Team:BroadRun-NorthernVA/Overview

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The Problem

With pressing environmental issues such as rising temperatures, climate change, deforestation, and pollution, affecting the Earth, many industries are taking new initiatives to reduce their footprint on the world; by changing their materials and manufacturing processes, to reduce environmental degradation.

One such example is Armstrong. Armstrong is a global construction manufacturer; they mass produce ceiling tiles, and floorboards.

Recently, as part of an initiative to be more sustainable and environmentally conscientious, Armstrong switched from using raw trees to produce their ceiling tiles, to recycling paper products. This change would be enable usage already available resources, repurposing old paper to create something new, rather than cutting down new trees to produce their ceiling tiles.

But this well-intentioned change has unforeseen consequences.

Production of paper-based ceiling tiles is a “wet” process. A million gallon water system is run through the system to produce the tiles, treated and then reused.

The switch to paper products, which have already been processed once, caused short chain cellulose fibers that were not incorporated into the tiles to be carried away in the water system. A variety of microbes feed off these cellulose fibers in the water system. Some of these of bacteria strains produce butyric acid as a byproduct of anaerobic fermentation. Butyric acid has an highly unpleasant smell, as it is the major distinctive smell in human vomit, and can be detected by humans at concentrations as low as 10 parts per million.

The million gallon water system is difficult to completely regulate the conditions of the system. As such, often the water system changes from its targeted oxygen level of 2.0 mg/L, to nearly 0 mg/L oxygen, resulting in an anaerobic system. When this occurs, it activates an anaerobic fermentation pathway, in which butyric acid is an end product.

Currently Armstrong remediates this issue by periodically using biocides when the butyric acid concentration is too high, chemicals that eliminate all microbial growth in the water system. This solution has many drawbacks. First, it is a short term solution, it kills all the microbial growth, but the butyric-acid-producing bacteria re-emerge, except now some may even be resistant to the biocides. It is expensive; costing thousands each time the biocides must be used. Lastly, it poses an environmental safety concern. If the biocides were to leave the closed factory system and contaminate the natural ecosystem; and the consequences to wildlife and their natural habitats would be devastating.

Faced with a dilemma, Armstrong presented us with this real world industrial problem with environmental consequences, asking us if we could use the power of synthetic biology to create a cost effective, scalable, environmentally safe, long term, and sustainable solution that could be applied immediately in their factory.

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
   • 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 Notebook section.

Issue?

Recently, after Armstrong switched to a more environmentally friendly and sustainable way of making ceiling tiles, their closed water system started to smell. At first, they didn't know what was causing brand new ceiling tiles to smell like old shoes and wet dog... They eventually found out that there was a greater amount of starch in their water than usual. They found out that the additional starch was being eaten by anaerobic bacteria when the system's pump broke down and there was no oxygen getting into the water.