Overview

• Target Construct

1. Attempt to add additional proteins into the vector and test for functionality
2. Vary linker lengths
3. Make a simpler version for trouble shooting the secretion system: [6x-His]-[NheI]-[prtB]
4. Transfer to a vector with the same origin and resistance as described in the original secretion system (pBR322+Carb).
5. Add a lacI into the expression and target vectors so repression is hard-coded into the vector and expression can be induced regardless of the cell line.

• Secretion System

1. Transfer to a Chlor resistance, p15A origin vector as used in the original papers
2. Add original upstream DNA (50bp) before the native RBS to ensure proper function
3. Add an arabanose inducible promoter for better control over secretion system activation
4. Combine with target vector so entire secretion system is contained in one plasmid.

• Display System

1. Construct a new modular display system: CDS
2. Use computational protein design to create a monomeric protein that binds tightly to biotin: FoldIt