Our first challenge in making Ampersand was to develop a system that limits basal expression of our peptide, due to its toxicity to the host. Several expression systems have been designed in the past, including the T7 promoter and T7 RNA Polymerase system, to suppress protein expression. The current BL21 (DE3) strain is leaky due to the weak suppressing promoter lacUV5 that drives T7 RNA polymerase in the DE3 strains. As a result, low levels of toxic protein are constitutively expressed, ultimately killing the host it was made in and in turn lowering the overall yield of the protein produced.

In order to reduce the expression of T7 RNA polymerase and create an efficient system for the expression of recombinant proteins in E. coli we chose to introduce two levels of regulation:

• Transcriptional Regulation: We will use pZE21 of the pZ system of vectors developed by Lutz and Buschard with the P_LlacO promoter to inhibit the expression of T7 RNA polymerase.
• Translational Regulation: We will use the artificial riboregulatory elements devised by Isaacs et al. to restrict translation of the mRNA sequence encoding T7 RNA Polymerase.⁹ The cis-repressing RNA (crRNA) sequence will be inserted downstream of the promoter driving T7 RNA Polymerase and upstream of the ribosomal binding site (RBS). The crRNA is complimentary to the RBS and forms a stem loop at the 5’ end of the mRNA segment, blocking ribosomal docking and translation. A second promoter, P_LtetO will express the trans-activating RNA (taRNA) capable of undergoing a linear-loop interaction that will expose the RBS and allow for translation of T7 RNA Polymerase. The ribo-regulated T7 RNA Polymerase will be ultimately incorporated into the genome of strain capable of high protein production. A second pZ plasmid will contain the gene of interest expressed by a T7 promoter.

In summary, the benefits to this type of system are its robustness, portability, and efficiency in that it does not require the cell to expend more energy on the constitutive synthesis of another protein. We theorize that by utilizing these two levels of control we will be able to reduce the expression of T7 RNA polymerase and produce a system with zero basal expression of the gene of interest and permit stable cloning of toxic recombinant proteins.

The ability for mussels to survive the harsh conditions of the intertidal zone is directly related to their capability to bind to both organic and inorganic surfaces that they come in contact with. As a result, these mussels have evolved to secrete “adhesion proteins” that enable formation of powerful bonds in an otherwise deleterious aqueous environment. The quantity known as “work of adhesion (W_A)” amongst interfaces in an aqueous environment is significantly less favorable due to the water-surface interactions that must be outcompeted by the adhesive if binding is to occur.¹⁰ Therefore, these mussel adhesion proteins offer a promising avenue towards enhanced bioadhesives that retain functionality in aqueous environments.

The structure responsible for the binding of mussels to these surfaces is known as the byssus, which is composed of a well-characterized assembly of approximately 25-30 proteins.¹⁰ The byssus manifests itself as an outgrowth from the mussel’s “foot” composed of threads, which terminate in plaques deposited on the surface. Thus far, a total of 5 proteins, known as mussel foot proteins (mfp’s), were shown to be unique to the plaque. Of these 5 plaque proteins, mfp-3 and mfp-5 have elicited the most investigation for adhesive applications, as both are localized at the interface between the plaque and surface. Additionally, it was shown that all such mfp’s have a uniquely high concentration of tyrosine, which is post-translationally modified to 3,4-dihydroxy-L-phenylalanine (L-Dopa). Atomic force microscopy studies focusing on a single dopa residue showed that its interaction with a metal oxide surface involves a remarkably high strength, reversible coordination bond.¹¹ It has been shown that catechol oxidase enzymes present in mfp secretions are able to convert the catechol groups of L-dopa into reactive orthoquinone functionalities, which undergo irreversible covalent bonding with organic surfaces.^11,12 Ultimately, the high degree of surface affinity such dopa-containing peptides possess makes them novel tools in the development of biomimetic adhesive peptides.