Difference between revisions of "Team:Oxford/Description"

 
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        <h3>PROJECT</h3>
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                         <h2>Overview</h2>
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<div class="container-fluid page-heading" style="background-image: url(https://static.igem.org/mediawiki/2015/6/61/Ox_biofilmdrawing.jpeg)">
 +
    <h3>Description</h3>
 +
</div>
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<div class="container-fluid">
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    <div class="row">
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        <div class="col-md-9">
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            <div class="section" id="overview">
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                    <h2>Overview</h2>
 +
                     <div class="quote quote-full">
 +
                        <p>
 +
                            "Years from now we will treat most infections with bacteria and not antibiotics."
 +
                        </p>
 +
                        <h3>Professor James Malone-Lee<br>Barlow Professor of Geriatric Medicine</h3>
 +
                    </div>
 +
                    <p>
 +
                        A solution is urgently needed for the increasing number of infections caused by antibiotic-resistant bacteria. The engineering of bacterial cells to fight human pathogens is a promising alternative to administering traditional antibiotics. Our project involves the use of synthetic biology to engineer living therapeutics that have the potential to treat urinary tract infections (UTIs), which are a major public health concern in developed countries. This is largely due to growing antibiotic resistance.
 +
                    </p>
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                </div>
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            </div>
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            <div class="section" id="problem">
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                    <h2>The Problem</h2>
 +
                    <ul>
 +
                         <li>Antibiotics have negative side effects and their resistance is a growing problem</li>
 +
                        <li>UTIs are the <strong>most common hospital-acquired infection</strong> and the bacteria that cause them are frequently <strong>resistant to antibiotics</strong></li>
 +
                        <li>Antibiotic resistance in UTIs is caused by <strong>biofilms</strong></li>
 +
                        <li>Biofilms are currently estimated to be responsible for over 65% of all hospital-acquired infections</li>
 +
                        <li><strong>Current UTI treatments are ineffective</strong> and fail to prevent recurrent infections</li>
 +
                    </ul>
 +
                </div>
 +
            </div>
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            <div class="section-spacer"></div>
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            <div class="section" id="antibio">
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                    <h2>Problem with Antibiotics</h2>
 +
                    <p>
 +
                         Antibiotic use is associated with numerous negative side effects, allergies and reactions. The most common side effects of antibiotics all impact the digestive system and occur in around one in ten people. <strong>Around one person in fifteen has an allergic reaction to antibiotics</strong>, especially penicillin and cephalosphorins [<a href="#references">10</a>]. Half of the patients we spoke to during our project wanted us to find an alternative to using antibiotics, owing to the severity of the negative side effects that they experience.
 +
                    </p>
 +
                    <p>
 +
                        Despite their negative side effects, antibiotics have been used so widely and for so long that the <strong>targeted microbes have adapted to become resistant</strong>, reducing the effectiveness of the drugs. A vicious cycle ensues in which ineffective antibiotic treatments leads to overprescription and overexposure, which amplifies the problem of antibiotic resistance. Global antibiotic consumption grew by 30% between 2000 and 2010. [<a href="#references">27</a>] Described by the Chief Medical Officer of England as “a threat equal to that of terrorism”, the growing resistance to antibiotics is hindering the effective prevention and treatment of an ever increasing range of infections.
 +
                    </p>
 +
                    <div class="quote quote-full">
 +
                        <p>
 +
                            antibiotic resistance poses a catastrophic threat. If we don't act now, any one of us could go into hospital in 20 years for minor surgery and die because of an ordinary infection that can't be treated by antibiotics.
 +
                        </p>
 +
                        <h3>Professor Dame Sally Davies<br>Chief Medical Officer<br>March 2013</h3>
 +
                    </div>
 +
                    <p>
 +
                        The World Health Organisation’s antibiotic resistance Global Report on Surveillance, reports increasing worldwide incidences of antimicrobial resistance, in particular antibiotic resistance. This highlights very high rates of resistance in bacteria that cause common healthcare associated and community-acquired infections, such as UTIs [<a href="#references">1</a>]. UTIs account for over 7 million doctor visits per year. <strong>Catheter associated UTIs (CAUTIs) are the most commonly acquired infection in hospitals, and there is a high incidence of antibiotic resistance in the bacteria that cause UTIs globally</strong> [<a href="#references">2</a>].
 +
                    </p>
 +
                </div>
 +
            </div>
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            <div class="section-spacer"></div>
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            <div class="section" id="biofilms">
 +
                <div class="slim">
 +
                    <h2>Biofilms</h2>
 +
                        <div class="image image-full">
 +
                            <img src="https://static.igem.org/mediawiki/2015/3/3b/Oxford-biofilm.jpeg" />
 
                             <p>
 
                             <p>
−
                                 "No action today means no cure tomorrow."
+
                                 Major structural elements of bacterial biofilms.
 
                             </p>
 
                             </p>
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                            <h3>Dr Margaret Chan<br>WHO Director General, 2011</h3>
 
 
                         </div>
 
                         </div>
 +
                    <p>
 +
                        Biofilms are aggregates of surface-associated microorganisms that are encased in a matrix of highly-hydrated extracellular polymeric substances, which include extracellular polysaccharides, extracellular DNA, as well as proteins [<a href="#references">6</a>]. Van Leeuwenhoek, using his simple microscopes in 1684, first observed microorganisms on tooth surfaces and can be credited with the discovery of microbial biofilms. "The number of these animalcules in the scurf of a man's teeth are so many that I believe they exceed the number of men in a kingdom." - Leeuwenhoek
 +
                    </p>
 +
                    <p>
 +
                        We now know a great deal more about biofilms. Environmental changes are responsible for the transition from planktonic growth to biofilm [<a href="#references">13</a>] and cause changes in the expression of surface molecules, virulence factors, and metabolic status. This allows the bacteria to acquire properties that enable their survival in unfavourable conditions [<a href="#references">14,15</a>], such as in the presence of antibiotics.
 +
                    </p>
 +
                    <p>
 +
                        The low nutrient and oxygen levels at the bottom of the biofilm give rise to metabolically-inactive bacteria, better known as persister cells. <strong>These persister cells are rendered unsusceptible to most traditional antibiotics, which rely on bacterial metabolism to exert cell-killing effect</strong> [<a href="#references">7</a>].
 +
                    </p>
 +
                </div>
 +
            </div>
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            <div class="section-spacer"></div>
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            <div class="section" id="utis">
 +
                <div class="slim">
 +
                    <h2>UTIs</h2>
 +
                    <p>
 +
                        Biofilms are currently estimated to be responsible for over 65% of nosocomial infections and 80% of all microbial infections [<a href="#references">16</a>]. Bacterial biofilms play an important role in UTIs. UTIs are caused by the pathogenic invasion of the urinary tract, which causes an inflammatory response of the urothelium.
 +
                    </p>
 +
                    <p>
 +
                        It is estimated that approximately 40% of women have had a UTI at some time in their lives [<a href="#references">10</a>]. UTIs may be caused by a variety of different organisms, most commonly bacteria. The most frequent cause of UTI in adult women is <em>Escherichia coli</em>, accounting for approximately 85% of community-acquired UTIs and 25-50% of hospital-acquired UTIs. Nosocomial infections may involve more aggressive organisms such as <em>Pseudomonas aeruginosa</em> and <em>Enterobacter</em> species.
 +
                    </p>
 +
                </div>
 +
            </div>
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            <div class="section-spacer"></div>
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            <div class="section" id="solution">
 +
                <div class="slim">
 +
                    <h2>The Solution</h2>
 +
                    <ul>
 +
                        <li>Break down bacterial biofilms to liberate the bacteria encased within and reduce the dose of antibiotics required</li>
 +
                        <li>Directly kill the bacteria encased within the biofilms to provide an alternative to antibiotics</li>
 +
                    </ul>
 +
                </div>
 +
                <div class="image-massive">
 +
                    <img src="https://static.igem.org/mediawiki/2015/4/47/Ox_Ecolidrawing.jpeg"/>
 +
                </div>
 +
                <div id="solution-overview">
 +
                    <div class="slim">
 +
                        <h3>Overview</h3>
 
                         <p>
 
                         <p>
−
                             Antimicrobial resistance is a complex problem driven by many interconnected factors. As such, single, isolated interventions have little impact. Coordinated action is required to minimize emergence and spread of antimicrobial resistance. The aim of our project is to contribute to the growing body of research into providing a solution to the threat of antimicrobial resistance.
+
                             Our solution is focused on providing a treatment for UTIs because conventional antibiotics are unable to treat these and other biofilm-associated infections. Given the prevalence of such infections, there is a growing need for alternative therapeutic agents that can specifically degrade biofilms and kill the bacteria encased within. The use of synthetic biology to produce enzymes is the most effective way to achieve this specificity based on current technology. Our solution aims to investigate how bacterial biofilm disrupting proteins and antimicrobial proteins can be exported from <em>E. coli</em> and subsequently retain their antibiofilm/antimicrobial function. Using this secretion device we seek to create a system that offers persistent protection against biofilm formation.
 +
                           
 
                         </p>
 
                         </p>
 
                         <p>
 
                         <p>
−
                             The World Health Organisation have recently (May 2015) endorsed a global action plan to tackle antimicrobial resistance.
+
                             We have designed a device that can exert antibiofilm and antimicrobial activity against <em>E. coli</em> and <em>P. aeruginosa</em>, the two leading causes of CAUTIs [<a href="#references">4</a>]. A nonpathogenic laboratory strain of <em>E. coli</em> is used as the expression host for the production of these enzymes as a proof-of-concept. The antibiofilm enzymes that we are using are Dispersin B and Micrococcal DNase, and the antimicrobial proteins that we are using are Art-175 and Microcin S.
 
                         </p>
 
                         </p>
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                    </div>
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                </div>
 +
                <div id="solution-degrading-biofilm">
 +
                    <div class="slim">
 +
                        <h3>Degrading the Biofilm</h3>
 
                         <p>
 
                         <p>
−
                             The plan sets out 5 objectives:
+
                             Prof. Malone-Lee stressed to us that sensitivity is a greater problem than complete antibiotic resistance. “Many more strains of bacteria are just insensitive to low doses of antibiotics, many can be overcome by high doses over long periods of time. Resistance is definitely not absolute.” Breaking down the biofilm increases the sensitivity of the bacteria embedded within it. Planktonic bacteria are metabolically active and are thus prone to antibiotics, meaning that lower doses are required.
 
                         </p>
 
                         </p>
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                        <ol>
 
−
                            <li><p>Improve awareness and understanding of antimicrobial resistance</p></li>
 
−
                            <li><p>Strengthen surveillance and research</p></li>
 
−
                            <li><p>Reduce the incidence of infection</p></li>
 
−
                            <li><p>Optimize the use of antimicrobial medicines</p></li>
 
−
                            <li><p>Ensure sustainable investment in countering antimicrobial resistance</p></li>
 
−
                        </ol>
 
 
                         <p>
 
                         <p>
−
                             Our solution considers the first two objectives of this plan: human practices to improve education and awareness of the problem that antibiotic resistance poses and laboratory work to further research into finding alternatives to administering antibiotics. We want to use synthetic biology to provide a solution.
+
                             DspB (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1659200">BBa_K1659200</a>) is an enzyme produced by <em>Aggregatibacter actinomycetemcomitans</em>, a species of bacteria found in the human oral cavity that grows almost exclusively in the form of biofilms. Structural analysis of Dispersin B showed that the enzyme only works specifically against the β-1,6-glycosidic linkages found in poly-N-acetylglucosamine, which is a polysaccharide structural element found in the biofilms of <em>E. coli</em> but not in <em>P. aeruginosa</em>. An additional enzyme would need to be used to target the polysaccharide component of <em>P. aeruginosa</em> biofilms.
 
                         </p>
 
                         </p>
 
                         <p>
 
                         <p>
−
                             Although bacteria are generally thought of as causing infection, most bacteria that live inside the human body are non-pathogenic and some of them can be turned, after proper engineering, into ‘smart’ living therapeutics that have the potential to treat a diverse range of diseases. We are focused specifically on treating UTIs and, by employing the power of engineered E. coli, we are creating a system that offers persistent protection against biofilm formation in the urinary tract and on the surface of catheters without the use of antibiotics.
+
                             Micrococcal DNase (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1659300">BBa_K1659300</a>) is an endo-exonuclease that non-specifically catalyzes the hydrolysis of single- and double-stranded DNA under basic conditions and in the presence of Ca<sup>2+</sup> ions, and is known to be able to speed up DNA hydrolysis by up to 1016 times [<a href="#references">17</a>]. We are using DNase to break down the extracellular DNA component of biofilms.
 
                         </p>
 
                         </p>
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                        <div id="overview-what">
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                    </div>
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                            <h3>What?</h3>
+
                </div>
 +
                <div id="solution-killing-bacteria">
 +
                    <div class="slim">
 +
                        <h3>Killing the Bacteria</h3>
 +
                        <p>
 +
                            Although antibiotic resistance is not absolute, it does pose a very big threat to the effective treatment of many infections. The insensitivity of bacteria to antibiotics can also be attributed to increasing antibiotic resistance. As described above, antibiotics also have many side effects that reduce patient quality of life and decrease the likelihood of completing a course of antibiotics. With all of this in mind, our solution does not only break down the biofilm, but also kills the bacteria embedded within so as to provide an alternative to antibiotics.
 +
                        </p>
 +
                        <div id="solution-killing-bacteria-art175">
 +
                            <h4>Art-175</h4>
 
                             <p>
 
                             <p>
−
                                 The threat of antimicrobial resistance is a serious global public health concern. One of the ways bacteria protect themselves against antimicrobial drugs is by growing biofilms. The biological definition for a biofilm: “an assemblage of surface-associated microorganisms that secrete a mucilaginous protective coating in which they are encased.” Van Leeuwenhoek, using his simple microscopes, first observed microorganisms on tooth surfaces and can be credited with the discovery of microbial biofilms. These bacterial slimes are responsible for a whole host of medical, industrial and environmental problems that are very costly and technically challenging to remedy. Biofilms are involved in catheter and implant infections, dental plaque formation as well as infections in cystic fibrosis patients.  Biofilm can be found in the urothelium, prostate stones, and implanted foreign bodies. In industry and infrastructure, biofilms are also the main culprit behind the fouling of various plants and pipelines for aquaculture, water treatment, and food production.
+
                                 Art-175 (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1659000">BBa_K1659000</a>) derive their name from “artificial endolysins”. Endolysins are bacteriophage-encoded peptidoglycan hydrolases that pass through the cytoplasmic membrane, degrading the peptidoglycan layer and inducing the lysis of the infected cell.
 
                             </p>
 
                             </p>
−
                            <p>
 
−
                                Our solution is focused on providing a treatment for urinary tract infections (UTIs). 90% of urinary infections are caused by uropathogenic E. coli (UPEC) and the biofilm that they form in the urinary tract. Resistance to one of the most widely used antibacterial drugs for the oral treatment of urinary infections caused by E. coli – fluoroquinolones – is now widespread and, with UTIs being the most commonly acquired infection at hospital, there is a huge need to find a solution for the treatment of UTIs and resistance to antimicrobial resistance.
 
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                            </p>
 
−
                            <p>
 
−
                                <em>See UTIs facts and figures for more information.</em>
 
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                            </p>
 
−
                            <p>
 
−
                                Both antimicrobial resistance and the other problems associated with biofilm formation are big issues in their own right but are especially problematic when they’re combined. The bacteria, already constantly evolving to afford themselves more innate resistance against antibiotics, produce biofilms as protective layers that shield them from the drugs even more comprehensively.
 
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                            </p>
 
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                        </div>
 
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                        <div id="overview-why">
 
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                            <h3>Why?</h3>
 
−
                            <ul>
 
−
                                <li>UTIs are the most common nosocomial infection</li>
 
−
                                <li>There is growing resistance to the antibiotics currently used to treat urinary infections</li>
 
−
                                <li>Biofilms are major problem both in health and industry </li>
 
−
                                <li>Antibiotics have a negative effect on the beneficial gut flora</li>
 
−
                                <li>There is a current failure to prevent recurrent infections</li>
 
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                            </ul>
 
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                            <div class="quote quote-full">
 
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                                <p>
 
−
                                    "Antimicrobial resistance poses a catastrophic threat. If we don't act now, any one of us could go into hospital in 20 years for minor surgery and die because of an ordinary infection that can't be treated with antibiotics."
 
−
                                </p>
 
−
                                <h3>Professor Dame Sally Davies<br>Chief Medical Officer, March 2013</h3>
 
−
                            </div>
 
 
                         </div>
 
                         </div>
−
                         <div id="overview-how">
+
                         <div id="solution-killing-bacteria-mccs">
−
                             <h3>How?</h3>
+
                             <h4>MccS</h4>
 
                             <p>
 
                             <p>
−
                                 There is currently no commercial antibiotic that specifically targets bacterial biofilms, but researchers have identified a range of bacterially-derived biomolecules that degrade and destroy biofilms. Our solution aims to investigate how bacterial biofilm disrupting proteins and antimicrobial proteins can be exported from E. coli.
+
                                 MccS (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1659100">BBa_K1659100</a>) is a type of microcin, a subclass of antibacterial proteins known as bacteriocins. Microcins are small, enterobacteria-produced bacteriocins that exert antibacterial activity against closely-related species, and MccS is produced by <em>E. coli</em> present in the probiotic drug Symbioflor 2 that has been shown to successfully treat gastrointestinal disorders.
 
                             </p>
 
                             </p>
 
                             <p>
 
                             <p>
−
                                 The proteins DispersinB, MicrocinS, DNase and Endolysin will be fused with secretion tags to target them to normal bacterial secretion pathways. By hijacking the natural processes by which E. coli secrete proteins, we can target our anti-biofilm agents out of E. coli and onto a biofilm infected surface. Additionally, our E. coli will lyse upon sensing the presence of a biofilm, releasing a pulse of proteins from the cytoplasm on detection of a high target cell density.
+
                                 For more information, please visit our <a href="https://2015.igem.org/Team:Oxford/Parts">Parts</a> page.
−
                            </p>
+
−
                            <p>
+
−
                                The beauty of the anti-biofilm agents we plan to use is that they have been shown not to induce resistance in the target bacteria, meaning that having them continually produced at a low level will not be nearly bad as with traditional antibiotics. Our system is applicable to a whole host of biofilm environments and with a simple design that can be used in multiple sectors, we hope to get a step further in providing a novel approach to treating microbial infections. In terms of product formulation and design, we hope to ultimately arrive at a functional proof-of-concept e.g. an enzyme-secreting infection-clearing catheter or a modular system that continuously and cheaply cleans out pipelines.
+
 
                             </p>
 
                             </p>
 
                         </div>
 
                         </div>
 +
                        <p>
 +
                            Current clinically-relevant pathogens have not been seen to exhibit resistance against our antimicrobial proteins of choice. Art-175 has been experimentally shown to be not susceptible to resistance development, likely because it targets the structural element of the bacterial cell wall that is highly conserved across species and difficult to mutate [<a href="#references">8</a>]. The mechanism by which Microcin S exerts antimicrobial activity is still currently unknown, but no bacterial strains except for the original strain of probiotic <em>E. coli</em> which produces Microcin S has been shown to be resistant to it thus far [<a href="#references">9</a>].
 +
                        </p>
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
−
                 <div class="section-spacer"></div>
+
                 <div id="solution-secreting">
−
                <div class="section" id="what-next">
+
 
                     <div class="slim">
 
                     <div class="slim">
−
                         <h2>What Next?</h2>
+
                         <h3>Secreting the Proteins</h3>
 
                         <p>
 
                         <p>
−
                             For us, the Oxford iGEM team, it has been a long, hard and very rewarding summer. We wish we could continue our project further, but now, time is our greatest enemy. We hope that our research will pave the way for other groups, both within and without iGEM, to take the reins in the battle against catheter-associated UTIs, and the wider assault on antibiotic resistance.
+
                             The proven secretion of folded, functional proteins across both bacterial cell membranes is a challenge for present day microbiologists. Our solution requires that we can export DspB, DNase, Art-175 and MccS out of the expression host and into the local biofilm environment. To achieve this, signal sequences are fused to the enzymes to target them for export through natural <em>E. coli</em> secretion pathways. Using this mechanism we can direct our anti-biofilm and antimicrobial agents at a biofilm infected surface as they are being produced.
 
                         </p>
 
                         </p>
−
                        <p>
+
                         <div id="solution-sectreting-dsba">
−
                            We hope this page will be a useful guide to teams (in particular UK teams) with medically-orientated projects, who intend to progress from laboratory work into clinical trials. We have also written a brief guide to the process of patenting, and how this would apply to our project in the future.
+
                             <h4>DsbA</h4>
−
                        </p>
+
−
                         <div id="what-next-preclinical-stage">
+
−
                             <h3>Preclinical stage</h3>
+
 
                             <p>
 
                             <p>
−
                                 Like most iGEM teams with projects in the Health and Medicine track, we have spent the summer developing our project in the preclinical stage. This is the 'laboratory' stage, where the different components of the project are constructed, tested, and optimised, without the use of human volunteers. The first goal is to discover if the team's invention is feasible; past that point, the preclinical goal is to improve the invention to point the point at which its efficacy and safety of use is maximised.
+
                                 DsbA is a oxidoreductase protein found predominantly in Gram-negative bacteria, which functions as a protein-folding factor [<a href="#references">19, 20</a>]. The 2-19 peptide sequence of DsbA is a signal sequence that can direct passenger proteins for co-translational export via the signal recognition particle pathway [<a href="#references">21, 22</a>]. It has recently been shown that the DsbA signal sequence is capable of mediating passenger protein secretion under a selection of different induction temperatures [<a href="#references">23</a>].
 
                             </p>
 
                             </p>
−
                        </div>
 
−
                        <div id="what-next-progression-to-clinical-trials">
 
−
                            <h3>Progression to clinical trials</h3>
 
 
                             <p>
 
                             <p>
−
                                 Extensive preclinical (lab) data is needed to support the use of the treatment on humans in clinical trials. As described above, the researchers must do their best to minimise the level of risk associated with the treatment, whilst maximising its efficacy, through lab models.
+
                                 Parts:  <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1659002">BBa_K1659002</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1659201">BBa_K1659201</a>,<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1659301">BBa_K1659301</a>
−
                            </p>
+
−
                            <h4>Review bodies</h4>
+
−
                            <p>
+
−
                                Any group wishing to trial their treatment on volunteers in the UK must abide by the Medicines for Human Use (Clinical Trials) Regulations of 2004. To abide by these terms, researchers must be granted a clinical trial authorisation (CTA) by the Medicines and Healthcare Products Regulatory Agency (MHRA). For a CTA application to be granted, the treatment must be reviewed and certified by a number of different review bodies, where each body scrutinises a different aspect of the project. We will use our project as an example here.
+
−
                            </p>
+
−
                            <p>
+
−
                                Most medically-orientated projects require approval from a <strong>Research Ethics Committee</strong> (REC). The committee makes sure that the volunteers have been well briefed (by the researchers) with regards to the trial, including; the possible benefits and risks of the treatment, the goals of the treatment, and who they should contact if they have any further questions. There are several RECs, found across the UK, who deal with clinical trial authorisations. There are several borough-based RECs in London - the iGEM team would most likely apply to one of these bodies.
+
−
                            </p>
+
−
                            <p>
+
−
                                As our treatment involves the use of a novel device (our 'custom catheter'), we need to apply to the MHRA, for a <strong>Notice of No Objection</strong>, to review the safety of the device. If granted, the notice would allow the device to be used in volunteers.
+
−
                            </p>
+
−
                            <p>
+
−
                                Potentially, we may attempt to model the effect our bacterial proteins have on human cells. To get hold of human tissue, we would need a license from the <strong>Human Tissue Authority</strong> (HTA). This includes extraction of tissue from a cadaver and a live participant.
+
−
                            </p>
+
−
                            <p>
+
−
                                In addition to the above approvals, we would need an organisation, such as a hospital or research institute, to host our trials. There are several funding bodies to which we could apply; however, it would be ideal to have funding from the NHS, as this is where we would most probably intend to host our trials - this is called <strong>Management Permission</strong>. Ideally, we would like our trials to be hosted by the NHS! At the time of our Management Permission application, we would most likely apply to the NHS for funding as well.
+
 
                             </p>
 
                             </p>
 
                         </div>
 
                         </div>
−
                         <div id="what-next-clinical-trials">
+
                         <div id="solution-sectreting-yebf">
−
                             <h3>Clinical trials</h3>
+
                             <h4>YebF</h4>
 
                             <p>
 
                             <p>
−
                                 We would be very pleased if our project managed to get this far! However, there would still be a long way to go before our product could go on the market. The clinical trials are split into 3 phases, with each phase more rigorous than the last.
+
                                 YebF is a 13kDa protein of unknown function that is perhaps the only protein that has been conclusively documented to be secreted into the extracellular medium by a laboratory <em>E. coli</em> strain. At the N-terminus, YebF has a 2.2 kDa sec-leader sequence which mediates its translocation through the bacterial inner membrane via the Sec pathway, and is cleaved upon translocation into the periplasm to give the 10.8 kDa "mature" form [<a href="#references">24</a>]. Export from periplasm into the extracellular space takes places via the Omp pathway. YebF has been used successfully to mediate the secretion of recombinant proteins [<a href="#references">25,26</a>].
 
                             </p>
 
                             </p>
−
                            <h4>Phase I</h4>
 
 
                             <p>
 
                             <p>
−
                                 Phase I trials, also known as 'first-in-human' trials, present the first time a treatment is tested on human volunteers; as a result, there is an unavoidable element of risk involved.
+
                                 Part: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1659003">BBa_K1659003</a>
−
                                A typical phase I trial might involve 10 or fewer healthy student volunteers. However, due to the nature of our treatment, the likely candidates for our phase I trials would be people who already require the use of a catheter, but potentially not those who have a urinary tract infection (UTI).
+
−
                            </p>
+
−
                            <p>
+
−
                                This phase is primarily a risk assessment, so we would be looking for any signs of immune response to the introduction of our bacteria and their secreted proteins; in particular DNase and Dispersin B. In addition, we may be looking to see what range of protein dosages the volunteers might be able to tolerate before they experience significant side effects or discomfort.
+
−
                            </p>
+
−
                            <h4>Phase II</h4>
+
−
                            <p>
+
−
                                If the volunteers from phase I show few side effects and the treatment is deemed safe enough, then the trials can proceed to phase II. These involve up to approximately 100 volunteers. This time, all these people selected should be ill, ie have a CAUTI, and the results of their treatment (reduction in the biofilm size) against the best current treatment; in our case this is antibiotic treatment.
+
−
                            </p>
+
−
                            <h4>Phase III</h4>
+
−
                            <p>
+
−
                                If the results of the phase II trials suggest that the new treatment could be better than the current treatment, then the trials can proceed to phase III. As this is the last stage, the organisation hosting the trial must be sure, on a statistical level, that the new treatment is significantly better than the currently-administered treatment, for it to pass. Phase III trials therefore involve a very large group of ill volunteers, sometimes greater than 1000 people.
+
 
                             </p>
 
                             </p>
 
                         </div>
 
                         </div>
−
                         <div id="what-next-market-licensing">
+
                         <div id="solution-secreting-fla">
−
                            <h3>Market licensing</h3>
+
                             <h4>Fla</h4>
−
                            <p>
+
                             <p>Flagellin are the constituent subunits of the helical filament substructure of bacterial flagella. In the flagellar-building process, flagellin are exported out of the cell sequentially by the flagellum-specific export apparatus. F. Vonderviszt et al. demonstrated through their work that the signal sequence responsible for allowing the flagellar export system to identify and export Salmonella flagellin is its 26-47 amino acid residue segment [<a href="#references">18</a>].</p>
−
                                If <em>solUTIon</em> passed phase III, then we could officially license our treatment, and release it onto the market! Wouldn't that be swell?
+
−
                            </p>
+
−
                             <h4>Phase IV (optional)</h4>
+
−
                             <p>
+
−
                                This is an additional phase which is sometimes implemented; however it is not always required! Phase IV 'trials' are slightly different to the previous three, as they monitor the treatment success on real patients, ie <em>after</em> a marketing license has been granted. As the treatment has already been licensed by this point, phase IV simply involves analysis of data from patients receiving the treatment to check its safety and efficacy.
+
−
                            </p>
+
−
                        </div>
+
−
                        <div id="what-next-patenting">
+
−
                            <h3>Patenting</h3>
+
−
                            <h4>What is a patent?</h4>
+
−
                            <p>
+
−
                                A patent is a license, given to an inventor, which prevents any competitor from making, using or selling their invention in a certain territory for a specific time frame. The 'territory' of the patent may be a country, or even an entire continent, such as Europe. The patent 'time frame' is usually 20 years.
+
−
                            </p>
+
−
                            <h4>What is the point of a patent?</h4>
+
−
                            <p>
+
−
                                Patenting is essential to the healthy progression of scientific research; why would companies want to invest millions of dollars into an invention, only for it to be stolen by a competitor when completed? Therefore, patenting gives companies a chance to earn back the money they invest in their inventions. However, the patent holder may, during these 20 years, permit certain parties to make use of their product for a license fee.
+
−
                            </p>
+
−
                            <h4>What can you patent?</h4>
+
−
                            <p>
+
−
                                In synthetic biology, any organism (aside from a human) which has been genetically modified, and hence gives rise to "a new assembly of chemicals", can be patented. This includes novel bacterial strains, as well as transgenic plants and animals. Evidently, humans cannot be patented for ethical reasons.
+
−
                            </p>
+
−
                            <p>
+
−
                                However, there are additional factors that must be satisfied; for an invention to be applicable for patenting it must be both of the following:
+
−
                                <ol>
+
−
                                    <li>
+
−
                                        <strong>Novel</strong> - the invention you present should not already be part of the 'state of the art' (basically anything published in a paper, presented at a conference, or anything freely accessible to the scientific community).
+
−
                                    </li>
+
−
                                    <li>
+
−
                                        <strong>Inventive</strong> - ie the invention should not be 'obvious' to those skilled in the field. It is often difficult to define if an invention is 'obvious'; however, several 'obvious' inventions can arise from, for example, the correlation of two previously-unlinked scientific papers.
+
−
                                    </li>
+
−
                                </ol>
+
−
                            </p>
+
−
                            <h4>Can Oxford iGEM apply for a patent?</h4>
+
−
                            <p>
+
−
                                There are a number of aspects to our project which are unpatentable. For example, the BioBricks we have constructed cannot be patented; they have been submitted to <strong>The Registry of Standard Biological Parts</strong> ('The Registry' for short) as part of our open source agreement. The Registry is iGEM's alternative to patenting; it allows for easy, open distribution of the many hundreds of BioBricks that now exist. In addition, EU law states that <strong>methods</strong> of therapy, treatment and diagnosis cannot be patented, to avoid conflict with doctors.
+
−
                            </p>
+
 
                             <p>
 
                             <p>
−
                                 However, the device used <em>within</em> the method may be patented. Our method of tackling CAUTIs rests heavily upon the design of our custom-catheter, which is both novel and inventive; we therefore intend to apply for a patent on this device.
+
                                 Part: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1659001">BBa_K1659001</a>
 
                             </p>
 
                             </p>
 
                         </div>
 
                         </div>
Line 216: Line 238:
 
                 </div>
 
                 </div>
 
             </div>
 
             </div>
−
             <div class="col-md-3 contents-sidebar">
+
             <div class="section-spacer"></div>
−
                <ul id="sidebar" class="nav nav-stacked sm-hidden xs-hidden" data-spy="affix">
+
            <div class="section" id="delivery">
−
                     <li>
+
                <div class="slim">
−
                         <a href="#overview">Overview</a>
+
                    <h2>Delivery</h2>
−
                         <ul class="nav nav-stacked">
+
                    <p>
−
                             <li><a href="#overview-what">What?</a></li>
+
                        A major part of our project involves investigating what is the best way to deliver our biofilm-degrading and antimicrobial enzymes to the site of infection in the urinary tract.
−
                            <li><a href="#overview-why">Why?</a></li>
+
                    </p>
−
                            <li><a href="#overview-how">How?</a></li>
+
                    <p>
−
                         </ul>
+
                        As we have mentioned above, patients with recurrent, complicated cases of UTI often get their infections from an already-inserted catheter which may have to be there and cannot be removed for a variety of other medical reasons. In view of that, we decided to conceptualize an initial delivery method which was centered on the catheter.
−
                     </li>
+
                    </p>
−
                     <li>
+
                    <p>
−
                         <a href="#what-next">What Next?</a>
+
                        Our AlgiBeads design involves encapsulating our therapeutic, enzyme-secreting bacteria in sodium alginate beads. These beads are immobilized in a modified section of a catheter, from which the bacteria can secrete the therapeutic enzymes into the infected urinary tract. On our <a href="https://2015.igem.org/Team:Oxford/Design">Design</a> page, thorough consideration was given to the AlgiBeads delivery method, including issues of safety and practicality.
−
                        <ul class="nav nav-stacked">
+
                     </p>
−
                            <li>
+
                    <p>
−
                                <a href="#what-next-preclinical-stage">Preclinical stage</a>
+
                         However, based on some preliminary data obtained for gene expression and diffusion rates, our <a href="https://2015.igem.org/Team:Oxford/Modeling">computational models</a> predicted that the equilibrium concentration of enzymes in solution based on the AlgiBeads delivery method would be too low when compared against the known concentrations required for biofilm degradation.
−
                            </li>
+
                    </p>
−
                            <li>
+
                    <p>
−
                                <a href="#what-next-progression-to-clinical-trials">Progression to clinical trials</a>
+
                         As such, we have had to instead consider an alternative delivery method - the introduction of our enzyme-releasing therapeutic engineered bacteria into the urinary microbiome, whereby the problem of low enzyme concentration in solution will be overcome by the close proximity between the therapeutic bacteria and the pathogenic bacteria. Another benefit of having therapeutic bacteria as part of the microbiome is of course that the treatment becomes preventive in nature, with the therapeutic bacteria now part of the bacterial community in the body constantly releasing pathogen-killing enzymes.
−
                            </li>
+
                    </p>
−
                            <li>
+
                    <p>
−
                                <a href="#what-next-clinical-trials">Clinical trials</a>
+
                        Of course, altering the microbiome comes with its own set of hazards, and we hope to mitigate it at least in part by doubling up the pathogen-killing mechanism as a population control mechanism for the engineered bacteria as well:
−
                            </li>
+
                    </p>
−
                            <li>
+
                    <div class="image image-full" style="object-align:center">
−
                                <a href="#what-next-market-licensing">Market licensing</a>
+
                        <img src="https://static.igem.org/mediawiki/2015/7/79/Oxford-animatiom.gif">
−
                            </li>
+
                        <p>
−
                            <li>
+
                             How our 3-part engineered microbe works:
−
                                <a href="#what-next-patenting">Patenting</a>
+
                                <br>1. Constant secretion of biofilm-degrading enzyme
−
                            </li>
+
                                <br>2. Production and accumulation of antibacterial Art-175
−
                        </ul>
+
                                <br>3. Detection of pathogenic bacteria via quorum sensing
−
                     </li>
+
                                <br>4. Permeabilization of inner membrane by T4 Holin
−
                 </ul>
+
                                <br>5. Access and lysis of host cell wall by Art-175
 +
                                <br>6. Release of Art-175 and lysis of target cell
 +
                           
 +
                        </p>
 +
                    </div>
 +
                    <p>
 +
                        Art-175 is normally prevented from reaching the cell wall of the expression host by the inner membrane. When a large amount of pathogenic bacteria is present, the quorum sensing signals trigger the production of T4 Holin, which permeabilizes the inner membrane, allowing Art-175 to reach the cell wall and degrade it. This causes lysis of the host cell and releases the accumulated Art-175 in a single high-concentration pulse, killing the pathogenic bacteria and achieving population control of the expression host at the same time.
 +
                    </p>
 +
                    <p>
 +
                        Other safety aspects of this microbiome-modification design, including issues on immunogenicity, can be found <a href="https://2015.igem.org/Team:Oxford/UTB#Urinary_Tract_Biome">here</a>.
 +
                    </p>
 +
                </div>  
 +
            <div class="section-spacer"></div>
 +
            <div class="section" id="results">
 +
                <div class="slim">
 +
                    <h2>Results</h2>
 +
                    <p>
 +
                        Through our experimental work we were able to obtain preliminary evidence suggesting the validity of these points:
 +
                    </p>
 +
                    <ul>
 +
                        <li>DsbA-DNase and DsbA-DspB can be secreted in a fully folded and functional state</li>
 +
                        <li>Both DNase and DspB are able to degrade biofilms</li>
 +
                        <li>Art-175 is able to exert cell lytic activity on planktonic <i>E. coli</i> and <i>P. putida</i></li>
 +
                        <li>Art-175 is able to kill a portion of biofilm-encased <i>P. putida</i> cells</li>
 +
                    </ul>
 +
                    <br>
 +
                    <p>
 +
                        The results and in-depth discussion of our experimental work can be found on the <a href="https://2015.igem.org/Team:Oxford/Experiments">Experiments</a> page.
 +
                    </p>
 +
                   
 +
                </div>
 +
            </div>
 +
            <div class="section-spacer"></div>
 +
            <div class="section" id="improving-part-function">
 +
                <div class="slim">
 +
                    <h2>Improving Part Function</h2>
 +
                    <p>
 +
                         Improving the function of another team’s part: BBa_K729004
 +
                    </p>
 +
 
 +
                    <p>
 +
                        Team UCL 2012 also had a part comprising Staphylococcal DNase with a DsbA tag upstream of it. We were interested in finding out:
 +
                     </p>
 +
                     <ul>
 +
                        <li>Whether the DsbA 2-19 sequence is able to facilitate the export of this part of expression host organism E. coli MG1655</li>
 +
                         <li>Whether the Staphylococcal nuclease can degrade E. coli biofilms (it was shown to degrade S. aureus biofilms in <a href="http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005822">Mann et al, 2009</a>)
 +
                    </ul>
 +
                   
 +
                    <div class="image image-full">
 +
                        <img src="https://static.igem.org/mediawiki/parts/f/fd/Oxford15-dnase_SDS-PAGE_copycopy.jpg">
 +
                        <p>Figure 14: SDS-PAGE of <i>E. coli</i> MG1655 BBa_K729004 [pBAD], 0% ara supernatant (A) and E. coli MG1655 BBa_K729004 [pBAD], 0.2% ara supernatant (B)</p>
 +
                    </div>
 +
                   
 +
                    <p> Figure 14 shows the successful DsbA-directed secretion of DNase across both cell membranes. </p>
 +
                    <p> A is the supernatant of uninduced E. coli MG1655 BBa_K729004 [pBAD], whilst B is the supernatant of 0.2% induced E. coli MG1655 BBa_K729004 [pBAD]. The band is approximately 21 kDa, corresponding to the size of DsbA-DNase.</p>
 +
                   
 +
                    <div class="image image-full">
 +
                        <img src="https://static.igem.org/mediawiki/parts/5/5b/Oxford15-Bbak729004.png">
 +
                        <p>Figure 15: Expression host MG1655 BBa_K729004 [pBAD] biofilm growth assay </p>
 +
                    </div>
 +
                   
 +
                    <p> Figure 15 shows the effect of inducing the expression of BBa_K729004 [pBAD] on the ability of the host to form biofilms. The control (MG1655, pBAD/HisB, 0.2% ara) and MG1655, BBa_K729004[pBAD], 0% ara are both able to grow biofilms, as shown by the intensity of the crystal violet staining. When BBa_K729004[pBAD] is expressed, the intensity of the crystal violet staining is reduced, showing a diminished ability to grow biofilm. This data suggests that the secretion of DNase is able to inhibit biofilm formation. </p>
 +
                </div>
 +
            </div>
 +
            <div class="section-spacer"></div>
 +
            <div class="section" id="conclusion">
 +
                <div class="slim">
 +
                    <h2>Conclusion</h2>
 +
                    <p>
 +
                        Through our experimental work, we have successfully created and submitted 12 sequence-confirmed BioBrick parts, 7 of which we rigorously characterized for antibacterial and/or antibiofilm function. We validated that Art-175 and Microcin S are both potent antibacterials, the former of which is shown to be even capable of killing antibiotic-resistant biofilm-encased bacteria. On the antibiofilm side of things, we not only showed that the enzymes of interest, DNase and DspB, were successfully exported across both membrane layers of <i>E. coli</i> following our modification of them with secretion tags, but also proved that the enzymes are able to refold properly post-secretion such that they retain their enzymatic function.
 +
                    </p>
 +
                    <p>
 +
                        In conclusion, we achieved our aim of creating bacterial "living therapeutics" - strains of bacteria genetically engineered to secrete functional antibiofilm and antimicrobial proteins towards the treatment of UTIs.
 +
                    </p>
 +
                </div>
 +
            </div>
 +
            <div class="section-spacer"></div>
 +
            <div class="section" id="future">
 +
                <div class="slim">
 +
                    <h2>Future</h2>
 +
                    <p>
 +
                        To develop our project beyond a proof-of-concept design, we would adopt a more suitable chassis, such as <em>Lactococcus lactis</em>. <em>L. lactis</em> has been widely used as a expression host for the production of proteins in both the medical and food industries. Being a Gram-positive species of bacteria, it is less likely to be killed by the same mechanisms as major Gram-negative pathogens such as <i>E. coli</i> and <i>P. aeruginosa</i> (e.g. Art-175's peptidoglycan lysis ability is specific for Gram-negative bacteria). On top of that, being Gram-positive means that it will not pose the problems of endotoxicity brought about by the outer membranes of Gram-negative bacteria. Using <em>E. coli</em> as our host was purely a starting point, in view of its ease-of-use as well as availability of pre-existing resources.
 +
                    </p>
 +
                    <p>
 +
                        In addition to secreting antibiofilm/antimicrobial proteins, a comprehensive treatment for UTIs would be a bacteria engineered to also sense and move towards biofilms. We conducted extensive literature review on this in the early stages of the project but, due to the time restraints of a summer project, could not put our ideas into practice. With further work, we would incorporate both a sensing and chemotaxis mechanism into our design.
 +
                    </p>
 +
                    <p>
 +
                        Nurses, doctors and professors all raised to us the issue of targeting the multiple bacterial and fungal species that are involved in UTIs, highlighting the fact that the problem extends further than <em>E. coli</em> and <em>P. aeruginosa</em>. We have explored how we would approach this in the <a href="https://2015.igem.org/Team:Oxford/Practices">Practices</a> page.
 +
                    </p>
 +
                    <p>
 +
                        Beyond the scientific issues of implementation, thinking seriously about the questions of ethics and public acceptance is also crucial for the further development of synbio-based medical therapies especially in view of the fact that it is currently illegal to even bring genetically-modified organisms outside of the laboratory environment. We have explored this theme also in the <a href="https://2015.igem.org/Team:Oxford/Practices">Practices</a> page.
 +
                    </p>
 +
                </div>
 +
            </div>
 +
            <div id="references">
 +
                <h2>References</h2>
 +
                <ol class="references">
 +
                    <li>Global Report on Surveillance of Antimicrobial Resistance: 2014. WHO.</li>
 +
                    <li>Johnson, J.R., 2004. Laboratory diagnosis of urinary tract infections in adult patients. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 39(6), p.873; author reply 873–874.</li>
 +
                    <li>Zalewska-Piatek, B. et al., 2013. Biochemical characteristic of biofilm of uropathogenic Escherichia coli Dr+ strains. Microbiological Research, 168, pp.367–378.</li>
 +
                    <li>Sievert, D.M. et al., 2013. antibiotic-resistant pathogens associated with healthcare-associated infections: summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2009-2010. Infection control and hospital epidemiology : the official journal of the Society of Hospital Epidemiologists of America, 34(1), pp.1–14. Available at: <a href="http://www.ncbi.nlm.nih.gov/pubmed/23221186">http://www.ncbi.nlm.nih.gov/pubmed/23221186</a>.</li>
 +
                    <li>Fux, C. a. et al., 2005. Survival strategies of infectious biofilms. Trends in Microbiology, 13(1), pp.34–40.</li>
 +
                    <li>Flemming, H.-C. & Wingender, J., 2010. The biofilm matrix. Nature reviews. Microbiology, 8(9), pp.623–633. Available at: <a href="http://dx.doi.org/10.1038/nrmicro2415">http://dx.doi.org/10.1038/nrmicro2415</a>.</li>
 +
                    <li>Høiby, N. et al., 2010. Antibiotic resistance of bacterial biofilms. International Journal of antibiotic Agents, 35(4), pp.322–332.</li>
 +
                    <li>Briers, Y. et al., 2014. Art-175 is a highly efficient antibiotic against multidrug-resistant strains and persisters of Pseudomonas aeruginosa. antibiotic Agents and Chemotherapy, 58(7), pp.3774–3784.</li>
 +
                    <li>Gunzer, F., 2013. Bacterially-formed microcin S, a new antibiotic peptide, effective against pathogenic microorganisms, e.g. enterohaemorrhagic Escherichia coli (EHEC), European Patent EP2557163A1.</li>
 +
                    <li>Antibiotics - Side effects. Avaolable from: <a href="http://www.nhs.uk/Conditions/Antibiotics-penicillins/Pages/Side-effects.aspx">http://www.nhs.uk/Conditions/Antibiotics-penicillins/Pages/Side-effects.aspx</a> [5/06/2015]</li>
 +
                    <li>C. M. Kunin, “Urinary tract infections in females,” Clinical Infectious Diseases, vol. 18, no. 1, pp. 1–12, 1994.</li>
 +
                    <li>J. W. Warren, “Catheter-associated urinary tract infections,” Infectious Disease Clinics of North America, vol. 11, no. 3, pp. 609–622, 1997</li>
 +
                     <li>A. P. Lenz, K. S. Williamson, B. Pitts, P. S. Stewart, and M. J. Franklin, “Localized gene expression in Pseudomonas aeruginosa biofilms,” Applied and Environmental Microbiology, vol. 74, no. 14, pp. 4463–4471, 2008.</li>
 +
                    <li>L. Zhang and T. Mah, “Involvement of a novel efflux system in biofilm-specific resistance to antibiotics,” Journal of Bacteriology, vol. 190, no. 13, pp. 4447–4452, 2008.</li>
 +
                    <li>J. Klebensberger, A. Birkenmaier, R. Geffers, S. Kjelleberg, and B. Philipp, “SiaA and SiaD are essential for inducing autoaggregation as a specific response to detergent stress in Pseudomonas aeruginosa,” Environmental Microbiology, vol. 11, no. 12, pp. 3073–3086, 2009</li>
 +
                    <li>U. Römling and C. Balsalobre, “Biofilm infections, their resilience to therapy and innovative treatment strategies,” Journal of Internal Medicine, vol. 272, no. 6, pp. 541–561, 2012</li>
 +
                    <li>Hale, S.P., Poole, L.B. & Gerlt, J. a, 1993. Mechanism of the reaction catalyzed by staphylococcal nuclease: identification of the rate-determining step. Biochemistry, 32(29), pp.7479–7487</li>
 +
                    <li>Vondervizst, F., Sajó, R., Dobó, J., & Závodszky, P. (2012). The Use of a Flagellar Export Signal for the Secretion of Recombinant Proteins in Salmonella. In: Recombinant Gene Expression - Reviews and Protocols, Methods in Molecular Biology, 824, 131-143.</li>
 +
                    <li>Guddat, L.W., Bardwell, J.C. & Martin, J.L., 1998. Crystal structures of reduced and oxidized DsbA: investigation of domain motion and thiolate stabilization. Structure (London, England : 1993), 6(6), pp.757–767.</li>
 +
                    <li>Heras, B. et al., 2009. DSB proteins and bacterial pathogenicity. Nature reviews. Microbiology, 7(3), pp.215–225.</li>
 +
                    <li>Schierle, C.F. et al., 2003. The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway. Journal of Bacteriology, 185(19), pp.5706–5713.</li>
 +
                    <li>Steiner, D. et al., 2006. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. Nature biotechnology, 24(7), pp.823–831.</li>
 +
                    <li>Božić, N. et al., 2013. The DsbA signal peptide-mediated secretion of a highly efficient raw-starch-digesting, recombinant α-amylase from Bacillus licheniformis ATCC 9945a. Process Biochemistry, 48(3), pp.438–442.</li>
 +
                    <li>Zhang, G., Brokx, S. & Weiner, J.H., 2006. Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli. Nature biotechnology, 24(1), pp.100–104.</li>
 +
                    <li>Fisher, A.C. et al., 2011. Production of secretory and extracellular N-linked glycoproteins in Escherichia coli. Applied and Environmental Microbiology, 77(3), pp.871–881.</li>
 +
                    <li>Hwang, I.Y. et al., 2014. Reprogramming microbes to be pathogen-Seeking killers. ACS Synthetic Biology, 3(4), pp.228–237.</li>
 +
                    <li>Dramatic rise seen in antibiotic use. Available from: <a href="http://www.nature.com/news/dramatic-rise-seen-in-antibiotic-use-1.18383?WT.mc_id=TWT_NatureNews">http://www.nature.com/news/dramatic-rise-seen-in-antibiotic-use-1.18383?WT.mc_id=TWT_NatureNews</a> [17/09/2015]</li>
 +
                 </ol>
 
             </div>
 
             </div>
 +
        </div>
 +
</div>
 +
        <div class="col-md-3 contents-sidebar">
 +
            <ul id="sidebar" class="nav nav-stacked" data-spy="affix">
 +
                <li><a href="#overview">Overview</a></li>
 +
                <li><a href="#problem">The Problem</a></li>
 +
                <li><a href="#antibio">The Problem with Antibiotics</a></li>
 +
                <li><a href="#biofilms">Biofilms</a></li>
 +
                <li><a href="#utis">UTIs</a></li>
 +
                <li>
 +
                    <a href="#solution">The Solution</a>
 +
                    <ul class="nav nav-stacked">
 +
                        <li><a href="#solution-overview">Overview</a></li>
 +
                        <li><a href="#solution-degrading-biofilm">Degrading the Biofilm</a></li>
 +
                        <li>
 +
                            <a href="#solution-killing-bacteria">Killing the Bacteria</a>
 +
                            <ul class="nav nav-stacked">
 +
                                <li><a href="#solution-killing-bacteria-art175">Art-175</a></li>
 +
                                <li><a href="#solution-killing-bacteria-mccs">MccS</a></li>
 +
                            </ul>
 +
                        </li>
 +
                        <li>
 +
                            <a href="#solution-secreting">Secreting the Proteins</a>
 +
                            <ul class="nav nav-stacked">
 +
                                <li><a href="#solution-sectreting-dsba">DspA</a></li>
 +
                                <li><a href="#solution-sectreting-yebf">YebF</a></li>
 +
                                <li><a href="#solution-secreting-fla">Fla</a></li>
 +
                            </ul>
 +
                        </li>
 +
                    </ul>
 +
                </li>
 +
                <li>
 +
                    <a href="#delivery">Delivery</a>
 +
                </li>
 +
                <li>
 +
                    <a href="#results">Results</a>
 +
                </li>
 +
                <li><a href="#improving-part-function">Improving Part Function</a></li>
 +
                <li><a href="#conclusion">Conclusion</a></li>
 +
                <li><a href="#future">Future</a></li>
 +
            </ul>
 
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{{:Team:Oxford/Templates/Foot}}
 
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Latest revision as of 11:29, 20 November 2015

Description

Overview

"Years from now we will treat most infections with bacteria and not antibiotics."

Professor James Malone-Lee
Barlow Professor of Geriatric Medicine

A solution is urgently needed for the increasing number of infections caused by antibiotic-resistant bacteria. The engineering of bacterial cells to fight human pathogens is a promising alternative to administering traditional antibiotics. Our project involves the use of synthetic biology to engineer living therapeutics that have the potential to treat urinary tract infections (UTIs), which are a major public health concern in developed countries. This is largely due to growing antibiotic resistance.

The Problem

  • Antibiotics have negative side effects and their resistance is a growing problem
  • UTIs are the most common hospital-acquired infection and the bacteria that cause them are frequently resistant to antibiotics
  • Antibiotic resistance in UTIs is caused by biofilms
  • Biofilms are currently estimated to be responsible for over 65% of all hospital-acquired infections
  • Current UTI treatments are ineffective and fail to prevent recurrent infections

Problem with Antibiotics

Antibiotic use is associated with numerous negative side effects, allergies and reactions. The most common side effects of antibiotics all impact the digestive system and occur in around one in ten people. Around one person in fifteen has an allergic reaction to antibiotics, especially penicillin and cephalosphorins [10]. Half of the patients we spoke to during our project wanted us to find an alternative to using antibiotics, owing to the severity of the negative side effects that they experience.

Despite their negative side effects, antibiotics have been used so widely and for so long that the targeted microbes have adapted to become resistant, reducing the effectiveness of the drugs. A vicious cycle ensues in which ineffective antibiotic treatments leads to overprescription and overexposure, which amplifies the problem of antibiotic resistance. Global antibiotic consumption grew by 30% between 2000 and 2010. [27] Described by the Chief Medical Officer of England as “a threat equal to that of terrorism”, the growing resistance to antibiotics is hindering the effective prevention and treatment of an ever increasing range of infections.

antibiotic resistance poses a catastrophic threat. If we don't act now, any one of us could go into hospital in 20 years for minor surgery and die because of an ordinary infection that can't be treated by antibiotics.

Professor Dame Sally Davies
Chief Medical Officer
March 2013

The World Health Organisation’s antibiotic resistance Global Report on Surveillance, reports increasing worldwide incidences of antimicrobial resistance, in particular antibiotic resistance. This highlights very high rates of resistance in bacteria that cause common healthcare associated and community-acquired infections, such as UTIs [1]. UTIs account for over 7 million doctor visits per year. Catheter associated UTIs (CAUTIs) are the most commonly acquired infection in hospitals, and there is a high incidence of antibiotic resistance in the bacteria that cause UTIs globally [2].

Biofilms

Major structural elements of bacterial biofilms.

Biofilms are aggregates of surface-associated microorganisms that are encased in a matrix of highly-hydrated extracellular polymeric substances, which include extracellular polysaccharides, extracellular DNA, as well as proteins [6]. Van Leeuwenhoek, using his simple microscopes in 1684, first observed microorganisms on tooth surfaces and can be credited with the discovery of microbial biofilms. "The number of these animalcules in the scurf of a man's teeth are so many that I believe they exceed the number of men in a kingdom." - Leeuwenhoek

We now know a great deal more about biofilms. Environmental changes are responsible for the transition from planktonic growth to biofilm [13] and cause changes in the expression of surface molecules, virulence factors, and metabolic status. This allows the bacteria to acquire properties that enable their survival in unfavourable conditions [14,15], such as in the presence of antibiotics.

The low nutrient and oxygen levels at the bottom of the biofilm give rise to metabolically-inactive bacteria, better known as persister cells. These persister cells are rendered unsusceptible to most traditional antibiotics, which rely on bacterial metabolism to exert cell-killing effect [7].

UTIs

Biofilms are currently estimated to be responsible for over 65% of nosocomial infections and 80% of all microbial infections [16]. Bacterial biofilms play an important role in UTIs. UTIs are caused by the pathogenic invasion of the urinary tract, which causes an inflammatory response of the urothelium.

It is estimated that approximately 40% of women have had a UTI at some time in their lives [10]. UTIs may be caused by a variety of different organisms, most commonly bacteria. The most frequent cause of UTI in adult women is Escherichia coli, accounting for approximately 85% of community-acquired UTIs and 25-50% of hospital-acquired UTIs. Nosocomial infections may involve more aggressive organisms such as Pseudomonas aeruginosa and Enterobacter species.

The Solution

  • Break down bacterial biofilms to liberate the bacteria encased within and reduce the dose of antibiotics required
  • Directly kill the bacteria encased within the biofilms to provide an alternative to antibiotics

Overview

Our solution is focused on providing a treatment for UTIs because conventional antibiotics are unable to treat these and other biofilm-associated infections. Given the prevalence of such infections, there is a growing need for alternative therapeutic agents that can specifically degrade biofilms and kill the bacteria encased within. The use of synthetic biology to produce enzymes is the most effective way to achieve this specificity based on current technology. Our solution aims to investigate how bacterial biofilm disrupting proteins and antimicrobial proteins can be exported from E. coli and subsequently retain their antibiofilm/antimicrobial function. Using this secretion device we seek to create a system that offers persistent protection against biofilm formation.

We have designed a device that can exert antibiofilm and antimicrobial activity against E. coli and P. aeruginosa, the two leading causes of CAUTIs [4]. A nonpathogenic laboratory strain of E. coli is used as the expression host for the production of these enzymes as a proof-of-concept. The antibiofilm enzymes that we are using are Dispersin B and Micrococcal DNase, and the antimicrobial proteins that we are using are Art-175 and Microcin S.

Degrading the Biofilm

Prof. Malone-Lee stressed to us that sensitivity is a greater problem than complete antibiotic resistance. “Many more strains of bacteria are just insensitive to low doses of antibiotics, many can be overcome by high doses over long periods of time. Resistance is definitely not absolute.” Breaking down the biofilm increases the sensitivity of the bacteria embedded within it. Planktonic bacteria are metabolically active and are thus prone to antibiotics, meaning that lower doses are required.

DspB (BBa_K1659200) is an enzyme produced by Aggregatibacter actinomycetemcomitans, a species of bacteria found in the human oral cavity that grows almost exclusively in the form of biofilms. Structural analysis of Dispersin B showed that the enzyme only works specifically against the β-1,6-glycosidic linkages found in poly-N-acetylglucosamine, which is a polysaccharide structural element found in the biofilms of E. coli but not in P. aeruginosa. An additional enzyme would need to be used to target the polysaccharide component of P. aeruginosa biofilms.

Micrococcal DNase (BBa_K1659300) is an endo-exonuclease that non-specifically catalyzes the hydrolysis of single- and double-stranded DNA under basic conditions and in the presence of Ca2+ ions, and is known to be able to speed up DNA hydrolysis by up to 1016 times [17]. We are using DNase to break down the extracellular DNA component of biofilms.

Killing the Bacteria

Although antibiotic resistance is not absolute, it does pose a very big threat to the effective treatment of many infections. The insensitivity of bacteria to antibiotics can also be attributed to increasing antibiotic resistance. As described above, antibiotics also have many side effects that reduce patient quality of life and decrease the likelihood of completing a course of antibiotics. With all of this in mind, our solution does not only break down the biofilm, but also kills the bacteria embedded within so as to provide an alternative to antibiotics.

Art-175

Art-175 (BBa_K1659000) derive their name from “artificial endolysins”. Endolysins are bacteriophage-encoded peptidoglycan hydrolases that pass through the cytoplasmic membrane, degrading the peptidoglycan layer and inducing the lysis of the infected cell.

MccS

MccS (BBa_K1659100) is a type of microcin, a subclass of antibacterial proteins known as bacteriocins. Microcins are small, enterobacteria-produced bacteriocins that exert antibacterial activity against closely-related species, and MccS is produced by E. coli present in the probiotic drug Symbioflor 2 that has been shown to successfully treat gastrointestinal disorders.

For more information, please visit our Parts page.

Current clinically-relevant pathogens have not been seen to exhibit resistance against our antimicrobial proteins of choice. Art-175 has been experimentally shown to be not susceptible to resistance development, likely because it targets the structural element of the bacterial cell wall that is highly conserved across species and difficult to mutate [8]. The mechanism by which Microcin S exerts antimicrobial activity is still currently unknown, but no bacterial strains except for the original strain of probiotic E. coli which produces Microcin S has been shown to be resistant to it thus far [9].

Secreting the Proteins

The proven secretion of folded, functional proteins across both bacterial cell membranes is a challenge for present day microbiologists. Our solution requires that we can export DspB, DNase, Art-175 and MccS out of the expression host and into the local biofilm environment. To achieve this, signal sequences are fused to the enzymes to target them for export through natural E. coli secretion pathways. Using this mechanism we can direct our anti-biofilm and antimicrobial agents at a biofilm infected surface as they are being produced.

DsbA

DsbA is a oxidoreductase protein found predominantly in Gram-negative bacteria, which functions as a protein-folding factor [19, 20]. The 2-19 peptide sequence of DsbA is a signal sequence that can direct passenger proteins for co-translational export via the signal recognition particle pathway [21, 22]. It has recently been shown that the DsbA signal sequence is capable of mediating passenger protein secretion under a selection of different induction temperatures [23].

Parts: BBa_K1659002, BBa_K1659201,BBa_K1659301

YebF

YebF is a 13kDa protein of unknown function that is perhaps the only protein that has been conclusively documented to be secreted into the extracellular medium by a laboratory E. coli strain. At the N-terminus, YebF has a 2.2 kDa sec-leader sequence which mediates its translocation through the bacterial inner membrane via the Sec pathway, and is cleaved upon translocation into the periplasm to give the 10.8 kDa "mature" form [24]. Export from periplasm into the extracellular space takes places via the Omp pathway. YebF has been used successfully to mediate the secretion of recombinant proteins [25,26].

Part: BBa_K1659003

Fla

Flagellin are the constituent subunits of the helical filament substructure of bacterial flagella. In the flagellar-building process, flagellin are exported out of the cell sequentially by the flagellum-specific export apparatus. F. Vonderviszt et al. demonstrated through their work that the signal sequence responsible for allowing the flagellar export system to identify and export Salmonella flagellin is its 26-47 amino acid residue segment [18].

Part: BBa_K1659001

Delivery

A major part of our project involves investigating what is the best way to deliver our biofilm-degrading and antimicrobial enzymes to the site of infection in the urinary tract.

As we have mentioned above, patients with recurrent, complicated cases of UTI often get their infections from an already-inserted catheter which may have to be there and cannot be removed for a variety of other medical reasons. In view of that, we decided to conceptualize an initial delivery method which was centered on the catheter.

Our AlgiBeads design involves encapsulating our therapeutic, enzyme-secreting bacteria in sodium alginate beads. These beads are immobilized in a modified section of a catheter, from which the bacteria can secrete the therapeutic enzymes into the infected urinary tract. On our Design page, thorough consideration was given to the AlgiBeads delivery method, including issues of safety and practicality.

However, based on some preliminary data obtained for gene expression and diffusion rates, our computational models predicted that the equilibrium concentration of enzymes in solution based on the AlgiBeads delivery method would be too low when compared against the known concentrations required for biofilm degradation.

As such, we have had to instead consider an alternative delivery method - the introduction of our enzyme-releasing therapeutic engineered bacteria into the urinary microbiome, whereby the problem of low enzyme concentration in solution will be overcome by the close proximity between the therapeutic bacteria and the pathogenic bacteria. Another benefit of having therapeutic bacteria as part of the microbiome is of course that the treatment becomes preventive in nature, with the therapeutic bacteria now part of the bacterial community in the body constantly releasing pathogen-killing enzymes.

Of course, altering the microbiome comes with its own set of hazards, and we hope to mitigate it at least in part by doubling up the pathogen-killing mechanism as a population control mechanism for the engineered bacteria as well:

How our 3-part engineered microbe works:
1. Constant secretion of biofilm-degrading enzyme
2. Production and accumulation of antibacterial Art-175
3. Detection of pathogenic bacteria via quorum sensing
4. Permeabilization of inner membrane by T4 Holin
5. Access and lysis of host cell wall by Art-175
6. Release of Art-175 and lysis of target cell

Art-175 is normally prevented from reaching the cell wall of the expression host by the inner membrane. When a large amount of pathogenic bacteria is present, the quorum sensing signals trigger the production of T4 Holin, which permeabilizes the inner membrane, allowing Art-175 to reach the cell wall and degrade it. This causes lysis of the host cell and releases the accumulated Art-175 in a single high-concentration pulse, killing the pathogenic bacteria and achieving population control of the expression host at the same time.

Other safety aspects of this microbiome-modification design, including issues on immunogenicity, can be found here.

Results

Through our experimental work we were able to obtain preliminary evidence suggesting the validity of these points:

  • DsbA-DNase and DsbA-DspB can be secreted in a fully folded and functional state
  • Both DNase and DspB are able to degrade biofilms
  • Art-175 is able to exert cell lytic activity on planktonic E. coli and P. putida
  • Art-175 is able to kill a portion of biofilm-encased P. putida cells

The results and in-depth discussion of our experimental work can be found on the Experiments page.

Improving Part Function

Improving the function of another team’s part: BBa_K729004

Team UCL 2012 also had a part comprising Staphylococcal DNase with a DsbA tag upstream of it. We were interested in finding out:

  • Whether the DsbA 2-19 sequence is able to facilitate the export of this part of expression host organism E. coli MG1655
  • Whether the Staphylococcal nuclease can degrade E. coli biofilms (it was shown to degrade S. aureus biofilms in Mann et al, 2009)

Figure 14: SDS-PAGE of E. coli MG1655 BBa_K729004 [pBAD], 0% ara supernatant (A) and E. coli MG1655 BBa_K729004 [pBAD], 0.2% ara supernatant (B)

Figure 14 shows the successful DsbA-directed secretion of DNase across both cell membranes.

A is the supernatant of uninduced E. coli MG1655 BBa_K729004 [pBAD], whilst B is the supernatant of 0.2% induced E. coli MG1655 BBa_K729004 [pBAD]. The band is approximately 21 kDa, corresponding to the size of DsbA-DNase.

Figure 15: Expression host MG1655 BBa_K729004 [pBAD] biofilm growth assay

Figure 15 shows the effect of inducing the expression of BBa_K729004 [pBAD] on the ability of the host to form biofilms. The control (MG1655, pBAD/HisB, 0.2% ara) and MG1655, BBa_K729004[pBAD], 0% ara are both able to grow biofilms, as shown by the intensity of the crystal violet staining. When BBa_K729004[pBAD] is expressed, the intensity of the crystal violet staining is reduced, showing a diminished ability to grow biofilm. This data suggests that the secretion of DNase is able to inhibit biofilm formation.

Conclusion

Through our experimental work, we have successfully created and submitted 12 sequence-confirmed BioBrick parts, 7 of which we rigorously characterized for antibacterial and/or antibiofilm function. We validated that Art-175 and Microcin S are both potent antibacterials, the former of which is shown to be even capable of killing antibiotic-resistant biofilm-encased bacteria. On the antibiofilm side of things, we not only showed that the enzymes of interest, DNase and DspB, were successfully exported across both membrane layers of E. coli following our modification of them with secretion tags, but also proved that the enzymes are able to refold properly post-secretion such that they retain their enzymatic function.

In conclusion, we achieved our aim of creating bacterial "living therapeutics" - strains of bacteria genetically engineered to secrete functional antibiofilm and antimicrobial proteins towards the treatment of UTIs.

Future

To develop our project beyond a proof-of-concept design, we would adopt a more suitable chassis, such as Lactococcus lactis. L. lactis has been widely used as a expression host for the production of proteins in both the medical and food industries. Being a Gram-positive species of bacteria, it is less likely to be killed by the same mechanisms as major Gram-negative pathogens such as E. coli and P. aeruginosa (e.g. Art-175's peptidoglycan lysis ability is specific for Gram-negative bacteria). On top of that, being Gram-positive means that it will not pose the problems of endotoxicity brought about by the outer membranes of Gram-negative bacteria. Using E. coli as our host was purely a starting point, in view of its ease-of-use as well as availability of pre-existing resources.

In addition to secreting antibiofilm/antimicrobial proteins, a comprehensive treatment for UTIs would be a bacteria engineered to also sense and move towards biofilms. We conducted extensive literature review on this in the early stages of the project but, due to the time restraints of a summer project, could not put our ideas into practice. With further work, we would incorporate both a sensing and chemotaxis mechanism into our design.

Nurses, doctors and professors all raised to us the issue of targeting the multiple bacterial and fungal species that are involved in UTIs, highlighting the fact that the problem extends further than E. coli and P. aeruginosa. We have explored how we would approach this in the Practices page.

Beyond the scientific issues of implementation, thinking seriously about the questions of ethics and public acceptance is also crucial for the further development of synbio-based medical therapies especially in view of the fact that it is currently illegal to even bring genetically-modified organisms outside of the laboratory environment. We have explored this theme also in the Practices page.

References

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