A biofilm is a community of bacteria attached to a surface that exhibit high resistance to antibiotics and human immunity. Biofilm formation poses a serious threat to the medical and shipping industries in the following ways:

1. Medical Industry
   • Protein adsorption, cell-adhesion, and subsequent biofilm formation have been found to lead to failure of medical implants and infection in patients.¹
2. Shipping Industry
   • Government and industry spend upwards of $5.7 billion annually in the control of marine biofouling. High levels of biofouling result in increased drag and the subsequent loss of hydrodynamic performance.²
     

   
   
   Figure X. (A) Biofilm formation leads to failure of implanted cardiac devices and heart valve infection. (B) Biofilm formation on the hulls of ships leads to loss of optimal hydrodynamic performance and structural failures.

To address this issue, we aimed to develop an anti-microbial adhesive peptide composed of two components. We envision these domains can be modulated to suit a variety of functional adhesive applications:

1. Component 1: Adhesive Domain
   • Because biofilm formation affects both organic and inorganic substrates, the anti-biofilm coating should show strong adhesion to a variety of surfaces. Mussel adhesive proteins (MAPs), which are secreted by the mussel to help it anchor and survive in the harsh conditions of the intertidal zone, would be ideal for this application. MAP adhesion has been well-characterized and has been investigated in biomimetic adhesive applications in the past. We intend to broaden the scope of their application by looking at their inclusion in the first anti-biofilm adhesive recombinant protein.³
2. Component 2: Anti-Microbial Domain
   • As our anti-microbial domain, we selected LL-37, a member of the cathelicidin family of peptides, due to the potency of its lipid bilayer disruption by toroidal pore formation. Because this peptide is toxic to the E. coli in which we intend to produce it, we designed a controlled, inducible system that limits basal expression. A novel T7 riboregulation system that controls expression at both the transcriptional and translational levels was designed. This improved system is a precise synthetic switch for the expression of cytotoxic substances.^4,5
3. Environmental Concerns
   • Concerns of environmental toxicity often arise in materials being investigated for anti-fouling activity such as copper paints and Muntz metal. Therefore, we set out to develop an anti-fouling coating with strong adhesive activity to limit leachants into the environment. Additionally, the selection of a MAP, found in a biological organism, as our adhesive domain is crucial to maintaining the soundness of our product's eco-friendliness.

1. Control expression of anti-microbial peptides:
   • Since we intend to synthesize an anti-microbial peptide, it is possible that the peptide will be toxic to the E. coli used in our synthetic route. To improve our overall protein yield, we designed a plasmid with specific locks in place to control expression of the T7 RNA polymerase, an RNA polymerase from the T7 bacteriophage. When the T7 RNA polymerase is expressed, it can then specifically target the T7 Promoter located in a different plasmid upstream of our coding sequence, initiating protein translation. The specific mechanism of our T7 riboregulation system is outlined in a section below.^6,7
2. Modular construct design:
   • As our adhesive domain, we selected the mussel foot protein (mefp) consensus sequence mefp 1-mgfp 5-mefp-1, which was found to be effective in Lee et al., 2008. At the N-terminus, we included a the twin Strep-FLAG tag, used in the purification and isolation of our construct and that can be readily cleaved. The LL-37 antimicrobial peptide, which is short enough to be inserted via primer overhang, is linked via a 36 residue linker, which we believe is long enough not to engender any unforeseen structural interaction between our domains. On the other side of the foot protein, we included an sfGFP connected by a shorter linker, which will be used to assay presence and yield of construct. Using targeted primers, the construct can be amplified in its entirety, or only with the anti-microbial or GFP segment. Note that the entire construct was designed so that a variety of functional peptide domains can be substituted for LL-37 if desired. A diagram of our entire construct is presented below:
   
   
   Figure X. A diagram illustrating the components in our final construct. The black domain is our anti-microbial peptide, LL-37, while the blue domain represents the recombinant mussel foot protein adhesive component. All other components are labeled accordingly and restriction sites are highlighted to emphasize the modularity of each separate region.
   

3. Characterize peptide's adhesion and anti-microbial properties:
   • Adhesion:
     • Subject peptide coated surfaces to liquid erosion:
       • A number of ASTM assays used in industrial coating testing were investigated, but none offered the level of quantitation desired for our applications. Therefore, an original rig was designed and built to introduce liquid based erosion by laminar flow through a bath. This system directly mimics the drag that a coated surface might experience on a ship's hull. Precise specifications of the rig are provided in a separate section below.
       
       
       Figure X. A diagram illustrating the configuration of the erosion rig developed to introduce coated surfaces to liquid erosion.
       
       
     • Assay presence of peptide on eroded surfaces:
       • Quartz Crystal Microbalance (QCM): A QCM is capable of measuring mass per unit area on a very sensitive scale. The QCM used in these experiments recorded masses with ±1 ng/cm² uncertainty. The way this instrument works is by measuring change in the resonance frequency, which is converted into a mass estimate on the basis that resonance frequency will decrease with increasing mass. We intend to subject the quartz crystals to varying levels of erosion and determining coating retention from the QCM mass measurement. Alternatively, various flow cell and in-situ erosion techniques can be coupled to the QCM to show the real-time changes in resonance frequency due to loss of mass. Several labs and facilities assisted with planning and execution of this measurement, including Dr. Michael Rooks at the Yale Institute for Nanoscience and Quantum Engineering and Dr. Islam Mosa in the lab of Dr. James Rusling in the Department of Chemistry at the University of Connecticut.
       • Total Protein Staining and Fluorescence Imaging: Coomassie Blue was used as a total protein stain to determine presence of coating on eroded slides. Adsorbed protein content can theoretically be determined visually from density of stain. Since our construct was designed with an sfGFP domain, we intend to assay presence of our peptide with fluorescence.
       • Contact Angle Measurement: A contact angle measurement of protein coated silica substrates was conducted as an indicator for presence of peptide, protein hydrophilicity/hydrophobicity, and surface energy. Wetting surfaces show a shallow contact angle, while hydrophobic surfaces show a larger contact angle. A contact angle characterizes the wettability of a surface and Young's equation can be used to determine interfacial energies between the three phases in equilibrium, given below. Note that γ_XY corresponds to the interfacial energy between phase X and phase Y.
         0=γ_SG – γ_SL – γ_LSCos(θ_C)
         This measurement was conducted with the assistance of Dr. Raphael Sarfati, Dr. Katharine Jensen, and Dr. Rostislav Boltyanskiy in the lab of Dr. Eric Dufresne in the Yale Department of Mechanical Engineering.
       • Fourier Transform Infrared Spectroscopy (FTIR): As a further test to determine if material is adhered to surfaces, we will use Fourier Transform Infrared Spectroscopy (FTIR). The cured adhesive film should exhibit a different spectrum than the uncured adhesive. A notable difference would speak to a change in vibrational bond energies caused by coordination or bonding to our surface.
     • Assay peptide adhesion strength:
       • Atomic Force Microscopy (AFM): The standard for measurement of the force of adhesion of MAPs is AFM. This type of measurement is known as a "pull-off" force determination and involves depressing an AFM cantilever functionalized with a 20 µm bead until it comes in contact with a coated substrate surface. The instrument then determines the force required to remove the cantilever from the substrate.
         
         
         Figure X. This is an AFM cantilever with a 20 µm silica bead fixed to the tip. By functionalizing the tip, we can control the adhesion interface for which we test our MAP adhesives. In this case, we intend to use a silica bead to measure the adhesion of our coating to a silica interface. This measurement was conducted with the assistance of Dr. Michael Rooks at the Yale Institute for Nanoscience and Quantum Engineering.

         
         

       • Optical Tweezers: While AFM has been used in many MAP studies successfully to measure MAP adhesion force, it comes with some limitations. Inevitably, there is some significant contact area, which makes the adhesion measurement read the adhesive force of multiple proteins. However, the technology exists to measure adhesion on the individual protein level. Some studies have measured the adhesion force of L-DOPA on the single molecule level by chemically linking the L-DOPA residue to the AFM cantilever. However, no such study have looked at adhesion force on the single protein level. Using high intensity lasers, one can engender a repulsive force between two beads in relation to their refractive indices. We intend to link our MAP to a biotin functionalized bead and measure its adhesion to a silica bead substrate.
         
         Figure X. This diagram illustrates how a DNA handle can be linked to a protein of interest to bind the protein to an optical tweezer bead into which the high intensity laser can be fired to engender a pull force. We intend to conduct a similar protocol with our adhesive peptide. ⁸ This measurement was conducted with the assistance of Dr. Junyi Jiao in the lab of Dr. Yongli Zhang from the Yale Department of Cell Biology.

1. An Improved T7 System Expression System

   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.

   
   Figure X. This diagram illustrates the T7 riboregulation system developed to ensure low basal levels of protein expression.⁹

2. Why Mussel Adhesion 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.

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