Difference between revisions of "Team:Oxford/Design"

 
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    <div class="container-fluid page-heading" style="background-image: url(https://static.igem.org/mediawiki/2015/1/10/Ox_DesignHeader.jpeg)">
 +
        <h3>Design</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">
 
                 <div class="slim">
 
                 <div class="slim">
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                     <h2>Introduction</h2>
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                     <div class="section" id="preface">
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                    <p>
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                        <h2>Preface</h2>
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                        Our project relies on a three way conversation between the team, the public and experts. It touches every aspect of the project, from our choice of application to the details of our delivery system. We promoted Synthetic Biology and iGEM through outreach programs to inspire the next generation.
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                        <p>
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                    </p>
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                            This page comprehensively discusses the various aspects of our initial therapeutic delivery design idea, the AlgiBeads. Our subsequent and final delivery idea, the microbiome-modification design, can be found <a href="https://2015.igem.org/Team:Oxford/Description#delivery">here</a>, along with its safety considerations <a href="https://2015.igem.org/Team:Oxford/UTB">here</a>.
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                    <p>
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                        </p>
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                        Urinary tract infections are a huge problem globally with millions of cases reported each year. We’re producing a guide for everything you need to know about urinary tract infections, as well as a treatment to beat antibiotics, which are rapidly becoming ineffective.
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                    </p>
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                    <div class="section" id="introduction">
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                    <p>
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                        <h2>Introduction</h2>
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                        We want to make our website as accessible as possible to all readers, regardless of their level of expertise. Words with a <a class="definition" title="Dotted Blue Underline" data-content="Yep, just like this one.">dotted blue underline</a> will show a definition when you hover over them.
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                        <p>
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                    </p>
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                            Following on from all of the safety research we conducted, we put that knowledge into designing a catheter in order to get our proteins (DNase and DspB) into the urinary tract where we want them.
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                        </p>
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                        <p>
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            <div class="section-spacer"></div>
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                            Designing a novel method of getting our bacteria into the urinary tract was a major consideration during the beginning stages of our project. The most effective approach would probably be to deliver our bacteria directly through the catheter into the bladder. However, we found that the biofilm also forms on the outside of the catheter, so in our design we attempted to fight the biofilm from both outside and within.
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            <div class="section" id="project-choice">
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                        </p>
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                <div class="slim">
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                        <div class="quote quote-full">
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                    <h2>Project Choice</h2>
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                    <div id="project-choice-approaching-the-public">
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                            <h3>Approaching the Public</h3>
+
 
                             <p>
 
                             <p>
−
                                 To decide on our project idea, we sent out an initial questionnaire to the public to hear about what they thought about synthetic biology. We asked what big problems they wanted solving. We took the questionnaire to schools, to the streets and to our friends.
+
                                 The ability to disperse biofilms formed by multidrug-resistant bacteria adds a major new weapon to the limited arsenal of therapies available today.
 
                             </p>
 
                             </p>
 +
                            <h3>Neville Kallenbach<br> Professor of Chemistry at New York University NYC</h3>
 +
                        </div>
 +
                        <p>
 +
                            While our bacteria could potentially be applied industrially in various pipes to tackle a growing world problem with biofilms, we instead decide to focus our efforts on a medical application for them. We chose to tackle the problem of urinary tract infections as a member of our team, George Driscoll, had seen first hand the extreme impact it can have on people’s lives – especially women.
 +
                        </p>
 +
                        <p>
 +
                            Our catheter would have a three-pronged attack on the biofilm. Firstly it would be able to eliminate the biofilm forming in the lining of the bladder, then prevent biofilm from forming on the outside of the catheter itself, and finally by attacking the biofilm trying to form on the inside walls of the catheter.
 +
                        </p>
 +
                        <p>
 +
                            Our initial research into the current designs of catheter began online, where we began to get a better understanding of the scale we were working with. We looked into the problems of insertion, removal and general life with having a catheter in place. Throughout the design process we constantly kept these issues in mind in order to create something that would not only help with infection, but would also be practical for the patient, doctor and manufacturer.
 +
                        </p>
 +
                        <p>
 +
                            To get some more first hand experience of how small a catheter is, we purchased our very own. This struck home for us how small our containment method would have to be, with the typical internal volume being 43mm<sup>3</sup> and 23mm<sup>3</sup> for female and male catheters respectively. As UTIs mainly affect women we decided to buy a 14F female Foley catheter, which gave us a much better idea of the size we were working with: i.e. very small. Knowing this informed our choice of chemical for making containment beads for our bacteria. We also managed to obtain a few catheters from the local hospital. This opened our eyes to the sheer range of different catheter forms there were: different sizes, different materials, and different pipe configurations. Ultimately this lead us to choose to design a 3-way catheter.
 +
                        </p>
 +
                        <div class="image image-full">
 +
                            <img src="https://static.igem.org/mediawiki/2015/e/e9/Ox_FrenchCatheterScale.png" />
 
                             <p>
 
                             <p>
−
                                 Examples of their suggestions for the applications of synthetic biology include bacteria which:
+
                                 Catheter diameters based on the French Catheter scale. Rangeing from 1mm to 1.13cm.
−
                            </p>
+
−
                            <ul>
+
−
                                <li>Remove carbon dioxide from the atmosphere</li>
+
−
                                <li>Target and kill cancerous cells</li>
+
−
                                <li>Help treat Alzheimer's disease</li>
+
−
                                <li>Produce energy</li>
+
−
                                <li>Sew up holes in clothes</li>
+
−
                                <li>Produce teeth glue</li>
+
−
                                <li>Indicate how long someone has been dead for</li>
+
−
                                <li>Combat antibiotic resistance</li>
+
−
                            </ul>
+
−
                            <p>
+
−
                                Of our responses, around 40 were related to Medicine and Health [<a href="#PRef1">1</a>]. This led us to choose that track for our project. However, it was our team member George Driscoll’s work at the UTI clinic in London which helped us to select UTIs as a specific cause. Due to the un-aesthetic nature of the infection, it often receives less attention with regard to research.
+
−
                            </p>
+
−
                            <p>
+
−
                                A large proportion of our responses expressed concern for how Synthetic Biology would be used in society, with several references to the issues of contamination and exploitation for profit. With this in mind, we constructed a second questionnaire about our project, to test whether the public would get behind it.
+
 
                             </p>
 
                             </p>
 +
                        </div>
 +
                        <p>
 +
                            The idea of having a containment method as part of our project was first realised during the safety research as it would be dangerous to allow free bacteria into the human body. We started to incorporate the idea of a containment method into our project as we first realized during safety research that allowing free-living bacteria into the human body could potentially be dangerous. The bacteria could mutate, and lose and reacquire new genes, with no way for us to predict what could happen in several generations. There was a real chance of there being a negative impact on the patient’s health – perhaps fatally, depending on how badly we miscalculated our approach.
 +
                        </p>
 +
                        <p>
 +
                            However, if we chose to seal our beads within the catheter then we would not be able to access them again as long as the catheter was left in place – doing so could allow for foreign bacteria to enter the urinary tract and cause further infection. Therefore, any nutrients our bacteria needed would have to be present inside the beads, or be available from the urine running through the catheter.
 +
                        </p>
 +
                        <p>
 +
                            The bacteria would potentially need to be able to survive for 3 months – so ideally we would have been able to leave some beads for 3 months in the lab to test their longevity. Sadly the timescale of the project did not allow us to do that as it was only until the later stages of the project that we came up with the idea of bead encapsulation. We also would have liked to have tested whether the bead could retain its shape and integrity outside of the CaCl<sub>2</sub> for 3 months, as well as potential long term storage methods for the beads. Freezing the beads proved unsuccessful for us, but storing them in a cold room gave us promising results.
 +
                        </p>
 +
                        <p>
 +
                            Another important consideration is that the gel we used had to be non-toxic to humans. Many medical devices are made from silicone due to its inert nature and the fact that it does not cause any allergies or side effects – this material seemed ideal when bearing in mind the safety aspect of our project. The fact that catheters themselves are made from it shows that silicone polymers are safe for use inside the human body.
 +
                        </p>
 
                     </div>
 
                     </div>
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                        <div id="project-choice-approaching-the-public-initial-feedback">
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                </div>
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                              <h4>Initial Feedback</h4>
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                <div class="section-spcaer"></div>
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                              <p>
+
                <div class="image-massive">
−
                                  We sent a second questionnaire to find out more about whether the public would use a Solution from synthetic biology to treat Urinary tract infections. We asked more about whether they had heard of genetic engineering or synthetic biology, and how much they trust a recommended treatment by a doctor. In collaboration with UCL, we also filmed some of these responses on the street. The results were overwhelmingly positive.
+
                    <img src="https://static.igem.org/mediawiki/2015/4/46/Ox_RiaLarge.jpeg" />
−
                              </p>
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                </div>
 +
                <div class="section-spacer"></div>
 +
                <div class="section" id="catheter">
 +
                    <div class="slim">
 +
                        <h2>Catheter Design</h2>
 +
                        <p>
 +
                            There are 3 main parts in the design of a catheter; these parts are discrete and modular so therefore anything from one to all three could be implemented.
 +
                        </p>
 +
                        <div class="image image-full">
 +
                            <img src="https://static.igem.org/mediawiki/2015/b/b2/Ox_CatheterDesign.png" />
 
                         </div>
 
                         </div>
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                     <div id="project-choice-our-inspiration">
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                        <p>
−
                            <h3>Our Inspiration</h3>
+
                            This would be the overall design of the catheter with all three elements. It was designed while taking into account all the safety research we carried out.
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                            <p>
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                        </p>
−
                            </p>
+
                     </div>
 +
                </div>
 +
                <!-- <div class="slim"> -->
 +
                <div class="section-spacer"></div>
 +
                <div class="section" id="beads">
 +
                    <div class="slim">
 +
                        <h2>AlgiBeads</h2>
 
                     </div>
 
                     </div>
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                        <div id="project-choice-our-inspiration-jorge-talk">
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                    <div class="section" id="beads-intro">
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                             <h4>Jorge Talk</h4>
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                        <div class="slim">
 +
                             <h3>Introduction</h3>
 
                             <p>
 
                             <p>
 +
                                In order to stop the biofilm forming on the inside of the catheter it will contain beads that have our bacteria encapsulated inside of them however the protein is still able to diffuse out. This is needed as a way to contain the bacteria so they won’t be free in the urinary tract and therefore cause potential health problems for the patient.
 
                             </p>
 
                             </p>
−
                        </div>
 
−
                        <div id="project-choice-our-inspiration-churchill">
 
−
                            <h4>Churchill Hospital, Oxford</h4>
 
 
                             <p>
 
                             <p>
−
                                 Our first visit to the hospital was to the outpatient clinic during which we spoke with Jan, one of the nurses on the ward. Jan told us about a case of a person getting septicaemia as a result of a urinary infection. The patient had received antibiotics for seven days and had come back for a check up. Their urine sample was clear and all seemed fine but then the patient had started to shake. The bacteria were now in their blood as it had travelled back up the ureter to the kidney. Even though this was a rare case, it was shocking to hear about such a serious case and made our project feel very relevant.
+
                                 Below is a diagram of our cells secreting proteins out of the beads that they are contained in.
 
                             </p>
 
                             </p>
 +
                            <div class="image image-full">
 +
                                <img src="https://static.igem.org/mediawiki/2015/6/6a/OxiGEM_Beads_Description_Diagram.png" />
 +
                            </div>
 
                             <p>
 
                             <p>
−
                                 Jan also made the following points:
+
                                 These beads could also be contained outside the body inside a bag of sterile water; this bag would then be plugged into a 3-way Foley catheter. This solution would then be washed through the catheter and into the bladder, therefore tackling any infection that may be present in the lining of the bladder.
 
                             </p>
 
                             </p>
−
                             <ul>
+
                             <div class="image image-full">
−
                                 <li>People with infections have a catheter because they need a way to empty the bladder; else the urine travels up the ureter and back into the bladder</li>
+
                                 <img src="https://static.igem.org/mediawiki/2015/0/00/Ox_BeadDesign.jpeg" />
−
                                <li>If a patient becomes septic the catheter has to be removed or can be fatal</li>
+
                                 <p>First attempt at making the beads using Sodium Algniate</p>
−
                                <li>UTIs are not just contracted by the catheter and it is important to also consider community based UTIs</li>
+
                             </div>
−
                                <li>“UTIs are very common and can be quite painful”</li>
+
−
                                <li>No separate ward for UTIs – they are treated in every ward</li>
+
−
                                 <li>The protocol for treatment is to take a urine sample, see if there is an infection, and prescribe antibiotics that the bacteria are most sensitive to</li>
+
−
                                <li>Elderly hospital wards are likely to have many cases of UTIs</li>
+
−
                             </ul>
+
 
                             <p>
 
                             <p>
−
                                 We took a lot from this initial conversation. We went onto investigating the pros and cons of the current methods of treating urinary infections and compared these to what Solution could offer. We realized that we needed to consider the catheter more from a hospital/medical perspective as up to this point we had confused its function, thinking it was more to do with administering medication rather than emptying the bladder. Following this meeting, the design of the catheter became an integral part of the project.
+
                                 To see our process of designing and making the beads, look <a href="https://2015.igem.org/Team:Oxford/Beads">here.</a>
 
                             </p>
 
                             </p>
 
                         </div>
 
                         </div>
−
                        <div id="project-choice-our-inspiration-jr">
+
                    </div>
−
                            <h4>John Radcliffe Hospital, Oxford</h4>
+
                    <div class="section-spacer"></div>
−
                            <p>
+
                    <div class="section" id="beads-proof">
−
                                We still wanted to learn more about urinary infections as well as to get some feedback from nurses abourtour idea. We organized a trip to the Adams Ward (Geratology) to learn more about how urinary infections affect elderly people.
+
                        <div class="slim">
−
                             </p>
+
                             <h3>Proof of Principle</h3>
−
                             <div id="project-choice-our-inspiration-jr-evans">
+
                             <div id="beads-proof-chem">
−
                                 <h4>First interview with Laura Evans, Adams Ward</h4>
+
                                 <h4>Chemistry in Making the Beads</h4>
 
                                 <p>
 
                                 <p>
−
                                     What is the procedure for treating UTIs?
+
                                     The gel created to encapsulate the bacteria is made of Calcium Alginate; this is synthesised when aqueous Sodium Alginate solution is dropped into Calcium Chloride solution.
 
                                 </p>
 
                                 </p>
−
                                <ol class="interview-response">
 
−
                                    <li><em>Dip urine</em></li>
 
−
                                    <li><em>If the test comes back as positive, treat with a wide spectrum antibiotic</em></li>
 
−
                                    <li><em>Whether or not the catheter is inserted with prophylactic antibiotic treatment is the doctor’s decision</em></li>
 
−
                                </ol>
 
 
                                 <p>
 
                                 <p>
−
                                     What happens when a patient tests positive for a urinary infection?
+
                                     Calcium Alginate is a water-insoluble gelatinous polymer; therefore it forms beads when the sodium ions are exchanged with the calcium ions. Each calcium ion can bond with two alginate polymer chains; this is called cross-linking. As the sodium ions can only bind to one polymer this cross-linking doesn’t occur and the polymer is water soluble, so the gel does not form.
 
                                 </p>
 
                                 </p>
−
                                 <p class="interview-response">
+
                                 <div class="image image-full">
−
                                     <em>Whether or not the catheter is removed if a patient tests positive for a urinary infection depends largely on the reason that the catheter has been fitted. On the whole, the catheter remains fitted and the patient is treated with a large dose of antibiotics.</em>
+
                                     <img src="https://static.igem.org/mediawiki/2015/9/9b/Ox_Reaction_Beads.png" />
 +
                                </div>
 +
                            </div>
 +
                            <div class="section-spacer"></div>
 +
                            <div id="beads-proof-tute">
 +
                                <h4>Tutorial Video</h4>
 +
                                <video class="video" poster="https://static.igem.org/mediawiki/2015/7/79/Screen_Shot_2015-09-18_at_23.27.02_copy.jpg" controls>
 +
                                    <source src="https://static.igem.org/mediawiki/2015/6/6c/Ria_beads.mp4" type="video/mp4"/>
 +
                                </video>
 +
                            </div>
 +
                            <div class="section-spacer"></div>
 +
                            <div id="beads-proof-data">
 +
                                <h4>Experimental Data</h4>
 +
                                <p>
 +
                                    Firstly, we wanted to show that we could get the bacteria inside of the beads. To do this we created beads that contained fluorescent bacteria. The bacteria we used were from the interlab study: 20K MG, 20K ∆F, and 20K DH5, with MG(-) as a negative control.
 
                                 </p>
 
                                 </p>
 +
                                <div class="image image-left">
 +
                                    <img src="https://static.igem.org/mediawiki/2015/b/b0/Ox_beadfluorall.jpeg" />
 +
                                </div>
 
                                 <p>
 
                                 <p>
−
                                     Is antibiotic resistance a problem?
+
                                     We made sets of the beads for 5 days while measuring the fluorescence using the GFP protocol on the FLUOstar Omega plate reader.
 
                                 </p>
 
                                 </p>
−
                                 <p class="interview-response">
+
                                 <p>
−
                                     <em>Yes, particularly on this ward. As we treat elderly patients with recurring infections, their UTIs are frequently resistant to antibiotic treatment. We try different combinations of antibiotics but recurring infections are a significant problem.</em>
+
                                     The data shows that the wells that contain beads made with encapsulated fluorescent bacteria have a much higher fluorescence than the beads made without any bacteria and the beads made with the negative control bacteria. Therefore, we concluded that the bacteria are encapsulated inside of the beads.
 
                                 </p>
 
                                 </p>
 
                                 <p>
 
                                 <p>
−
                                     Our project involves designing a catheter that prevents the formation of a biofilm on its surface. What do you think of this idea?
+
                                     We then made beads using Crystal Violet dye in order to record the diffusion of the dye out of the bead. The data could then be related to the diffusion of the protein via dimensional analysis for our modelling.
 
                                 </p>
 
                                 </p>
−
                                 <p class="interview-response">
+
                                 <p>
−
                                     <em>A catheter like that would be useful, but it depends on how long your catheter would work for. Patients can have catheters fitted for 3 months or longer. Catheters are also removed for other reasons, for example if they become blocked. Blockage is particularly an issue when the patient is suffering from a urinary infection.</em>
+
                                     We collected the following spectrophotometric absorption data which we calibrated to prepared concentration standards.
 
                                 </p>
 
                                 </p>
 +
                                <div class="image image-full">
 +
                                    <img src="https://static.igem.org/mediawiki/2015/3/37/Ox_diffusionbead.jpeg" />
 +
                                </div>
 
                                 <p>
 
                                 <p>
−
                                     Laura’s response regarding how long a catheter remains in place spurred us into researching how we could keep our Solution bacteria alive. This is what we found.
+
                                     (Further analysis of this can found in our <a href="https://2015.igem.org/Team:Oxford/Modeling">modeling</a> section.)
 +
                                </p>
 +
                                <p>
 +
                                    Next we tried to determine whether the bacteria were alive inside the beads and could therefore grow and continue to secrete our proteins.
 +
                                </p>
 +
                                <p>
 +
                                    To do this we made up beads containing the 4 different types of bacteria and placed these bacteria into wells of a 96-well plate containing M9 media. We measured the fluorescence of each well every 15 minutes over 20 hours at 37˚C.
 +
                                </p>
 +
                                <p>
 +
                                    This experiment produced very promising data that suggests the bacteria are growing inside the beads and producing GFP. Therefore we can assume that our bacteria would be able to grow and produce our anti-biofilm agents while encapsulated. The graph shows that the fluorescence of the 3 beads containing the fluorescent bacteria increased over time, and that the bead containing no bacteria remain constantly low. The bead containing our negative control had a very slight increase in fluorescence over time but much less than the bacteria containing the gene for GFP. This could be due to some contamination during the process of making the beads.
 
                                 </p>
 
                                 </p>
−
                            </div>
 
−
                        </div>
 
−
                    <div id="project-choice-the-problem">
 
−
                            <h3>The Problem</h3>
 
−
                            <p>
 
−
                                Following our inspiring visits to the Churchill and John Radcliffe hospitals, we wanted to find out the public thought about the problem of antibiotic resistance. We posed the question:
 
−
                                <em>To what extent do you feel that antibiotic resistance is a problem that needs addressing in society today?</em>
 
 
                                 <div class="image image-full">
 
                                 <div class="image image-full">
−
                                     <img src="https://static.igem.org/mediawiki/2015/d/dd/Ox_Q2_antibiotic_resistance.png">
+
                                     <img src="https://static.igem.org/mediawiki/2015/7/74/Ox_BeadsGrowth.png" />
 
                                 </div>
 
                                 </div>
 
                                 <p>
 
                                 <p>
−
                                     Our survey clearly shows that, according to the general public, antibiotic resistance is an important problem that needs solving. Therefore we felf it was a useful area to direct our project towards. One of our team members, George, worked in a UTI clinic over the summer of 2014, so he knows first-hand that UTIs are a big problem for a lot of people and that, in severe cases, current methods of treatment are inadequate. When he brought this to our attention, we thought it was worthwile trying to find a solution to this problem.
+
                                     We put some consideration into the storage of our beads as hospitals would need to have a supply of them in order to carry out treatment effectively without having to make the beads and use them in a specific amount of time. This would cause delay in treatment and also a lot of waste of materials.
 
                                 </p>
 
                                 </p>
−
                            </p>
+
                                <p>
−
                    </div>
+
                                    Our first idea was to freeze the beads, however when they are frozen the structure of the alginate is compromised and the skeleton of the beads begin to break down. Therefore, we looked at storing the beads at 4˚C and then investigated whether or not the bacteria would again secrete GFP after being warmed up to 37˚C.
−
                        <div id="project-choice-the-problem-experts">
+
                                </p>
−
                            <h4>Feedback from Oxford Experts</h4>
+
                                <div class="image image-right">
−
                            <p>
+
                                    <img src="https://static.igem.org/mediawiki/2015/2/23/Ox_ColdBeads.png" />
−
                              To gain a further insight into the feasibility of Solution, we gave two talks during the summer, one at the termly Corpus Christi College Biochemistry talks and another to a group of alumni from the Oxford Biochemistry department. Two important questions arose from these talks:
+
                                </div>
−
                            </p>
+
                                <p>
−
                            <ul>
+
                                    From this we obtained some promising data. Beads stored in the cold room for 20 days were taken out and warmed to 37˚C. Then we measured the fluorescence of these beads every 15 minutes for 20 hours. The graph of this data shows that fluorescence does increase over time, indicating that the bacteria are growing inside of the beads and continuing to produce GFP.
−
                              <li>Have you considered whether the proteins you planning on secreting are immunogenic?</li>
+
                                </p>
−
                              <li>If you are to kill all of the pathogenic bacteria in the urinary tract, will that make fungal infections more likely?</li>
+
                                <p>
−
                             </ul>
+
                                    An initial issue with our beads was that the beads had to be kept immersed in media in order to ensure a supply of nutrients to the encapsulated bacteria. To get around this, we tried making beads with our media as the solvent so the beads would have media encapsulated inside them, and thus the bacteria could still survive if the beads were placed into water/urine/etcetera. This internal nutrient supply would help to increase the longevity of the bacteria over the duration of the treatment, with the result that the catheter would have to be replaced less frequently - thus reducing the discomfort and risk of infection for the patient.
 +
                                </p>
 +
                                <p>
 +
                                    The beads were made up using 30mL of LB + Chl and 0.36g of sodium alginate. We then placed the resulting beads into the plate reader and measured the fluorescence of these beads every 15 minutes for 20 hours. We plotted the data on the graph below:
 +
                                </p>
 +
                                <div class="image image-full">
 +
                                    <img src="https://static.igem.org/mediawiki/2015/4/46/Ox_mediagrowth.png" />
 +
                                </div>
 +
                                <p>
 +
                                    The graph demonstrates that the bacteria do grow inside of the beads, but they reach stationary phase much quicker than the beads what were surrounded by media. This could be due to the lower concentration of nutrients available.
 +
                                </p>
 +
                                <div class="image image-left">
 +
                                    <img src="https://static.igem.org/mediawiki/2015/0/0a/Ox_ConfocalBeads.png" />
 +
                                </div>
 +
                                <br>
 +
                                <p>
 +
                                    We then took a bead and looked at it underneath a confocal microscope in order see into the bead. This gave us some incredible images of the bacteria fluorescing within the beads.
 +
                                </p>
 +
                                <p>
 +
                                    This photo shows clearly that GFP-producing bacteria are encapsulated inside the bead. This is our main evidence that we can get bacteria to survive in the beads.
 +
                                </p>
 +
                                <p>
 +
                                    Using confocal microscopy, we also took images of multiple layers of a bead and stacked them together to build up a 3D image of the bead and the bacteria encased within.
 +
                                </p>
 +
                                <div class="image image-full">
 +
                                    <img src="https://static.igem.org/mediawiki/2015/f/f7/Ox_stacked.png" />
 +
                                </div>
 +
                                <p>
 +
                                    We then began to probe the edge of the bead with the miscroscope to see whether an bacteria had begun to leak out. From the image below we can see that there is a definition between the edge of the bead, where inside you can see the bacteria fluorescing and outside you cannot.
 +
                                </p>
 +
                                <div class="image image-full">
 +
                                    <img src="https://static.igem.org/mediawiki/2015/b/be/Ox_BeadDivide.png" />
 +
                                </div>
 +
                            </div>
 +
                        </div>
 +
                        <div class="section-spacer"></div>
 +
                        <div class="slim">
 +
                            <div id="beads-proof-next">
 +
                                <h4>The Next Steps</h4>
 +
                                <p>
 +
                                    If we had longer to work on our beads there are several things we could attempt in order to improve their viability.
 +
                                </p>
 +
                                <p>
 +
                                    The first would be to try and coat the beads and provide a second physical barrier to prevent the escape of the bacteria. We had the idea of coating them in a second layer of the calcium alginate, or to have another chemical in the Calcium Chloride bath that would instantly form a coating around the bead as it formed. However, this would require much more complex chemistry and material science - beyond what we could attempt in 10 weeks.
 +
                                </p>
 +
                                <p>
 +
                                    Ideally we would also make the beads much smaller, giving the beads a smaller volume, and so more would be able to fit inside of the catheter – while simultaneously improving the rate of diffusion out of the beads due to the relative increase in surface area. This would require a needle with a smaller lumen. The approach of making the beads could easily be scaled up so they could be produced in bulk.
 +
                                </p>
 +
                             </div>
 +
                        </div>
 +
                        <div class="image-massive">
 +
                            <img src="https://static.igem.org/mediawiki/2015/2/2c/Ox_BeadStacked.png" />
 
                         </div>
 
                         </div>
−
                    <div id="project-choice-our-solution">
 
−
                          <h3>Our Solution</h3>
 
−
                          <p>
 
−
                          </p>
 
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
−
            </div>
+
                <!-- </div> -->
−
            <div class="section-spacer"></div>
+
                <div class="section-spacer"></div>
−
            <div class="section" id="project-viability">
+
 
                 <div class="slim">
 
                 <div class="slim">
−
                    <h2>Project Viability</h2>
+
                     <div class="section" id="semi">
−
                    <p>
+
                         <h2>Semi-Permeable Membrane</h2>
−
                        Now that we had decided on our project choice, we needed to find out if it wolud be feasible and, if so, what obstacles we wolud need to overcome.
+
−
                    </p>
+
−
                     <div id="project-viability-bacterial-sustainability">
+
−
                         <h3>Bacterial Sustainability</h3>
+
 
                         <p>
 
                         <p>
−
                          We made our first steps in the right direction after visiting the Bedford Ward at the John Radcliffe Hospital. We spoke with one of the catheterised patients there called Mavis. She admitted to having had urinary infections in the past, but said she had not contracted a UTI since having a catheter fitted.
+
                            We would use a selectively permeable membrane to contain the beads within a compartment inside the catheter. This membrane would also provide an additional level of physical defence against our bacteria escaping into the urinary tract. The membrane would be similar to that used in a dialysis machine, and thus would allow urine and protein to diffuse through while preventing the larger bacteria from passing. Our largest protein is approximately 40kDa, so the diameter of the membrane pores would have to be able to accommodate this, as well as the larger components of urine (in order to prevent damning and a build up behind the membrane, causing complications). We would most likely be using one of the many materials already used for dialysis tubing membranes: polysulfone, polyethersulfone, etched polycarbonate, or collagen.
 
                         </p>
 
                         </p>
 
                         <p>
 
                         <p>
−
                          We found that Mavis would possibly use the same catheter for up to 10 weeks. This enforced the importance of being able to keep our bacteria alive for a sustained period of time. When we asked her about treating infection with bacteria she said she would be happy to if it had been recommended to her by a doctor and told us that it is not dissimilar to using antibiotics.
+
                            After considering the various options, we decided that for our catheter polysulfone would be best as it is most the commonly used in the medical profession and in dialysis machines. It is also reasonably simple to control the size of the pores during synthesis and thus produce the desired pore size.
−
                        </p>
+
−
                    </div>
+
−
                    <div id="project-viability-ethics">
+
−
                        <h3>Ethics</h3>
+
−
                        <p>
+
−
                            To find out the public opinion on our project, we sent out a questionnaire to over 150 people, asking: <em>'If your doctor recommended a treatment for an infection, which involved the use of bacteria that had been engineered to treat the infection, would you use it?'</em>. We also asked this question to a number of medical professionals during our visits to hospitals and clinics.
+
−
                        </p>
+
−
                        <p>
+
−
                            Whilst the majority of feedback was very positive, we did encounter some valuable criticism, as shown in the graph below. Most medical professionals we spoke to had positive responses, though one nurse did have certain reservations about our idea.
+
 
                         </p>
 
                         </p>
 
                         <div class="image image-full">
 
                         <div class="image image-full">
−
                             <img src="https://static.igem.org/mediawiki/2015/1/15/Ox_Q2_doctor_recommendation.png">
+
                             <img src="https://static.igem.org/mediawiki/2015/d/df/Ox_polysulfone.png" />
 
                         </div>
 
                         </div>
 +
                    </div>
 +
                </div>
 +
                <div class="section-spacer"></div>
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                <div class="slim">
 +
                    <div class="section" id="coating">
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                        <h2>Coating</h2>
 
                         <p>
 
                         <p>
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                             This proportion of negative feedback, although small, highlighted to us that our dialogue with the public needed to be improved. We believe that, through improving people's understanding of our project, we can convince pessimists that genetic engineering is now a force for good. This also led us onto holding talks to student groups, which you can find in the <a href="#project-viability-increasing-awareness">Increasing Awareness</a> section below.
+
                             To prevent biofilm formation on the outside of the catheter we would immobilize bacteria in a gel that would coat the outer surface. One option would be to form the gel into fibres that could be wrapped around the catheter and fixed in place with medical adhesive. The advantage to this is that the fibres would have a large surface area to volume ratio, ensuring greater diffusion of our DNase and DspB out of the gel and improving the anti-biofilm effect. A similar approach has been taken with previous catheter designs that are currently on the market, with coatings that contain silver nanoparticles, antibiotics etcetera.
 
                         </p>
 
                         </p>
 
                         <p>
 
                         <p>
−
                             Nevertheless, this is very encouraging data for our project, and again highlights the importance of gaining support from doctors, because without their backing, this project is likely never to become as common a treatment as antibiotics.
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                             An alternative would be to encase our bacteria in a thin sol-gel film; keeping our bacteria confined to the catheter surface but still allowing them to maintain their activity from within the gel. The properties of the gel could be adjusted so that it would be hydrophilic, aiding the insertion of the catheter and improving the comfort for the patient while in place. Our kill-switch design would be particularly useful here as it could be used to kill and bacteria that escaped the gel matrix. Engineering the kill-switch would take too long when compared to the timescale of our project so is not something we have attempted; however there is published literature that has shown it to work.
 
                         </p>
 
                         </p>
 
                     </div>
 
                     </div>
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                     <div id="project-viability-the-uti-clinic">
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                </div>
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                         <h3>The UTI Clinic</h3>
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                <div class="section-spacer"></div>
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                <div class="slim">
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                     <div class="section" id="man">
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                         <h2>Manufacturing</h2>
 
                         <p>
 
                         <p>
 +
                            Currently catheters are very cheap to produce, and thus as part of our project we decided to look into how we could introduce the beads into the pipe during the manufacturing process. We spoke to a mechanical engineer, Steve Dinsdale, and he helped us with a design for a machine that could do this. The machine would use the technique of extrusion to make the tube and then a second cooled inflow tube would introduce the beads. This would hopefully keep the bacteria cool enough so they are not killed, while the polymer at a high enough temperature to remain thermoplastic and form the tube.
 
                         </p>
 
                         </p>
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                    </div>
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                         <div class="image image-full">
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                    <div id="project-viability-delivery-method">
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                            <img src="https://static.igem.org/mediawiki/2015/b/b5/Ox_Steve.PNG" />
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                         <h3>Delivery Method</h3>
+
                             <p>Manufacturing idea from mechanical engineer Steven Dinsdale</p>
−
                        <p>
+
−
                            Now that we had focused our project towards treatment of CAUTIs, we needed a suitable catheter design. We needed some method by which we could expose the catheter, and bladder, to our anti-microbial proteins, and in a safe manner.
+
−
                        </p>
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−
                        <p>
+
−
                            First, we considered the idea of having our bacteria implanted within the patient, in the confines of the catheter. It was of paramount importance that we make sure the bacteria would be safely contained, and not be able to escape into the bladder to cause further infection.
+
−
                        </p>
+
−
                        <p>
+
−
                            Our team member Ria Dinsdale made the suggestion of using sol-gel as a possible method of containment. Despite being a very convincing idea, we found that it would be too difficult to design under the time restraints of the summer.
+
−
                        </p>
+
−
                        <p>
+
−
                            However, we subseqently found an alternative containment method,
+
−
                            which we resurrected from the Oxford iGEM 2014 project 'DCMation'. Our predecessors' project also had to find a method of bacterial containment.
+
−
                        </p>
+
−
                    </div>
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−
                        <div id="project-viability-delivery-method-biobeads">
+
−
                            <h4>BioBeads</h4>
+
−
                            <div class="image image-right">
+
−
                                    <img src="https://static.igem.org/mediawiki/2015/6/66/Ox_Beads_design.JPG" />
+
−
                                    <p>First attempt at making the beads using Sodium Alginate</p>
+
−
                            </div>
+
−
                             <p>
+
−
                                The 2014 team encapsulated their bacteria in agarose to form BioBeads. These BioBeads were then given an outer coating of cellulose acetate, to allow flow of molecules into and out of the bead, whilst preventing the escape of their bacteria.
+
−
                            </p>
+
−
                            <p>
+
−
                                This year, we have been able to significantly improve the BioBead concept, by changing our inner material to sodium alginate, and our outer material to polystyrene; something which is still a work in progress. For full details on our developements on BioBeads, check out our <a href="https://2015.igem.org/Team:Oxford/Design">Design</a> page!
+
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                            </p>
+
 
                         </div>
 
                         </div>
−
                        <div id="project-viability-delivery-method-catheter-design">
 
−
                            <h2>Catheter design</h2>
 
−
                            <p>
 
−
                                Currently catheters are very cheap to produce, find out how much, therefore as part of our project we decided to look into how we could introduce the beads into the pipe during the manufacturing process. We spoken to a mechanical engineer, Steven Dinsdale, and he help us with a design for a machine that could do this. The machine would use the technique of extrusion to make the tube and then a second cooled inflow tube would introduce the beads. This would hopefully keep the bacteria cool enough so they are not killed yet the polymer would be hot enough to melt and then form the tube.
 
−
                            </p>
 
−
                            <div class="image image-full">
 
−
                                <img src="https://static.igem.org/mediawiki/2015/b/b5/Ox_Steve.PNG" />
 
−
                                <p>Manufacturing idea from mechanical engineer Steven Dinsdale</p>
 
−
                            </div>
 
−
                            <p>
 
−
                                Potential problems:
 
−
                            </p>
 
−
                            <ul>
 
−
                                <li>If this design were used for catheter production the bacteria would potentially not survive the sterilisation process at the end of the manufacturing.</li>
 
−
                                <li>Steps further down in the manufacturing process could harm out bacteria.</li>
 
−
                                <li>The bacteria may not survive in storage due to the length of time stored, temperature, etc</li>
 
−
                            </ul>
 
−
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                    <div id="project-viability-increasing-awareness">
 
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                        <h3>Increasing Awareness</h3>
 
 
                         <p>
 
                         <p>
−
                             To promote Synthetic Biology and iGEM, we’ve used a variety of approaches.
+
                             Potential problems:
 
                         </p>
 
                         </p>
−
                         <div id="project-viability-increasing-awareness-uniq-workshop">
+
                         <ul>
−
                                <h4>UNIQ Workshop</h4>
+
                            <li>If this design were used for catheter production the bacteria could potentially not survive the sterilisation process at the end of the manufacturing.</li>
−
                                <p>
+
                            <li>Steps further down in the manufacturing process could harm our bacteria.</li>
−
                                    We met with 40 prospective Oxford students to teach them about Synthetic Biology. The students had in interest in Biochemistry but knew nothing about iGEM. We hammered home the key message of Synthetic Biology - that we achieve more progress by expanding a registry of standardised biological parts - through a 15 minute introductory presentation on BioBricks. We then split them into groups and gave each one a mentor from our iGEM team. We worked through questions to test their understanding in a tutorial style and asked them to explain the constructs of previous iGEM teams. They finished by presenting their findings to each other.
+
                            <li>The bacteria may not survive in storage due to the length of time stored, temperature, etc</li>
−
                                </p>
+
                         </ul>
−
                        </div>
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                        <div id="project-viability-increasing-awareness-utc-oxfordshire">
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                                <h4>UTC Oxfordshire</h4>
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                                <p>
+
−
                                    A couple of us gave a presentation on antibiotic resistance to a class of GCSE students from UTC Oxfordshire (a local school specialising in science) at the Natural History Museum in Oxford, The Pitt Rivers Museum. Our talk covered the discovery of antibiotics, the advantages of them (including their use in laboratory work), how they work, and how bacteria can evolve to gain resistance to them, as well as concepts such as horizontal gene transfer and the consequences of antibiotic resistance on our everyday lives. It also covered our project outline, and pros and cons of Solution, showing how it should help combat antibiotic resistance. At the end, we held a discussion between the students and our team about antibiotic resistance, and their perception of the concern. We also asked how they would feel about using our engineered bacteria, and the response was positive, with most of the students saying that if their doctor recommended the treatment, they would be open to using it.
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                                </p>
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                        </div>
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                        <div id="project-viability-increasing-awareness-bbc-radio-oxford">
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                            <h4>BBC Radio Oxford</h4>
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                         </div>
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                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
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            <div class="col-md-3 contents-sidebar">
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                <ul id="sidebar" class="nav nav-stacked" data-spy="affix">
 +
                    <li><a href="#introduction">Introduction</a></li>
 +
                    <li><a href="#catheter">Catheter Design</a></li>
 +
                    <li>
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                        <a href="#beads">AlgiBeads</a>
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                            <li><a href="#beads-intro">Introduction</a></li>
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                            <li>
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                                <a href="#beads-proof">Proof of Principle</a>
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                                    <li><a href="#beads-proof-chem">Chemistry in Making the Beads</a></li>
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                                    <li><a href="#beads-proof-tute">Tutorial Video</a></li>
 +
                                    <li><a href="#beads-proof-data">Experimental Data</a></li>
 +
                                    <li><a href="#beads-proof-next">The Next Steps</a></li>
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                                </ul>
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                            </li>
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                        </ul>
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                    </li>
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                    <li><a href="#semi">Semi-Permeable Membrane</a></li>
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                    <li><a href="#coating">Coating</a></li>
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                    <li><a href="#man">Manufacturing</a></li>
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                <li><a href="#introduction">Introduction</a></li>
 
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                <li><a href="#project-choice">Project Choice</a>
 
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                    <ul class="nav nav-stacked">
 
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                        <li><a href="#project-choice-approaching-the-public">Approaching the Public</a>
 
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                                <li><a href="#project-choice-approaching-the-public-initial-feedback">Initial Feedback</a></li>
 
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                        </li>
 
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                        <li><a href="#project-choice-our-inspiration">Our Inspiration</a>
 
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                                <li><a href="#project-choice-our-inspiration-jorge-talk">Jorge Talk</a></li>
 
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                                <li><a href="#project-choice-our-inspiration-churchill">Churchill Hospital, Oxford</a></li>
 
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                                <li><a href="#project-choice-our-inspiration-jr">John Radcliffe Hospital, Oxford</a>
 
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                                    <ul class="nav nav-stacked">
 
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                                      <li><a href="#project-choice-our-inspiration-jr-evans">First Interview with Laura Evans, Adams Ward<li></a>
 
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                                    </ul>
 
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                                </li>
 
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                            </ul>
 
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                        </li>
 
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                        <li><a href="#project-choice-the-problem">The Problem</a>
 
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                                <li><a href="#project-choice-the-problem-experts">Feedback from Oxford Experts</a></li>
 
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                        <li><a href="#project-choice-our-solution">Our Solution</a></li>
 
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                <li><a href="#project-viability">Project Viability</a>
 
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                          <li><a href="#project-viability-bacterial-sustainability">Bacterial Sustainability </a></li>
 
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                          <li><a href="#project-viability-ethics">Ethics</a></li>
 
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                          <li><a href="#project-viability-the-uti-clinic">The UTI Clinic</a></li>
 
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                                  <li><a href="#project-viability-delivery-method-biobeads">BioBeads</a></li>
 
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                                  <li><a href="#project-viability-delivery-method-catheter-design">BioBeads</a></li>
 
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                          </li>
 
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                                  <li><a href="#project-viability-increasing-awareness-uniq-workshop">UNIQ Workshop</a></li>
 
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                                  <li><a href="#project-viability-increasing-awareness-utc-oxfordshire">UTC Oxfordshire</a></li>
 
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                                  <li><a href="#project-viability-increasing-awareness-bbc-radio-oxford">BBC Radio Oxford</a></li>
 
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{{:Team:Oxford/Templates/Foot}}
 
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Latest revision as of 11:05, 20 November 2015

Design

Preface

This page comprehensively discusses the various aspects of our initial therapeutic delivery design idea, the AlgiBeads. Our subsequent and final delivery idea, the microbiome-modification design, can be found here, along with its safety considerations here.

Introduction

Following on from all of the safety research we conducted, we put that knowledge into designing a catheter in order to get our proteins (DNase and DspB) into the urinary tract where we want them.

Designing a novel method of getting our bacteria into the urinary tract was a major consideration during the beginning stages of our project. The most effective approach would probably be to deliver our bacteria directly through the catheter into the bladder. However, we found that the biofilm also forms on the outside of the catheter, so in our design we attempted to fight the biofilm from both outside and within.

The ability to disperse biofilms formed by multidrug-resistant bacteria adds a major new weapon to the limited arsenal of therapies available today.

Neville Kallenbach
Professor of Chemistry at New York University NYC

While our bacteria could potentially be applied industrially in various pipes to tackle a growing world problem with biofilms, we instead decide to focus our efforts on a medical application for them. We chose to tackle the problem of urinary tract infections as a member of our team, George Driscoll, had seen first hand the extreme impact it can have on people’s lives – especially women.

Our catheter would have a three-pronged attack on the biofilm. Firstly it would be able to eliminate the biofilm forming in the lining of the bladder, then prevent biofilm from forming on the outside of the catheter itself, and finally by attacking the biofilm trying to form on the inside walls of the catheter.

Our initial research into the current designs of catheter began online, where we began to get a better understanding of the scale we were working with. We looked into the problems of insertion, removal and general life with having a catheter in place. Throughout the design process we constantly kept these issues in mind in order to create something that would not only help with infection, but would also be practical for the patient, doctor and manufacturer.

To get some more first hand experience of how small a catheter is, we purchased our very own. This struck home for us how small our containment method would have to be, with the typical internal volume being 43mm3 and 23mm3 for female and male catheters respectively. As UTIs mainly affect women we decided to buy a 14F female Foley catheter, which gave us a much better idea of the size we were working with: i.e. very small. Knowing this informed our choice of chemical for making containment beads for our bacteria. We also managed to obtain a few catheters from the local hospital. This opened our eyes to the sheer range of different catheter forms there were: different sizes, different materials, and different pipe configurations. Ultimately this lead us to choose to design a 3-way catheter.

Catheter diameters based on the French Catheter scale. Rangeing from 1mm to 1.13cm.

The idea of having a containment method as part of our project was first realised during the safety research as it would be dangerous to allow free bacteria into the human body. We started to incorporate the idea of a containment method into our project as we first realized during safety research that allowing free-living bacteria into the human body could potentially be dangerous. The bacteria could mutate, and lose and reacquire new genes, with no way for us to predict what could happen in several generations. There was a real chance of there being a negative impact on the patient’s health – perhaps fatally, depending on how badly we miscalculated our approach.

However, if we chose to seal our beads within the catheter then we would not be able to access them again as long as the catheter was left in place – doing so could allow for foreign bacteria to enter the urinary tract and cause further infection. Therefore, any nutrients our bacteria needed would have to be present inside the beads, or be available from the urine running through the catheter.

The bacteria would potentially need to be able to survive for 3 months – so ideally we would have been able to leave some beads for 3 months in the lab to test their longevity. Sadly the timescale of the project did not allow us to do that as it was only until the later stages of the project that we came up with the idea of bead encapsulation. We also would have liked to have tested whether the bead could retain its shape and integrity outside of the CaCl2 for 3 months, as well as potential long term storage methods for the beads. Freezing the beads proved unsuccessful for us, but storing them in a cold room gave us promising results.

Another important consideration is that the gel we used had to be non-toxic to humans. Many medical devices are made from silicone due to its inert nature and the fact that it does not cause any allergies or side effects – this material seemed ideal when bearing in mind the safety aspect of our project. The fact that catheters themselves are made from it shows that silicone polymers are safe for use inside the human body.

Catheter Design

There are 3 main parts in the design of a catheter; these parts are discrete and modular so therefore anything from one to all three could be implemented.

This would be the overall design of the catheter with all three elements. It was designed while taking into account all the safety research we carried out.

AlgiBeads

Introduction

In order to stop the biofilm forming on the inside of the catheter it will contain beads that have our bacteria encapsulated inside of them however the protein is still able to diffuse out. This is needed as a way to contain the bacteria so they won’t be free in the urinary tract and therefore cause potential health problems for the patient.

Below is a diagram of our cells secreting proteins out of the beads that they are contained in.

These beads could also be contained outside the body inside a bag of sterile water; this bag would then be plugged into a 3-way Foley catheter. This solution would then be washed through the catheter and into the bladder, therefore tackling any infection that may be present in the lining of the bladder.

First attempt at making the beads using Sodium Algniate

To see our process of designing and making the beads, look here.

Proof of Principle

Chemistry in Making the Beads

The gel created to encapsulate the bacteria is made of Calcium Alginate; this is synthesised when aqueous Sodium Alginate solution is dropped into Calcium Chloride solution.

Calcium Alginate is a water-insoluble gelatinous polymer; therefore it forms beads when the sodium ions are exchanged with the calcium ions. Each calcium ion can bond with two alginate polymer chains; this is called cross-linking. As the sodium ions can only bind to one polymer this cross-linking doesn’t occur and the polymer is water soluble, so the gel does not form.

Tutorial Video

Experimental Data

Firstly, we wanted to show that we could get the bacteria inside of the beads. To do this we created beads that contained fluorescent bacteria. The bacteria we used were from the interlab study: 20K MG, 20K ∆F, and 20K DH5, with MG(-) as a negative control.

We made sets of the beads for 5 days while measuring the fluorescence using the GFP protocol on the FLUOstar Omega plate reader.

The data shows that the wells that contain beads made with encapsulated fluorescent bacteria have a much higher fluorescence than the beads made without any bacteria and the beads made with the negative control bacteria. Therefore, we concluded that the bacteria are encapsulated inside of the beads.

We then made beads using Crystal Violet dye in order to record the diffusion of the dye out of the bead. The data could then be related to the diffusion of the protein via dimensional analysis for our modelling.

We collected the following spectrophotometric absorption data which we calibrated to prepared concentration standards.

(Further analysis of this can found in our modeling section.)

Next we tried to determine whether the bacteria were alive inside the beads and could therefore grow and continue to secrete our proteins.

To do this we made up beads containing the 4 different types of bacteria and placed these bacteria into wells of a 96-well plate containing M9 media. We measured the fluorescence of each well every 15 minutes over 20 hours at 37˚C.

This experiment produced very promising data that suggests the bacteria are growing inside the beads and producing GFP. Therefore we can assume that our bacteria would be able to grow and produce our anti-biofilm agents while encapsulated. The graph shows that the fluorescence of the 3 beads containing the fluorescent bacteria increased over time, and that the bead containing no bacteria remain constantly low. The bead containing our negative control had a very slight increase in fluorescence over time but much less than the bacteria containing the gene for GFP. This could be due to some contamination during the process of making the beads.

We put some consideration into the storage of our beads as hospitals would need to have a supply of them in order to carry out treatment effectively without having to make the beads and use them in a specific amount of time. This would cause delay in treatment and also a lot of waste of materials.

Our first idea was to freeze the beads, however when they are frozen the structure of the alginate is compromised and the skeleton of the beads begin to break down. Therefore, we looked at storing the beads at 4˚C and then investigated whether or not the bacteria would again secrete GFP after being warmed up to 37˚C.

From this we obtained some promising data. Beads stored in the cold room for 20 days were taken out and warmed to 37˚C. Then we measured the fluorescence of these beads every 15 minutes for 20 hours. The graph of this data shows that fluorescence does increase over time, indicating that the bacteria are growing inside of the beads and continuing to produce GFP.

An initial issue with our beads was that the beads had to be kept immersed in media in order to ensure a supply of nutrients to the encapsulated bacteria. To get around this, we tried making beads with our media as the solvent so the beads would have media encapsulated inside them, and thus the bacteria could still survive if the beads were placed into water/urine/etcetera. This internal nutrient supply would help to increase the longevity of the bacteria over the duration of the treatment, with the result that the catheter would have to be replaced less frequently - thus reducing the discomfort and risk of infection for the patient.

The beads were made up using 30mL of LB + Chl and 0.36g of sodium alginate. We then placed the resulting beads into the plate reader and measured the fluorescence of these beads every 15 minutes for 20 hours. We plotted the data on the graph below:

The graph demonstrates that the bacteria do grow inside of the beads, but they reach stationary phase much quicker than the beads what were surrounded by media. This could be due to the lower concentration of nutrients available.


We then took a bead and looked at it underneath a confocal microscope in order see into the bead. This gave us some incredible images of the bacteria fluorescing within the beads.

This photo shows clearly that GFP-producing bacteria are encapsulated inside the bead. This is our main evidence that we can get bacteria to survive in the beads.

Using confocal microscopy, we also took images of multiple layers of a bead and stacked them together to build up a 3D image of the bead and the bacteria encased within.

We then began to probe the edge of the bead with the miscroscope to see whether an bacteria had begun to leak out. From the image below we can see that there is a definition between the edge of the bead, where inside you can see the bacteria fluorescing and outside you cannot.

The Next Steps

If we had longer to work on our beads there are several things we could attempt in order to improve their viability.

The first would be to try and coat the beads and provide a second physical barrier to prevent the escape of the bacteria. We had the idea of coating them in a second layer of the calcium alginate, or to have another chemical in the Calcium Chloride bath that would instantly form a coating around the bead as it formed. However, this would require much more complex chemistry and material science - beyond what we could attempt in 10 weeks.

Ideally we would also make the beads much smaller, giving the beads a smaller volume, and so more would be able to fit inside of the catheter – while simultaneously improving the rate of diffusion out of the beads due to the relative increase in surface area. This would require a needle with a smaller lumen. The approach of making the beads could easily be scaled up so they could be produced in bulk.

Semi-Permeable Membrane

We would use a selectively permeable membrane to contain the beads within a compartment inside the catheter. This membrane would also provide an additional level of physical defence against our bacteria escaping into the urinary tract. The membrane would be similar to that used in a dialysis machine, and thus would allow urine and protein to diffuse through while preventing the larger bacteria from passing. Our largest protein is approximately 40kDa, so the diameter of the membrane pores would have to be able to accommodate this, as well as the larger components of urine (in order to prevent damning and a build up behind the membrane, causing complications). We would most likely be using one of the many materials already used for dialysis tubing membranes: polysulfone, polyethersulfone, etched polycarbonate, or collagen.

After considering the various options, we decided that for our catheter polysulfone would be best as it is most the commonly used in the medical profession and in dialysis machines. It is also reasonably simple to control the size of the pores during synthesis and thus produce the desired pore size.

Coating

To prevent biofilm formation on the outside of the catheter we would immobilize bacteria in a gel that would coat the outer surface. One option would be to form the gel into fibres that could be wrapped around the catheter and fixed in place with medical adhesive. The advantage to this is that the fibres would have a large surface area to volume ratio, ensuring greater diffusion of our DNase and DspB out of the gel and improving the anti-biofilm effect. A similar approach has been taken with previous catheter designs that are currently on the market, with coatings that contain silver nanoparticles, antibiotics etcetera.

An alternative would be to encase our bacteria in a thin sol-gel film; keeping our bacteria confined to the catheter surface but still allowing them to maintain their activity from within the gel. The properties of the gel could be adjusted so that it would be hydrophilic, aiding the insertion of the catheter and improving the comfort for the patient while in place. Our kill-switch design would be particularly useful here as it could be used to kill and bacteria that escaped the gel matrix. Engineering the kill-switch would take too long when compared to the timescale of our project so is not something we have attempted; however there is published literature that has shown it to work.

Manufacturing

Currently catheters are very cheap to produce, and thus as part of our project we decided to look into how we could introduce the beads into the pipe during the manufacturing process. We spoke to a mechanical engineer, Steve Dinsdale, and he helped us with a design for a machine that could do this. The machine would use the technique of extrusion to make the tube and then a second cooled inflow tube would introduce the beads. This would hopefully keep the bacteria cool enough so they are not killed, while the polymer at a high enough temperature to remain thermoplastic and form the tube.

Manufacturing idea from mechanical engineer Steven Dinsdale

Potential problems:

  • If this design were used for catheter production the bacteria could potentially not survive the sterilisation process at the end of the manufacturing.
  • Steps further down in the manufacturing process could harm our bacteria.
  • The bacteria may not survive in storage due to the length of time stored, temperature, etc