Team:CIDEB-UANL Mexico/WetLab-Planning

Construction plan

The protein Vip3ca3 is not in the parts registry catalog so we decided to synthesize that protein in order to work with it in the lab.

The circuit is divided in two parts. The work in the lab has 3 main objectives: 1. To test the Vip3ca3 protein production with insects. 2. To test the Vip3ca3 protein production with a reporter. 3. To test the regulation of the Vipca3 production with the repressor.

In order to achieve those objectives, we made a construction plan: The parts that were synthesized are named as “PUC-57” and “PCC1”.

For the first objective: The part “PCC1” contains the Vip3ca3 protein and it has a lambda cI promoter. The part “PCC1” is in a vector with ampicillin resistance so we have to cut the vector with restriction enzymes in the sites of EcoRI and PstI. As we are going to pass it to another vector it needs to have a different resistance so we choose kanamycine. The new vector is also cut in the same sites: EcoRI and PstI. Then we make a digestion with the plasmid and the insert.

For the second objective: We are going to bind the “PCC1” part with a reporter. This reporter is the GFP and it has degradation tag LVA. The “PCC1” and the GFP have ampicillin resistance so we choose a vector with kanamycine resistance. We cut the vector with EcoRI and PstI restriction enzymes. The insert that goes to the right, in this case the “PCC1”, is cut with EcoRI and SpeI and the other insert, which goes to the left, is cut with XbaI and PstI. Then the 2 inserts and the vector are joined with the ligase.
The same process goes for the third objective but the insert 1 is “PUC-57” and the insert 2 is the repressor lambda cI. Both parts have ampicillin resistance so the new vector must have a resistance different from ampicillin and kanamycine because it is already used. That’s why we choose a vector with chloramphenicol resistance.
Finally, the two plasmids will be inserted in the bacteria as the final product.