Biofilm formation on surfaces is an issue in the medical field, naval industry, and other areas.
We developed an anti-fouling peptide with two modular components: a mussel adhesion protein (MAP) anchor and LL-37, an antimicrobial peptide. MAPs can selectively attach to metal and organic surfaces via L-dopamine (L-DOPA), a nonstandard amino acid that was incorporated 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, the antimicrobial surface-binding peptide was assayed for functionality.

Project Goals

1. Create a T7 Riboregulation System to control the expression of our proteins:
We are dealing with antimicrobial 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).

Introduction

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).

T7 Riboregulation System: Experimental Design

Strains, Plasmids, and Reagents
E. coli strains used in this study included BL21(E. coli B F- dcm ompT hsdS(rB- mB-) gal [malB+]K-12(λS)), BL21(DE3)( F– ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])), ECNR2(ΔmutS:cat.Δ(ybhB-bioAB): [λcI857.Δ(cro-ea59):tetR-bla]), Mach1(ΔrecA1398 endA1 tonA Φ80ΔlacM15 ΔlacX74 hsdR(rK- mK+)), and 730. Strains used for transformation were grown in LB min (Cold Spring Harbor Protocols 2006). Cells used for cloning and mini-prep were grown in selective medium of 2XYT (2xYt Medium (7281) 2010) with either kanamycin (American Bioanalytical) or spectinomycin (Sigma-Aldrich). Kanamycin and streptomycin were used at 30 mg/mL and 95 mg/mL respectively.
One percent agarose gels were made with 0.5% TBE obtained from American Bio and stained with either ethidium bromide (Sigma-Aldrich) in the case of screening or SYBR Safe (Invitrogen) in the case of cloning. Gel extraction and purification was completed with QIAprep Gel Extraction Kit following the protocol provided. PCR purification was accomplished with the QIAquick PCR Purification Kit, following the protocol provided. Plasmid purification was accomplished using the QIAprep Spin Miniprep Kit and the protocol provided. For all DNA kits provided by QIAgen we used Denville Spin Columns for Nucleic Acid Purification. The concentration of DNA was measured using a Biotek Synergy HT Multi-Mode microplate Reader with accompanying Take3 Microvolume plates. All restriction enzymes, and Gibson Assembly Master Mix are from New England Biolabs. Hifi HotStart Readymix and 2GFAST Readymix with loading dye for PCR were obtained from KAPA Biosystems.

Two Levels of Regulation for T7 Polymerase Expression
Our goal is to reduce the expression of T7 RNA polymerase and create an efficient system for the expression of heterologous proteins in E. coli. In order to carry this out, we chose to introduce two levels of regulation. The first level of regulation will be at the transcriptional level. We will use pZE21 of the pZ system of vectors developed by Lutz and Buschard with the PLlacO promoter to inhibit the expression of T7 RNA polymerase. The PLlacO promoter controls the expression of the crRNA (cis repressing RNA) and is induced by IPTG (isopropyl-beta-D-thiogalactopyranoside). The second level of regulation will occur at the translational level. We will use the artificial riboregulatory elements (Figure 2) devised by Isaacs et al. to restrict translation of the mRNA sequence encoding the T7 RNA Polymerase (Isaacs et al., 2004). 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, PLtetO, which is induced by ATC, 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. Once the T7 RNA Polymerase is expressed, it can then bind to the T7 Promoter and lead to the expression of the gene of interest, such as the antimicrobial peptide (Figure 3).
The ribo-regulated T7 RNA Polymerase (formally known as α12c) and the TolC selection marker will be ultimately incorporated into a conjugative plasmid and into the genome of E.coli to control for copy number (Figure 2a and 2b). The reason why it is important to control for copy number is that the copy number of the pZE21 backbone is fairly large. This means that it will be more challenging for the cell to regulate protein expression, so a low copy number would enable better cell regulation of proteins. A second pZ plasmid will contain the gene of interest expressed by a T7 promoter. Finally, the third plasmid will contain the orthogonal translation system (Figure 3a and 3b). The benefit of this type of system is that it is robust and can be easily re-engineered, portable in the form of plasmids, compatible across multiple E.coli strains, and efficient in that it does not require the cell to expend more energy on the constitutive synthesis of another protein. We hypothesize 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.

Anti-Fouling Peptide Construct: Experimental Design

We hypothesize that we can develop an improved version of the current adhesives by developing a fusion protein of Mgfp-5 with Mefp-1 as the anchoring region for the anti-biofouling peptide. An integral part of developing this peptide is to co-translationally insert L-DOPA into our peptide, which has never been done before with mussel foot proteins (Figure 5). In this process of orthogonal translation, we first will get rid of the UAG stop codon and then transform the strain to synthesize tRNA and tRNA transferase that corresponds to the UAG codon and the L-DOPA non-standard amino acid to develop the GRO. The advantage of this procedure is that we have the ability to skip the time-consuming and inefficient tyrosinase enzyme treatment step.

Protein Purification We plan to purify the protein by using the Twin Strep Tag in tandem with the Flag tag, which was included in out master construct of the anti-biofouling peptide (Figure 6). The Flag tag is perfectly cleavable by the enzyme enterokinase. The FLAG tag is made up of 8 amino acids and works well for low-abundance proteins. It is hydrophilic, so it will most likely not interfere with protein folding and function of the target protein. The Strep tag is also made up of 8 amino acids that will not disturb the protein’s functions. We chose the FLAG tag because it is perfectly cleavable. Info on LL-37 and N-terminus? The protein will be purified in a Strep-Tactin® Sepharose® column. In order to address the L-DOPA adhesive L-DOPA component, our final step is to elute with a base to reduce the amount of the anti-biofouling peptide that sticks to the column due to L-DOPA adhesion (Figure 7).
Flag Tag Sequence: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
Strep Tag Sequence: Sequence: Trp-Ser-His-Pro-Gln-Phe-Glu-Lys

Characterization of Coating Adhesion Properties

We hypothesize that we can develop an improved version of the current adhesives by developing a fusion protein of Mgfp-5 with Mefp-1 as the anchoring region for the anti-biofouling peptide. An integral part of developing this peptide is to co-translationally insert L-DOPA into our peptide, which has never been done before with mussel foot proteins (Figure 5). In this process of orthogonal translation, we first will get rid of the UAG stop codon and then transform the strain to synthesize tRNA and tRNA transferase that corresponds to the UAG codon and the L-DOPA non-standard amino acid to develop the GRO. The advantage of this procedure is that we have the ability to skip the time-consuming and inefficient tyrosinase enzyme treatment step.

Protein Purification We plan to purify the protein by using the Twin Strep Tag in tandem with the Flag tag, which was included in out master construct of the anti-biofouling peptide (Figure 6). The Flag tag is perfectly cleavable by the enzyme enterokinase. The FLAG tag is made up of 8 amino acids and works well for low-abundance proteins. It is hydrophilic, so it will most likely not interfere with protein folding and function of the target protein. The Strep tag is also made up of 8 amino acids that will not disturb the protein’s functions. We chose the FLAG tag because it is perfectly cleavable. Info on LL-37 and N-terminus? The protein will be purified in a Strep-Tactin® Sepharose® column. In order to address the L-DOPA adhesive L-DOPA component, our final step is to elute with a base to reduce the amount of the anti-biofouling peptide that sticks to the column due to L-DOPA adhesion (Figure 7).
Flag Tag Sequence: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
Strep Tag Sequence: Sequence: Trp-Ser-His-Pro-Gln-Phe-Glu-Lys