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| − | <h1 style="color: white;
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| − | font-weight: bold;
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| − | font-size: 40pt;">Project Description</h1>
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| − | </div>
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| − | <div class="collapse navbar-collapse" id="myNavbar">
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| − | <ul class="nav nav-pills flex-column">
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| − | <li><a href="#section1">Problem</a></li>
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| − | <li><a href="#section2">Inspiration</a></li>
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| − | <li class="dropdown">
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| − | <a class="dropdown-toggle" data-toggle="dropdown" href="#">Solution </a>
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| − | <li><a href="#section31">Sensing</a></li>
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| − | <li><a href="#section32">Motility</a></li>
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| − | <li><a href="#section33">Killing</a></li>
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| − | <li><a href="#section4">Impact</a></li>
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| − | <li><a href="#section5">References</a></li>
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| − | <div id="section1" class="col-xs-12">
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| − | <h1>Problem</h1>
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| − | <p>
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| − | <i>Staphylococcus aureus</i> is a type of bacteria commonly found in hospitals, sports facilities,
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| − | and even the bodies of healthy individuals (1). As many as one third of the population carries
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| − | <i>S. aureus</i> in their nose, and about 20% of people carry <i>S. aureus</i> on the skin (1). While <i>S. aureus</i>
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| − | does not normally cause severe problems, it can cause infections of the skin, blood, and soft tissues,
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| − | with approximately 20,000 deaths reported in the United States in 2017 as a result of <i>S. aureus</i> infection (2).
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| − | Methicillin-resistant <i>Staphylococcus aureus</i>, or MRSA, is a potent strain of <i>S. aureus</i> that is resistant to
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| − | common antibiotic treatments (1). This resistance to conventional treatment makes MRSA infections difficult to
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| − | combat, turning <i>S. aureus</i> into a much more deadly pathogen. To overcome antibiotic resistance, the medical
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| − | community must find new ways to combat bacterial infection.
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| − | </p>
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| − | </div>
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| − | </div>
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| − | <div class="row">
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| − | <div id="section2" class="col-xs-12">
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| − | <h1>Inspiration</h1>
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| − | <p>
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| − | Our team was inspired by a 2013 paper by Hwang et al. titled “Reprogramming Microbes to be Pathogen-Seeking Killers” (3).
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| − | Hwang et al. engineered <i>E. coli</i> to detect and fight <i>Pseudomonas aeruginosa</i> infections using a seek-and-kill technique.
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| − | Their design is modular, which makes it possible to use their approach to target different bacteria. We found that the
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| − | rise of antibiotic resistant bacteria makes it increasingly vital to find new treatments, which inspired us to adapt the
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| − | system devised by Hwang et al. to target methicillin-resistant <i>Staphylococcus aureus</i>, or MRSA.
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| − | </p>
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| − | </div>
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| − | </div>
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| − | <div class="row">
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| − | <div id="section31" class="col-xs-12">
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| − | <h1>Sensing</h1>
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| − | <p>
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| − | Quorum sensing is a method of cell communication that uses small molecules to regulate gene expression (4). Gram-positive
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| − | and Gram-negative use different small molecules to accomplish quorum sensing; Gram-positive species use autoinducing peptides
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| − | (AIP) while Gram-negative species use acyl homoserine lactones (AHL) (4). Our target species, <i>Staphylococcus aureus</i>,
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| − | uses four forms of AIP in the accessory gene regulator (<i>agr</i>) quorum sensing system. In the <i>agr</i> system, the genes agrA and
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| − | agrC code for proteins that detect AIP produced by neighboring <i>S. aureus</i> and activate the P2 promoter in response (5).
| |
| − | </p>
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| − | <p>
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| − | Our project builds on BBa_K1022100, a BioBrick that combines agrA and agrC from the <i>agr</i> sensing system with green
| |
| − | fluorescent protein under a pBAD promoter. We found that the wild-type AgrA and AgrC proteins contain amino acids not
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| − | present in BBa_K1022100, and we aim to modify and potentially improve the existing part by introducing these missing
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| − | amino acids. The P2 promoter used in BBa_K1022100 is also missing base pairs found in the binding site region of the
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| − | wild-type P2 sequence. We plan to reintroduce these binding sites by using a more complete P2 promoter sequence to
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| − | improve GFP production in the original BioBrick. Lastly, sarA is a transcriptional activator found in <i>S. aureus</i>
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| − | that is believed to activate expression of genes in the <i>agr</i> system (6). We will introduce the sarA gene to further
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| − | improve the effectiveness of the <i>agr</i> system.
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| − | </p>
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| − | </div>
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| − | </div>
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| − | <div class="row">
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| − | <div id="section32" class="col-xs-12">
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| − | <h1>Motility</h1>
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| − | <p>
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| − | Chemotaxis is the movement of a cell towards or away from a chemical stimulus based on concentration, either from
| |
| − | high to low or from low to high (7). In our system, AIP will provide the chemical gradient necessary to move <i>E. coli</i>
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| − | in the direction of <i>S. aureus</i>. Chemotaxis is influenced by the post-translation modulation of the CheY and CheZ
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| − | proteins; however, there is an issue of potential overexpression that can lead to the inhibition of chemotaxis (3).
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| − | In order to combat this issue, we will be implementing a degron, a sequence of amino acids responsible for protein
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| − | degradation, to control CheZ expression (3). We will attach the YbaQ degron to CheZ’s C-terminus, much like the
| |
| − | application used by the Hwang et al. study in 2013 (3). To demonstrate the functionality of chemotaxis in our system,
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| − | we will first use a pBAD promoter to trigger chemotaxis. If successful, we will replace the pBAD promoter with a P2
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| − | promoter which is activated by the <i>agr</i> system. This will ignite chemotaxis in the presence of AIP, moving the <i>E. coli</i> towards <i>S. aureus.</i>
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| − | </p></div>
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| − | </div>
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| − | <div class="row">
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| − | <div id="section33" class="col-xs-12">
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| − | <h1>Killing</h1>
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| − | <p>
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| − | Bacteriocins are small antimicrobial peptides (AMPs) produced by bacteria to kill or inhibit other bacteria.
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| − | Many AMPs are currently under study as potential alternatives to antibiotic treatment due to the rise of antibiotic
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| − | resistance. One such AMP is garvicin KS, a bacteriocin produced by <i>Lactococcus garvieae</i>, a bacterial species
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| − | found in raw milk. Garvicin KS is effective against <i>S. aureus</i> and other Gram-positive bacteria, and is more potent
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| − | than many other bacteriocins (8). Mature garvicin KS is composed of three polypeptides encoded by three genes:
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| − | GakA, GakB, and GakC (9). By placing these genes into an <i>E. coli</i> chassis, we plan to use <i>E. coli</i> to produce
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| − | garvicin KS. As <i>E. coli</i> is a Gram-negative bacterium, additional steps must be taken to ensure that the
| |
| − | garvicin KS peptides can be secreted from the cell. We have chosen a novel signal peptide first constructed
| |
| − | in a 2017 paper by Han et al. to assist with secretion of the garvicin KS peptides (10). This peptide tags
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| − | proteins for secretion by the Sec pathway, a pathway used by <i>E. coli</i> to secrete proteins into the periplasm
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| − | and outer membrane (10).
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| − | </p>
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| − | </div>
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| − | </div> <div class="row">
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| − | <div id="section6" class="col-xs-12">
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| − | <h1>Impact</h1>
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| − | <p>
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| − | Our system has the potential to be used both in vivo and in vitro to combat MRSA planktonic cells and biofilms.
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| − | Many bacteria produce biofilms, which are collections of cells attached to a surface and the extracellular matrix
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| − | that encases these cells (11). Biofilms provide protection to the bacteria within and are often resistant to
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| − | antibiotic treatment, which further compounds the issue of antibiotic resistance (12). One possible solution
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| − | is the use of antimicrobial peptides such as garvicin KS, which have been shown to be more effective towards
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| − | biofilms than traditional antibiotics (13). Our three module system first uses quorum sensing to detect AIP
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| − | produced by MRSA biofilms and planktonic cells. Detection of AIP simultaneously triggers chemotaxis and the
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| − | production of garvicin KS. The use of chemotaxis allows our <i>E. coli</i> to seek out and move towards biofilms and
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| − | planktonic cells, releasing garvicin KS near the source and maximizing effectiveness. Potential applications
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| − | of the system include detecting and killing MRSA infections in the body as well as disinfecting medical or
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| − | sports equipment.
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| − | </p>
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| − | </div>
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| − | </div> <div class="row">
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| − | <div id="section7" class="col-xs-12">
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| − | <h1>References</h1>
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| − | <ol>
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| − | <li>
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| − | Staphylococcal infections [Internet]. Merck Manuals; [updated 2017 Sept; cited 2019 Jun 28].
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| − | Available from:
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| − | https://www.merckmanuals.com/professional/infectious-diseases/gram-positive-cocci/staphylococcal-infections
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| − | </a>
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| − | </li>
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| − |
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| − | <li>
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| − | Staph infections can kill [Internet]. Centers for Disease Control and Prevention (US); [updated 2019 Mar 22; cited 2019 Jun 28].
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| − | Available from: https://www.cdc.gov/vitalsigns/staph/index.html">https://www.cdc.gov/vitalsigns/staph/index.html</a>
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| − | </li>
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| − |
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| − | <li>
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| − | Hwang IY, Tan MH, Koh E, Ho CL, Poh CL, Chang MW. Reprogramming microbes to be pathogen-seeking killers. ACS Synth Biol. 2013;3(4):228-237.
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| − | <a href="https://doi.org/10.1021/sb400077j"></a>
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| − | </li>
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| − |
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| − | <li>
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| − | Rutherford ST, Bassler, B. L. Bacterial quorum sensing: its role in virulence and possibilities for its control.
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| − | Cold Spring Harb Perspect Med. 2012;2(11).
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| − | </li>
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| − |
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| − | <li>
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| − | Tan L, Li SR, Jiang B, Hu, XM, Li S. Therapeutic targeting of the <i>Staphylococcus aureus</i> accessory gene regulator
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| − | (agr) system. Front Microbiol. 2018;9(55).
| |
| − | </li>
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| − |
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| − | <li>
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| − | Cheung AL, Zhang G. Global regulation of virulence determinants in <i>Staphylococcus aureus</i> by the SarA protein
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| − | family. Front Biosci. 2002;7:1825-1842.
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| − | </li>
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| − |
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| − | <li>
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| − | Wang Y, Chen CL, Iijima M. Signaling mechanisms for chemotaxis. Dev Growth Differ. 2011;53(4):495-502.
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| − | </li>
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| − |
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| − | <li>
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| − | Chi H, Holo H. Synergistic antimicrobial activity between the broad spectrum bacteriocin garvicin KS and nisin,
| |
| − | farnesol, and polymyxin B against Gram-positive and Gram-negative bacteria. Curr Microbiol. 2018;75(3):272-277.
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| − | </li>
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| − |
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| − | <li>
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| − | Ovchinnikov KV, Chi H, Mehmeti I, Holo H, Nes IF, Diep, DB. Novel group of leaderless multipeptide
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| − | bacteriocins from Gram-positive bacteria. Appl Environ Microbiol. 2016;82(17):5216-5224.
| |
| − | </li>
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| − |
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| − | <li>
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| − | Han S, Machhi S, Berge M, Xi G, Linke T, Schoner R. Novel signal peptides improve
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| − | the secretion of recombinant <i>Staphylococcus aureu</i>s alpha toxin H35L in <i>Escherichia coli</i>. AMB Expr. 2017;7(93).
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| − | </li>
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| − |
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| − | <li>
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| − | Lopez D, Vlamakis H, Kolter R. (2010). Biofilms. Cold Spring Harb Perspect Biol. 2010;2(7).
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| − | </li>
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| − |
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| − | <li>
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| − | Ciofu O, Rojo-Molinero E, Macia MD, Oliver A. (2017). Antibiotic treatment of biofilm infections. APMIS. 2017;125(4).
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| − | </li>
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| − |
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| − | <li>
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| − | Mathur H, Field D, Rea MC, Cotter PD, Hill C, Ross RP. (2018). Fighting biofilms with lantibiotics and other groups of
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| − | bacteriocins. NPJ Biofilms Microbiomes. 2018;4.
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| − | </li>
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| − | </ol>
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