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| - | <h1 style="margin-top:70px; 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> | + | <div style="margin-top:50px"> </div> |
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| + | <h1 style="margin-top:22px; font-size:44px;">Notebook</h1> |
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| - | <h1>The Problem</h1>
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| - | <strong>The Problem: </strong> <li>Biofilm formation is a deleterious process in the medical and shipping industries. Protein adsorption, cell-adhesion, and subsequent biofilm formation have been found to lead to failure of medical implants and infection in patients. 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> </tr> </li>
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| - | <h1>Our Solution</h1>
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| - | <strong>The Solution: </strong>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<sup>TM</sup>.
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| - | <h1>Project Goals</h1>
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| - | <strong>1. Create a T7 Riboregulation System to control the expression of our proteins:</strong>
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| - | 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)
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| - | <strong> 2. Design the anti-biofouling peptide using both a modular approach: </strong>
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| - | 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).
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| - | <strong>3. Develop an erosion rig to assess the strength of the adhesive peptide:</strong>
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| - | (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. </p>
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| - | <center><img src="https://static.igem.org/mediawiki/2014/thumb/d/d5/Erosion_Rig_Image.png/800px-Erosion_Rig_Image.png"></center>
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| - | <i><strong>Figure 8.</strong> A diagram illustrating the configuration of the erosion rig developed to introduce adhesive coated surfaces to liquid erosion.<i>
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Phone: 203.432.3783
igem@yale.edu 
