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, which we envision can be modulated to suit a variety of functional adhesive applications:

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. 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 configuration of the erosion rig developed to introduce coated surfaces to liquid erosion.

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.

         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.

Biofilm formation: A problem in clinics and cargo ships
Biofilms, heterologous three-dimensional arrays of bacteria, are responsible for a number of problems in industry and medicine (Figure 1). According to Shirtliff & Leid, 2009, 60% of infections associated with hospitals are due to biofilm formation. Furthermore, a 2011 study conducted by the Woods Hole Oceanic Institution states that biofilm formation causes increased frictional drag time in ships, directly costing the US Navy about $200 million per year and lowering the life spans of ships. Furthermore, biofilms are more resistant to antibiotics as well as to shearing force, making them difficult and often costly to remove.

An improved T7 Riboregulation System
We can create novel anti-biofouling peptides with non-standard amino acids through the process of orthogonal translation in genetically recombined organisms (GRO), such as E.coli. However, these peptides will be potentially toxic to the GRO that they are made in, so it is first necessary to develop a tightly controlled expression system. In this way, we are improving the expression system for all toxic proteins and, in the process, developing anti-biofouling peptides.
Thus, we first sought to develop a controlled T7 Expression System. 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.

A DOPA-containing peptide derived from mussel foot protein
In parallel, we wanted to design an anti-biofouling peptide (Figure 4). One of the components of this peptide is its ability to anchor to different regions. We thought the mussel foot proteins would be a great option for this function. These proteins are able to attach to surfaces using protein tethers that contain L-Dopamine (L-DOPA). The catechol side chain of L-DOPA allows for many types of chemical interactions, because it cross-links to surfaces. L-DOPA binds to metals reversibly in aqueous conditions through a noncovalent interaction (metal-oxygen coordination bond). If the pH is changed to basic, then the L-DOPA component of the peptide will come off, which could be useful in naval applications because the L-DOPA-containing peptide would bind to the metal coating of the ship, and when the L-DOPA coating is no longer necessary, a base could be introduced to wash off the adhesive. Nonmetals (organic surfaces) have irreversible binding because L-DOPA gets oxidized. Oxidation of the catechol side chain leads to quinones forming, which further cross-link to organic surfaces through aryl-aryl coupling or through a Michael-type addition reaction (the exact mechanism is still unknown).
We chose to focus on using a combination of two mussel adhesive proteins: Mytilus galloprovincialis Foot Protein type 5 (Mgfp-5) and Mytilus Edulis Foot Protein (Mefp-1). According to Lee et al, Mefp-1 has strong adhesive quality and, when combined with Mgfp-5, is less toxic to cells. Mgfp-5 has adhesive qualities comparable to Cell-Tak®, which is a current commercially available adhesive. Cell-Tak® is a fusion of Mefp-1 and Mefp-2. It is created by replacing a tyrosine for L-DOPA and then subsequently exposing the entire peptide to the enzyme tyrosinase to post-translationally convert it into L-DOPA. However, this method takes a long time and a large amount of protein is lost between steps. Another adhesive currently on the market is Poly-L-Lysine, which is a scaffold of lysines that are positively charged and are able to attach to the negatively charged cell surface. However, L-DOPA adhesion is far superior to that of lysine.

Anti-biofouling Peptide: LL-37
Our candidate anti-microbial peptide is LL-37 (Figure 4). This peptide prevents uncontrolled growth of microbes. It is amphipathic, contains an alpha helix, and is 37 residues long starting with two leucines. We improved on this Biobrick from BBa_K1162006, Utah State 2013.
Conventional cationic antimicrobials target bacteria. However, there are an increasing number of bacteria that are resistant to antibiotics. Thus, we instead want to focus on targeting the biofilm formation. Anti-biofilm peptides are very similar to the cationic antimicrobial peptides, containing both cationic and hydrophobic amino acids, but they differ in their structure-activity relationship and have less specificity than antibiotics. Of the antimicrobial peptides, LL-37 seems to the most promising.
LL-37 is comprised of anionic and zwitterionic bilayers, which are important anti-fouling traits. This is because the hydrophilicity caused by electrostatic interaction with water molecules makes the replacement of the water molecules bound to the surface enthalpically unfavorable for foulants. LL-37 acts on the surfaces of cells, forming a toroidal pore that pierces through the cells of biofilm-forming bacteria. Transcriptome and biochemical investigations have shown that LL-37 can act against the common biofilm strain P. aeruginosa and prevent uncontrolled growth of microbes (Nagant et al., 2012). Previous research has successfully conjugated LL-37 to a carbohydrate-binding module from Clostridium thermocellum, and has successfully shown LL-37 functionality in the conjugated state (Ramos et al., 2010).

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