLux/cI(R0065)+ E0840....(control)

(2)J23102+ RBS+ cI+ Term+ R0065+ E0840

(3)J23102+ RBS+ luxR+ RBS+ luxI+ Term+ R0065+ E0840

(4)J23102+ RBS+ luxR+ RBS+ luxI+ RBS+ cI+ Term+ R0065+ E0840

3. For testing our circuit regulation:

(1)pLux/cI+ RBS+ E0040+ RBS+ luxI+ RBS+ luxR+ Term

Our expectation for Testing

1.

(1)can see GFP under fluroscence

(2)can not see GFP under fluroscence

(3)can see GFP under fluroscence again

Conclusion： tetR coding sequence has the ability to inhibit pTet.</br>

2.

(1)can not see GFP under fluroscence

(2)can not see GFP under fluroscence

(3)can see GFP under fluroscence

(4)can not see GFP under fluroscence

Conclusion： cI coding sequence is more dominant than LuxR/AHL, and cI coding sequence can well inhibit pLux/cI as well as LuxR& LuxI hibrid can initiate pLux/cI only when cI is absent.

Backbone selection

If we put this three circuit all in the high copy backbone, it probably does not work after the prediction of the modeling. Therefore, we decide to put sensing part on high copy backbone, regulation part on medium copy backbone, as well as killing part on low level copy backbone. In addition, since we want to be sure that these three different but related circuit all consumed by one E-coli, we have to choose three types antibiotics. We finally to select Amp, Cm and Kan for our backbone.