Flowchart of the iterative design process.

Design testing and refinement are integrated in a feedback process that used biophysical insights to reduce leakage or promote activation, as appropriate. In the feedback cycle where leakage is unacceptable, additional stabilization of the structure is required to prevent spontaneous downstream activation by uncleaved SCS molecules. There are a number of possible remedies, and it may take several experiments to identify which step is causing the problem.