Team:Yale/Project

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<h1 style="margin-top:22px; font-size:48px;">AMPersand: An Anti-Microbial Peptide Coating</h1> </div>
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<h1 style="margin-top:5px; font-size:50px;"><FONT COLOR="#00B585">Amp</FONT>ersand: an <FONT COLOR="#00B585">A</FONT>nti-<FONT COLOR="#00B585">M</FONT>icrobial <FONT COLOR="#00B585">P</FONT>eptide Coating</h1>
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<h1>The Problem</h1>
<h1>The Problem</h1>
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A <strong><i> biofilm</strong></i> 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:
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A <strong><i> biofilm</strong></i> is a community of bacteria attached to a surface that exhibits high resistance to antibiotics and human immunity. Biofilm formation poses a serious threat to the medical and shipping industries in the following ways:
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<ol type="I"><li><strong>Medical Industry</strong><ul style="list-style-type:square"> <li>Protein adsorption, cell-adhesion, and subsequent biofilm formation have been found to lead to failure of medical implants and infection in patients. </ul> <li> <strong> Shipping Industry </strong> <ul><li>  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.  <br /> </p> </ul>
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<ol type="I"><li><strong>Medical Industry</strong><ul style="list-style-type:square"> <li>Protein adsorption, cell-adhesion, and subsequent biofilm formation have been found to lead to failure of medical implants and infection in patients.<sup>1</sup> </ul> <li> <strong> Shipping Industry </strong> <ul><li>  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.<sup>2</sup> <br /> </p> </ul>
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<center><img src="https://static.igem.org/mediawiki/2014/2/2f/IGEM_Biofilm_Examples_Wiki.png"></center>
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<br><center><img src="https://static.igem.org/mediawiki/2014/2/2f/IGEM_Biofilm_Examples_Wiki.png"></center>
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                 <i><strong>Figure X.</strong> A diagram illustrating the configuration of the erosion rig developed to introduce coated surfaces to liquid erosion.</i>
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                 <i><strong>Figure 1.</strong> <strong>(A)</strong> Biofilm formation leads to failure of implanted cardiac devices and heart valve infection. <strong>(B)</strong> Biofilm formation on the hulls of ships leads to loss of optimal hydrodynamic performance and structural failures.</i>
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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:  
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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:  
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<ol type="I"><li><strong>Adhesive Domain</strong><ul style="list-style-type:square"> <li>
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<ol type="I"><li><strong>Component 1: Adhesive Domain</strong><ul style="list-style-type:square"> <li>
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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. </ul>
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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.<sup>3</sup> The functional residue in MAPs is L-3,4-dihydroxyphenylalanine (L-DOPA), which is generated by post-translational modification of tyrosine with tyrosinase. Since L-DOPA is a non-standard amino acid, it cannot be incorporated by standard translation systems. However, we intend to be the first group to use a genetically recoded organism (GRO) to incorporate L-DOPA in-situ, eliminating the need for any post-translational modification.</ul>
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<li><strong>Anti-Microbial Domain</strong> <ul><li>
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<li><strong>Component 2: Anti-Microbial Domain</strong> <ul><li>
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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. </ul>
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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 <i>E. coli</i> 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.<sup>4,5</sup> </ul>
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<li><strong>Environmental Concerns</strong> <ul><li> 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 an MAP, found in a biological organism, as our adhesive domain is crucial to maintaining the soundness of our product's eco-friendliness.  
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<li><strong>Addressing Environmental Concerns</strong> <ul><li> 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.  
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<td colspan = "12"><p><center><strong><h2 style = "border-bottom:none">Video Walkthrough of our Project</h2></strong>
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<ol type="I"><li><strong>Control expression of anti-microbial peptides:</strong><ul style="list-style-type:square"> <li>
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<ol type="I"><li><strong>Control Expression of Anti-Microbial Peptides Using an Improved T7 Riboregulation System:</strong><ul style="list-style-type:square"> <li>
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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. </ul>
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Since we intend to synthesize an anti-microbial peptide, it is possible that the peptide will be toxic to the <i>E. coli</i> 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.<sup>6,7</sup> </ul>
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<strong> Modular construct design: </strong> <ul><li>
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<strong>A Modular Anti-Microbial Construct based on Mussel Foot Protein:</strong> <ul><li>
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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: </ul>
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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 <i>et al.</i>, 2008.<sup>8</sup> At the N-terminus, we included a 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: </ul><br>
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                 <i><strong>Figure X.</strong> A diagram illustrating the configuration of the erosion rig developed to introduce coated surfaces to liquid erosion.</i>
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                 <i><strong>Figure 2.</strong> 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.</i>
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<strong>Characterize peptide's adhesion and anti-microbial properties:</strong>
<strong>Characterize peptide's adhesion and anti-microbial properties:</strong>
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<ul><li> We intend to perform a number of assays to test the erosion resistance of our adhesive coating using an original apparatus designed to introduce erosion by laminar flow through a liquid bath. The specific tests that we investigated for adhesion testing are detailed in the <a href = "https://2014.igem.org/Team:Yale/MaterialsMethods">Materials and Methods</a> section.
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<li> To assess the efficacy of our peptide in inhibiting biofilm formation, we intend to perform a minimum biofilm eradication concentration (MBEC) assay (Innovotech). Further information is provided in our <a href = "https://2014.igem.org/Team:Yale/MaterialsMethods">Materials and Methods</a>  section.
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<ul><li><strong><u>Adhesion:</u></strong> <ul><li><strong> Subject peptide coated surfaces to liquid erosion: </strong><ul><li>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.</ul><br>
 
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                <i><strong>Figure X.</strong> A diagram illustrating the configuration of the erosion rig developed to introduce coated surfaces to liquid erosion.</i>
 
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<li><strong>Assay presence of peptide on eroded surfaces:</strong><ul><li><u> Quartz Crystal Microbalance (QCM):</u> 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/cm<sup>2</sup> 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. <i>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. </i>
 
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<li><u>Total Protein Staining and Fluorescence Imaging:</u> 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.
 
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<li><u>Contact Angle Measurement:</u> 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<sub>XY</sub> corresponds to the interfacial energy between phase X and phase Y.
 
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<center>0=γ<sub>SG</sub> – γ<sub>SL</sub> – γ<sub>LS</sub>Cos(θ<sub>C</sub>)</center>
 
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<i>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. </i>
 
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<li><u>Fourier Transform Infrared Spectroscopy (FTIR):</u> 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. </ul>
 
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<li><strong>Assay peptide adhesion strength:</strong><ul><li><u> Atomic Force Microscopy (AFM):</u> 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. </p>
 
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                <i><strong>Figure X.</strong> 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.</i>
 
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<li><u>Optical Tweezers:</u> 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
 
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<h1>Introduction</h1>
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<h1><a href = "https://2014.igem.org/Team:Yale/MaterialsMethods">Materials and Methods</a> </h1>
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For a detailed description of our experimental design regarding the T7 expression system, anti-microbial peptide construct, and adhesion assays, see <a href = "https://2014.igem.org/Team:Yale/MaterialsMethods">materials and methods.</a>
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<h1>Materials and Methods</h1>
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<h1><a href = "https://2014.igem.org/Team:Yale/Results">Results</a> </h1>
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For a detailed description of our results, see <a href = "https://2014.igem.org/Team:Yale/Results">results.</a>
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<h1>Results</h1>
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<h1><a href = "https://2014.igem.org/Team:Yale/Project/modeling">Modeling</a> </h1>
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We modeled theoretical bacterial population survival rates based on various points of anti-microbial peptide induction. See <a href = "https://2014.igem.org/Team:Yale/Project/modeling">modeling.</a>
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<h1>References</h1>
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<h1><a href = "https://2014.igem.org/Team:Yale/Parts">Submitted Parts</a> </h1>
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Our collection of submitted biobricks consists of: </p><p>
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<li>Mussel foot protein (MFP) 1-5-1 sequence [combination of Mytilus galloprovincialis Foot Protein 5 (Mgfp-5) and Mytilus Edulis Foot Protein 1 (Mefp-1)].</li>
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<li>MFP with superfolder Green Fluorescence Protein (sfGFP).</li>
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<li>MFP with our anti-microbial peptide, LL-37.</li>
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<li>Entire construct of our anti-microbial adhesive peptide: 2XStrep_Flagtag--LL-37--Mussel Foot Protein--sfGFP.</li>
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<a href = "https://2014.igem.org/Team:Yale/Parts">See here for more information on our Biobricks!</a>
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<h1>Additional Background Information</h1>
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<ol type="1"><li><strong>Genomically Recoded <i>E. coli</i></strong><p>
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<strong>Biofilm formation: A problem in clinics and cargo ships</strong>
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Whole-genome recoding was reported for the first time in 2011 by a team including our advisor, Professor Farren Isaacs, when it described replacing all TAG codons in <i>E. coli</i> by TAA. <sup>9</sup>  Both TAG and TAA are stop codons (amber and ochre), but their transcripts are bound by different release factors (RF1 binds UAG and UAA, whereas RF2 binds UAA and UGA), not only making this recoding nonlethal but also allowing the deletion of RF1 (ΔprfA) from the recoded strain, given RF2’s response to both remaining stop codons. <p>
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This genome-wide recoding was enabled by lambda-Red recombineering<sup>10</sup> and multiplex automated genome engineering. <sup>11</sup>  lambda-Red recombineering uses the lambda prophage’s protein Beta to mediate the homologous recombination of an exogenous single-stranded oligonucleotide, delivered into the cell by electroporation, with the host genome, by annealing that oligonucleotide to single-stranded genomic DNA exposed on the lagging strand of the replication fork. <sup>12</sup>  A 30-bp region of homology at either end of a sequence is sufficient to drive recombination of the entire strand, including mismatched regions, and mismatches thus incorporated are passed to progeny at high efficiency (up to 30%) in strains lacking methyl-directed mismatch repair (ΔmutS).<sup>13</sup>  <p>
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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.
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Multiplex automated genome engineering (MAGE) using lambda-Red recombineering can then make many directed mutations across the host genome, as was necessary to replace all 314 TAG codons in the strain EcNR2 (<i>E. coli</i> MG1655 ΔmutS::cat Δ(ybhB-bioAB)::[λcI857 Δ(cro-ea59)::tetR-bla]).<sup>9</sup>  To allow recombineering, the lambda prophage was introduced into the host genome by P1 transduction. Because the probability of each target sequence being mutated in a given cycle is at most 30%, attempting to introduce all 314 mutations using a single pool of 314 mutagenic oligonucleotides would much sooner generate any of ~2314 other partially recoded strains before the fully recoded strain, and running enough MAGE cycles to obtain that strain would allow time for spontaneous point mutations to accumulate unchecked by the knocked-out mismatch repair system. To construct the strain we will use 32 separate strains were generated, each with a different recoded sector, and those sectors were assembled by hierarchical conjugation. The recoded strain was validated by sequencing, and its RF1 was deleted to free the TAG codon for reassignment to an orthogonal translation system.<sup>14</sup> <p><lu>
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<li><strong>Orthogonal Translation Systems</strong><p>
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An amino acid can be assigned to a particular codon by a two-part translation system: a tRNA with its anticodon loop complementary to the codon, and an aminoacyl synthetase (hereafter called a synthetase) able to charge this tRNA with the amino acid. This system is orthogonal to endogenous translation systems if and only if this synthetase aminoacylates only this tRNA and endogenous synthetases cannot aminoacylate this tRNA. Usually, an unnatural amino acid thus assigned should also be sufficiently dissimilar from naturally encoded amino acids that endogenous synthetases would not charge endogenous tRNAs with the unnatural amino acid, to avoid partially reassigning other codons and disrupting cell function.<sup>15</sup> <p>
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Many synthetases include a nonconserved loop specific to their associated tRNA’s anticodon, preventing them from binding a tRNA reassigned to CUA, but the archaeum Methanococcus jannaschii’s tyrosyl-tRNA synthetase (MjtRNAs) and its associated tRNA translation system still bind after the anticodon is mutated, and is an amber suppressor when transformed into <i>E. coli</i>.<sup>16</sup>  This MjtRNAs was still aminoacylated by endogenous synthetases, so its specificity for its synthetase was improved by random mutation at eleven positions not directly interacting with its synthetase, followed by negative selection against nonspecific acylation and positive selection for specific acylation. <sup>17</sup>
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<p>This orthogonal system has since been engineered by directed evolution of its synthetase to incorporate nonstandard amino acids, in place of tyrosine. <sup>18</sup> Diverse synthetases were generated by site-directed mutagenesis of five residues in the amino acid-binding pocket, and screened by positive selection, for incorporation of the amino acid into a protein conferring antibiotic resistance, and a negative screen, against mutants resistant to the antibiotic even in the absence of the nonstandard amino acid (by treating colonies on a replica plate lacking the amino acid and then observing which were killed). Selected mutants were then recombined and subjected to further mutagenesis. <p><lu>
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<li><strong>An Improved T7 Expression System</strong>
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<p> 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 <i>lacUV5</i> 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.</p>
 +
<p> In order to reduce the expression of T7 RNA polymerase and create an efficient system for the expression of recombinant proteins in <i>E. coli</i> we chose to introduce two levels of regulation:<ul style="list-style-type:square"> <li>
 +
<strong>Transcriptional Regulation:</strong> We will use pZE21 of the pZ system of vectors developed by Lutz and Buschard with the P<sub>LlacO</sub> promoter to inhibit the expression of T7 RNA polymerase.
 +
<li>
 +
<strong>Translational Regulation:</strong> We will use the artificial riboregulatory elements devised by Isaacs <i>et al.</i>. to restrict translation of the mRNA sequence encoding T7 RNA Polymerase.<sup>19</sup>  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<sub>LtetO</sub> 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. </ul>
 +
 
 +
<p>
 +
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.<br><br>
 +
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/T7system.png" width = "600" height ="auto"></center>
 +
                <center>
 +
                <i><strong>Figure 3.</strong> This diagram illustrates the T7 riboregulation system developed to ensure low basal levels of protein expression.<sup>19</sup> </i></center><p><p><br>
 +
<li><strong>Mussel Adhesion Proteins</strong>
 +
<p>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<sub>A</sub>)” 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.<sup>10</sup> Therefore, these mussel adhesion proteins offer a promising avenue towards enhanced bioadhesives that retain functionality in aqueous environments. </p>
 +
<p>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.<sup>20</sup> 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.<sup>21</sup> 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.<sup>21,22</sup> Ultimately, the high degree of surface affinity such dopa-containing peptides possess makes them novel tools in the development of biomimetic adhesive peptides.</p>
 +
 
 +
 
 +
 
 +
 
-
<p>
 
-
<strong>An improved T7 Riboregulation System</strong>
 
-
<br />
 
-
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.
 
-
<br/>
 
-
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. 
 
-
</p>
 
-
 
-
<p>
 
-
<strong>A DOPA-containing peptide derived from mussel foot protein</strong>
 
-
<br />
 
-
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).
 
-
<br />
 
-
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.
 
-
</p>
 
-
<p>
 
-
<strong>Anti-biofouling Peptide: LL-37</strong> <br />
 
-
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 <strong>BBa_K1162006</strong>, Utah State 2013.
 
-
<br />
 
-
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.
 
-
<br />
 
-
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).
 
-
</p>
 
</div>
</div>
</td>
</td>
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 +
 +
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<td colspan="12">
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<td colspan="12">
-
<h1 style="margin-top:25px; margin-bottom:45px; font-size:35px">T7 Riboregulation System: Experimental Design</h1>
+
<div class = "tinycall">
-
<p>
+
<h1>References</h1>
-
<strong>Strains, Plasmids, and Reagents</strong> <br />
+
</div>
-
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.  
+
<div class = "well">
-
<br />
+
 
-
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.
+
<ol type="1">
-
</p>
+
<li>Donlan, R. M. (2001). Biofilms and device-associated infections. Emerg Infect Dis, 7(2), 277-281.
-
<p>
+
<li>Hwang, D. S., Gim, Y., Yoo, H. J., & Cha, H. J. (2007). Practical recombinant hybrid mussel bioadhesive fp-151. Biomaterials, 28(24), 3560-3568.
-
<strong>Two Levels of Regulation for T7 Polymerase Expression</strong><br />
+
<li>Isaacs, F. J. (2012). Synthetic biology: Automated design of RNA devices. Nat Chem Biol, 8(5), 413-415.
-
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).
+
<li>Isaacs, F. J., Carr, P. A., Wang, H. H., Lajoie, M. J., Sterling, B., Kraal, L., <i>et al.</i>. (2011). Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science, 333(6040), 348-353.
-
<br />
+
<li>Nagant, C., Pitts, B., Stewart, P. S., Feng, Y., Savage, P. B., & Dehaye, J. P. (2013). Study of the effect of antimicrobial peptide mimic, CSA-13, on an established biofilm formed by Pseudomonas aeruginosa. Microbiologyopen, 2(2), 318-325.
-
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.
+
<li>Ramos, R., Domingues, L., and Gama, M. (2011) LL-37, a human antimicrobial peptide with immunomodulatory properties. 2, pp.693-1348, In: Science Against Microbial Pathogens: Communicating Current Research and Technological Advances. Formatex Research Center Publications. Badajoz, Spain.
-
</p>
+
<li>Salta, M., Wharton, J. A., Stoodley, P., Dennington, S.P., Goodes, L. R., & Werwinski, S., <i>et al.</i>. (2010). Designing biomimetic antifouling surfaces. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 368(1929), 4729-4754.
-
</td>
+
<li>H. Lee, S. M. Dellatore, W. M. Miller and P. B. Messersmith, "Mussel-Inspired Surface Chemistry for Multifunctional Coatings", Science, 318, 426-430, (2007).
 +
Endy, Drew. Standard Registry of Parts. Massachusetts Institute of Technology. partsregistry.org. 2008.
 +
<li>Isaacs, Farren J., <i>et al.</i> "Precise manipulation of chromosomes in vivo enables genome-wide codon replacement." <i>Science</i> 333.6040 (2011): 348-353. <p>
 +
<li>Sharan, Shyam K., Lynn C. Thomason, and Sergey G. Kuznetsov. "Recombineering: a homologous recombination-based method of genetic engineering." <i>Nature protocols</i> 4.2 (2009): 206-223. <p>
 +
<li>Wang, Harris H., <i>et al.</i> "Programming cells by multiplex genome engineering and accelerated evolution." <i>Nature</i> 460.7257 (2009): 894-898. <p>
 +
<li>Ellis, Hilary M., Daiguan Yu, and Tina DiTizio. "High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides." <i>Proceedings of the National Academy of Sciences</i> 98.12 (2001): 6742-6746. <p>
 +
 
 +
<li>Costantino, Nina. & Court, Donald L. “Enhanced levels of  Red-mediated recombinants in mismatch repair mutants.” <i>Proceedings of the National Academy of Sciences</i> 100.26 (2003): 15748–15753. <p>
 +
<li>Lajoie, M. J., Rovner, A. J., Goodman, D. B., Aerni, H. R., Haimovich, A. D., Kuznetsov, G., <i>et al.</i> (2013). Genomically recoded organisms expand biological functions. <i>Science</i>, 342(6156), 357-360. <p>
 +
<li>Levine, Melvin, and Harold Tarver. "Studies on ethionine III. Incorporation of ethionine into rat proteins." <i>Journal of Biological Chemistr</i>y 192.2 (1951): 835-850. <p>
 +
<li>Wang, Lei, <i>et al.</i> "A new functional suppressor tRNA/aminoacyl-tRNA synthetase pair for the in vivo incorporation of unnatural amino acids into proteins." <i>JOURNAL-AMERICAN CHEMICAL SOCIETY</i>122.20 (2000): 5010-5011. <p>
 +
<li>Wang, Lei, and Peter G. Schultz. "A general approach for the generation of orthogonal tRNAs." <i> amino acids</i> 3 (2001): 4. <p>
 +
<li>Wang, Lei, <i>et al.</i> "Expanding the genetic code of Escherichia coli." <i>Science</i> 292.5516 (2001): 498-500. <p>
 +
<li>Isaacs, F. J., <i>et al.</i> (2004). "Engineered riboregulators enable post-transcriptional control of gene expression." Nature biotechnology 22(7): 841-847.
 +
<li>BP Lee, PB Messersmith, JN Israelachvili, JH Waite. (2011) Mussel-Inspired Adhesives and Coatings. Annual Review of Materials Research; 41: 99-132.
 +
<li>H Lee , NF Scherer, PB Messersmith. (2006) Single-Molecule Mechanics of Mussel Adhesion. Proc Natl Acad Sci; 103:12999-3003.
 +
<li>M Yu, J Hwang, TJ Deming. (1999) Role of L-3,4-Dihydroxyphenylalanine in Mussel Adhesive Proteins. J. Am. Chem. Soc. 1999, 121, 5825-5826
 +
<li>Gao, Y., Zorman, S., Gundersen G., Xi, Z., Ma L., Sirinakis G., Rothman J.E., & Zhang Y. (2012) Single Reconstituted Neuronal SNARE Complexes Zipper in Three Distinct Stages. Science (New York, N.Y.) 337(6100):1340-1343
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<h1 style="margin-top:25px; margin-bottom:45px; font-size:35px">Anti-Fouling Peptide Construct: Experimental Design</h1>
 
-
<p>
 
-
<strong> 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. </strong> 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.
 
-
</p>
 
-
<p>
 
-
<strong>Protein Purification</strong>
 
-
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).
 
-
<br />
 
-
Flag Tag Sequence:  Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys <br />
 
-
Strep Tag Sequence: Sequence: Trp-Ser-His-Pro-Gln-Phe-Glu-Lys
 
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<h1 style="margin-top:25px; margin-bottom:45px; font-size:35px">Characterization of Coating Adhesion Properties</h1>
 
-
<p>
 
-
<strong> 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. </strong> 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.
 
-
</p>
 
-
<p>
 
-
<strong>Protein Purification</strong>
 
-
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).
 
-
<br />
 
-
Flag Tag Sequence:  Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys <br />
 
-
Strep Tag Sequence: Sequence: Trp-Ser-His-Pro-Gln-Phe-Glu-Lys
 
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Latest revision as of 03:55, 18 October 2014

AMPersand: An Anti-Microbial Peptide Coating

The Problem

A biofilm is a community of bacteria attached to a surface that exhibits 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 1. (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.

Our Solution

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 The functional residue in MAPs is L-3,4-dihydroxyphenylalanine (L-DOPA), which is generated by post-translational modification of tyrosine with tyrosinase. Since L-DOPA is a non-standard amino acid, it cannot be incorporated by standard translation systems. However, we intend to be the first group to use a genetically recoded organism (GRO) to incorporate L-DOPA in-situ, eliminating the need for any post-translational modification.
  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. Addressing 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.

Video Walkthrough of our Project


Project Goals

  1. Control Expression of Anti-Microbial Peptides Using an Improved T7 Riboregulation System:
    • 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. A Modular Anti-Microbial Construct based on Mussel Foot Protein:
    • 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., 2008.8 At the N-terminus, we included a 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 2. 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:
    • We intend to perform a number of assays to test the erosion resistance of our adhesive coating using an original apparatus designed to introduce erosion by laminar flow through a liquid bath. The specific tests that we investigated for adhesion testing are detailed in the Materials and Methods section.
    • To assess the efficacy of our peptide in inhibiting biofilm formation, we intend to perform a minimum biofilm eradication concentration (MBEC) assay (Innovotech). Further information is provided in our Materials and Methods section.

For a detailed description of our experimental design regarding the T7 expression system, anti-microbial peptide construct, and adhesion assays, see materials and methods.

For a detailed description of our results, see results.

We modeled theoretical bacterial population survival rates based on various points of anti-microbial peptide induction. See modeling.

Our collection of submitted biobricks consists of:

  • Mussel foot protein (MFP) 1-5-1 sequence [combination of Mytilus galloprovincialis Foot Protein 5 (Mgfp-5) and Mytilus Edulis Foot Protein 1 (Mefp-1)].
  • MFP with superfolder Green Fluorescence Protein (sfGFP).
  • MFP with our anti-microbial peptide, LL-37.
  • Entire construct of our anti-microbial adhesive peptide: 2XStrep_Flagtag--LL-37--Mussel Foot Protein--sfGFP.
  • See here for more information on our Biobricks!

Additional Background Information

  1. Genomically Recoded E. coli

    Whole-genome recoding was reported for the first time in 2011 by a team including our advisor, Professor Farren Isaacs, when it described replacing all TAG codons in E. coli by TAA. 9 Both TAG and TAA are stop codons (amber and ochre), but their transcripts are bound by different release factors (RF1 binds UAG and UAA, whereas RF2 binds UAA and UGA), not only making this recoding nonlethal but also allowing the deletion of RF1 (ΔprfA) from the recoded strain, given RF2’s response to both remaining stop codons.

    This genome-wide recoding was enabled by lambda-Red recombineering10 and multiplex automated genome engineering. 11 lambda-Red recombineering uses the lambda prophage’s protein Beta to mediate the homologous recombination of an exogenous single-stranded oligonucleotide, delivered into the cell by electroporation, with the host genome, by annealing that oligonucleotide to single-stranded genomic DNA exposed on the lagging strand of the replication fork. 12 A 30-bp region of homology at either end of a sequence is sufficient to drive recombination of the entire strand, including mismatched regions, and mismatches thus incorporated are passed to progeny at high efficiency (up to 30%) in strains lacking methyl-directed mismatch repair (ΔmutS).13

    Multiplex automated genome engineering (MAGE) using lambda-Red recombineering can then make many directed mutations across the host genome, as was necessary to replace all 314 TAG codons in the strain EcNR2 (E. coli MG1655 ΔmutS::cat Δ(ybhB-bioAB)::[λcI857 Δ(cro-ea59)::tetR-bla]).9 To allow recombineering, the lambda prophage was introduced into the host genome by P1 transduction. Because the probability of each target sequence being mutated in a given cycle is at most 30%, attempting to introduce all 314 mutations using a single pool of 314 mutagenic oligonucleotides would much sooner generate any of ~2314 other partially recoded strains before the fully recoded strain, and running enough MAGE cycles to obtain that strain would allow time for spontaneous point mutations to accumulate unchecked by the knocked-out mismatch repair system. To construct the strain we will use 32 separate strains were generated, each with a different recoded sector, and those sectors were assembled by hierarchical conjugation. The recoded strain was validated by sequencing, and its RF1 was deleted to free the TAG codon for reassignment to an orthogonal translation system.14

  2. Orthogonal Translation Systems

    An amino acid can be assigned to a particular codon by a two-part translation system: a tRNA with its anticodon loop complementary to the codon, and an aminoacyl synthetase (hereafter called a synthetase) able to charge this tRNA with the amino acid. This system is orthogonal to endogenous translation systems if and only if this synthetase aminoacylates only this tRNA and endogenous synthetases cannot aminoacylate this tRNA. Usually, an unnatural amino acid thus assigned should also be sufficiently dissimilar from naturally encoded amino acids that endogenous synthetases would not charge endogenous tRNAs with the unnatural amino acid, to avoid partially reassigning other codons and disrupting cell function.15

    Many synthetases include a nonconserved loop specific to their associated tRNA’s anticodon, preventing them from binding a tRNA reassigned to CUA, but the archaeum Methanococcus jannaschii’s tyrosyl-tRNA synthetase (MjtRNAs) and its associated tRNA translation system still bind after the anticodon is mutated, and is an amber suppressor when transformed into E. coli.16 This MjtRNAs was still aminoacylated by endogenous synthetases, so its specificity for its synthetase was improved by random mutation at eleven positions not directly interacting with its synthetase, followed by negative selection against nonspecific acylation and positive selection for specific acylation. 17

    This orthogonal system has since been engineered by directed evolution of its synthetase to incorporate nonstandard amino acids, in place of tyrosine. 18 Diverse synthetases were generated by site-directed mutagenesis of five residues in the amino acid-binding pocket, and screened by positive selection, for incorporation of the amino acid into a protein conferring antibiotic resistance, and a negative screen, against mutants resistant to the antibiotic even in the absence of the nonstandard amino acid (by treating colonies on a replica plate lacking the amino acid and then observing which were killed). Selected mutants were then recombined and subjected to further mutagenesis.

  3. An Improved T7 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 PLlacO 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.19 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 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 3. This diagram illustrates the T7 riboregulation system developed to ensure low basal levels of protein expression.19


  4. 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 (WA)” 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.20 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.21 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.21,22 Ultimately, the high degree of surface affinity such dopa-containing peptides possess makes them novel tools in the development of biomimetic adhesive peptides.

References

  1. Donlan, R. M. (2001). Biofilms and device-associated infections. Emerg Infect Dis, 7(2), 277-281.
  2. Hwang, D. S., Gim, Y., Yoo, H. J., & Cha, H. J. (2007). Practical recombinant hybrid mussel bioadhesive fp-151. Biomaterials, 28(24), 3560-3568.
  3. Isaacs, F. J. (2012). Synthetic biology: Automated design of RNA devices. Nat Chem Biol, 8(5), 413-415.
  4. Isaacs, F. J., Carr, P. A., Wang, H. H., Lajoie, M. J., Sterling, B., Kraal, L., et al.. (2011). Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science, 333(6040), 348-353.
  5. Nagant, C., Pitts, B., Stewart, P. S., Feng, Y., Savage, P. B., & Dehaye, J. P. (2013). Study of the effect of antimicrobial peptide mimic, CSA-13, on an established biofilm formed by Pseudomonas aeruginosa. Microbiologyopen, 2(2), 318-325.
  6. Ramos, R., Domingues, L., and Gama, M. (2011) LL-37, a human antimicrobial peptide with immunomodulatory properties. 2, pp.693-1348, In: Science Against Microbial Pathogens: Communicating Current Research and Technological Advances. Formatex Research Center Publications. Badajoz, Spain.
  7. Salta, M., Wharton, J. A., Stoodley, P., Dennington, S.P., Goodes, L. R., & Werwinski, S., et al.. (2010). Designing biomimetic antifouling surfaces. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 368(1929), 4729-4754.
  8. H. Lee, S. M. Dellatore, W. M. Miller and P. B. Messersmith, "Mussel-Inspired Surface Chemistry for Multifunctional Coatings", Science, 318, 426-430, (2007). Endy, Drew. Standard Registry of Parts. Massachusetts Institute of Technology. partsregistry.org. 2008.
  9. Isaacs, Farren J., et al. "Precise manipulation of chromosomes in vivo enables genome-wide codon replacement." Science 333.6040 (2011): 348-353.

  10. Sharan, Shyam K., Lynn C. Thomason, and Sergey G. Kuznetsov. "Recombineering: a homologous recombination-based method of genetic engineering." Nature protocols 4.2 (2009): 206-223.

  11. Wang, Harris H., et al. "Programming cells by multiplex genome engineering and accelerated evolution." Nature 460.7257 (2009): 894-898.

  12. Ellis, Hilary M., Daiguan Yu, and Tina DiTizio. "High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides." Proceedings of the National Academy of Sciences 98.12 (2001): 6742-6746.

  13. Costantino, Nina. & Court, Donald L. “Enhanced levels of Red-mediated recombinants in mismatch repair mutants.” Proceedings of the National Academy of Sciences 100.26 (2003): 15748–15753.

  14. Lajoie, M. J., Rovner, A. J., Goodman, D. B., Aerni, H. R., Haimovich, A. D., Kuznetsov, G., et al. (2013). Genomically recoded organisms expand biological functions. Science, 342(6156), 357-360.

  15. Levine, Melvin, and Harold Tarver. "Studies on ethionine III. Incorporation of ethionine into rat proteins." Journal of Biological Chemistry 192.2 (1951): 835-850.

  16. Wang, Lei, et al. "A new functional suppressor tRNA/aminoacyl-tRNA synthetase pair for the in vivo incorporation of unnatural amino acids into proteins." JOURNAL-AMERICAN CHEMICAL SOCIETY122.20 (2000): 5010-5011.

  17. Wang, Lei, and Peter G. Schultz. "A general approach for the generation of orthogonal tRNAs." amino acids 3 (2001): 4.

  18. Wang, Lei, et al. "Expanding the genetic code of Escherichia coli." Science 292.5516 (2001): 498-500.

  19. Isaacs, F. J., et al. (2004). "Engineered riboregulators enable post-transcriptional control of gene expression." Nature biotechnology 22(7): 841-847.
  20. BP Lee, PB Messersmith, JN Israelachvili, JH Waite. (2011) Mussel-Inspired Adhesives and Coatings. Annual Review of Materials Research; 41: 99-132.
  21. H Lee , NF Scherer, PB Messersmith. (2006) Single-Molecule Mechanics of Mussel Adhesion. Proc Natl Acad Sci; 103:12999-3003.
  22. M Yu, J Hwang, TJ Deming. (1999) Role of L-3,4-Dihydroxyphenylalanine in Mussel Adhesive Proteins. J. Am. Chem. Soc. 1999, 121, 5825-5826
  23. Gao, Y., Zorman, S., Gundersen G., Xi, Z., Ma L., Sirinakis G., Rothman J.E., & Zhang Y. (2012) Single Reconstituted Neuronal SNARE Complexes Zipper in Three Distinct Stages. Science (New York, N.Y.) 337(6100):1340-1343
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