Team:Hamburg/Demonstrate
We successfully implemented a new approach based on RNA regulation to transform E.Coli DH5α cells with multiple plasmids using only one antibiotic resistance. We tested this approach as an alternative to standard transformation procedures for which different antibiotics are needed. We could show that the transformation of E.Coli DH5α cells with our trigger and gate plasmids leads to the expression of CAT, because bacterial growth after triple transformation with all three different plasmid was visible. Therefore, we concluded that our RNA based logic gate worked. In contrast no bacterial growth was observed after single transformation of the trigger plasmids. Introduction
We used RNA toehold switches, which sequester the ribosome binding site of our downstream target gene and prevent translation. We used two triggers, which formed a trigger complex and bind to the gate sequence to linearize the toehold switch and start the translation.
For the setup of our experiments please visit our Design page. Further information on the parts we used can be obtained from our Part page. To have a look on our results see our Results.Translation of chloramphenicol acetyltransferase (CAT) by an AND-gate logic with two triggers
We observed bacterial growth after single transformation with the gate plasmid and investigated the leakage of the gate. Therefore, the antibiotic resistance is not only expressed when both triggers are present in the cell, but also when only the gate plasmid is transformed. We could show that at high chloramphenicol concentrations a triple transformation leads to more viable bacteria compared to single transformations with only the gate plasmid. We therefore conclude, that our RNA logic is working as expected at higher chloramphenicol concentrations.
With the previous results we could show that our RNA logic is working and that the triggers can open the gate as expected.