Throughout our project, we have successfully introduced a new cloning method, developed and validated several protocols, and collected data that matches our mathematical models.

3G Assembly has proven to be an accurate and efficient way to create circuits, not only in our lab, but in our collaborators’ as well. All twenty sequence confirmed circuits we are submitting to the registry were created using 3G assembly, demonstrating that it is a reliable method of DNA assembly.

Along with our 3G Protocol and description of Mixed 3G Assembly, we created several other procedures to improve experiments in our lab. First, we developed a protocol that allows for the freezing of cells before flow cytometry measurements are performed. This decreases time and labor costs and allows teams without flow cytometers to send their samples to other institutions for measurement. In addition, we designed and validated a method to dynamically remove inducer from media. Using this method, we biologically confirmed our mathematical model predicting that an incoherent feedforward loop will produce a stepwise output from a pulsatile input of inducer.