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.
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.
     

The Solution: To address this issue, we aimed to develop an anti-microbial peptide composed of two components, which we envision can be modulated to suit a variety of functional adhesive applications. The ideal anti-microbial peptide coating would exhibit both biofilm-inhibitory activity as well as strong adhesion to a variety of substrates. In addition, we sought to identify a biomimetic adhesive domain so as to enhance the environmental friendliness of our peptide. Mussel adhesive proteins (MAPs), which are secreted by the mussel to help it anchor and survive in the harsh conditions of the intertidal zone, are an effective and environmentally sound adhesive domain. As our anti-microbial domain, we selected LL-37, a member of the cathelicidin family of peptides, due to the potency of its toroidal pore mechanism of lipid bilayer disruption. MAPs selectively attach to inorganic and organic surfaces via L-dopamine (L-DOPA), which is generated by post-translational modification of tyrosine with tyrosinase (often secreted along with the MAPs by the mussels in an enzyme cocktail that assists with the crosslinking of MAPs). Our study sought to be the first to synthesize an entire recombinant anti-microbial adhesive peptide without the need for post-translational modifications. We incorporated L-DOPA, a nonstandard amino acid, into our construct using a genetically recoded organism (GRO). Because this peptide is toxic to the GRO in which it is produced, we designed a better controlled inducible system that limits basal expression. This was achieved through a novel T7 riboregulation system that controls expression at both the transcriptional and translational levels. This improved system is a precise synthetic switch for the expression of cytotoxic substances in the already robust T7 system. Lastly, a variety of tests were carried out to characterize our recombinant protein in comparison to the commercially available MAP-based adhesive, Cell-Tak^TM.

1. Create a T7 Riboregulation System to control the expression of our proteins:
We are dealing with anti-microbial peptides, so there is the possibility that the peptide we create would be toxic to E. coli which we are using to synthesize the peptide. We created a plasmid with specific locks in place so that we control when the T7 RNA polymerase, an RNA polymerase from the T7 bacteriophage, is expressed. Once the T7 RNA polymerase is expressed, it can then specifically target the T7 Promoter located in a different plasmid, which will lead to the expression of the specific peptide we want. (Show Figure 3)

2. Design the anti-biofouling peptide using both a modular approach:
In order to carry this out, we used the foot protein consensus sequence mefp 1-mgfp 5-mefp-1, which was found to be effective in Lee et al., 2008. At the N-terminus is the twin Strep-FLAG tag (using Strep tag for purification, and FLAG tag for easy cleavage). Then, the LL-37 antimicrobial peptide (AMPs are generally short enough to be inserted via primer overhang) is present on a long 36 residue linker. On the other side of the foot protein is sfGFP connected by a shorter linker. With targeted primers, the construct can be amplified in its entirety, or only with the AMP or GFP segment (Show Figure 6).

3. Develop an erosion rig to assess the strength of the adhesive peptide:
(Show figure 8) First, we will need to determine if we have adhered material present in various solutions and surfaces. In order to these this out, we will look at the contact angle measurement. Surfaces that are wet will have a very shallow contact angle because the surface absorbs the test liquid. Non-wetting surfaces will usually exhibit an obtuse contact angle because there is no absorption. This test will determine if our coating is present and does not dissolve when wet. As a further test to determine if the material is able to adhere to surfaces, we will use Fourier Transform Infrared Spectroscopy (FTIR). The adhesive should exhibit a different spectrum than uncured adhesive. This difference probably lies in the different vibrational bond energies caused by coordination or bonding to our surface. The next assessment will be to determine how much coating is retained under stress with atomic force microscopy (AFM). A probe will be applied to the sample to determine the force between the atoms of the sample and the atoms of the tip. Image contrast can then be generated by monitoring the forces of the interactions between the tip and the peptide’s surface.

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).