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

Circuit Regulation Video

Circuit Regulation Testing

1.For pTet (R0040)：

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

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

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

2. For pLux/cI hybrid (R0065)：

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

(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 our killing material concentration estimation:

(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.