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| | <div class="section-spacer"></div> | | <div class="section-spacer"></div> |
| − | <div class="section" id="delivery">
| |
| − | <div class="slim">
| |
| − | <h2>Delivery</h2>
| |
| − | <p>
| |
| − | 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.
| |
| − | </p>
| |
| − | <p>
| |
| − | 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.
| |
| − | </p>
| |
| − | <p>
| |
| − | 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.
| |
| − | </p>
| |
| − | <p>
| |
| − | 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.
| |
| − | </p>
| |
| − | <p>
| |
| − | 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.
| |
| − | </p>
| |
| − | <p>
| |
| − | 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:
| |
| − | </p>
| |
| − | <div class="image image-full" style="object-align:center">
| |
| − | <img src="https://static.igem.org/mediawiki/2015/7/79/Oxford-animatiom.gif">
| |
| − | <p>
| |
| − | How our 3-part engineered microbe works:
| |
| − | <br>1. Constant secretion of biofilm-degrading enzyme
| |
| − | <br>2. Production and accumulation of antibacterial Art-175
| |
| − | <br>3. Detection of pathogenic bacteria via quorum sensing
| |
| − | <br>4. Permeabilization of inner membrane by T4 Holin
| |
| − | <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"> | | <div id="references"> |
| | <h2>References</h2> | | <h2>References</h2> |
| | <ol class="references"> | | <ol class="references"> |
| − | <li>Global Report on Surveillance of Antimicrobial Resistance: 2014. WHO.</li> | + | <li>Reference one.</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
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| − | 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.
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| − | </li>
| + | |
| − | <li>Sievert, D.M. et al., 2013. antibiotic-resistant pathogens associated with
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| − | healthcare-associated infections: summary of data reported to the National Healthcare
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| − | Safety Network at the Centers for Disease Control and Prevention, 2009-2010. Infection
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| − | control and hospital epidemiology : the official journal of the Society of Hospital
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| − | Epidemiologists of America, 34(1), pp.1–14. Available at: <a
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| − | href="http://www.ncbi.nlm.nih.gov/pubmed/23221186">http://www.ncbi.nlm.nih.gov/pubmed/23221186</a>.
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| − | </li>
| + | |
| − | <li>Fux, C. a. et al., 2005. Survival strategies of infectious biofilms. Trends in
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| − | Microbiology, 13(1), pp.34–40.
| + | |
| − | </li>
| + | |
| − | <li>Flemming, H.-C. & Wingender, J., 2010. The biofilm matrix. Nature reviews.
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| − | Microbiology, 8(9), pp.623–633. Available at: <a
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| − | href="http://dx.doi.org/10.1038/nrmicro2415">http://dx.doi.org/10.1038/nrmicro2415</a>.
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| − | </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
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| − | and Chemotherapy, 58(7), pp.3774–3784.
| + | |
| − | </li>
| + | |
| − | <li>Gunzer, F., 2013. Bacterially-formed microcin S, a new antibiotic peptide, effective
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| − | against pathogenic microorganisms, e.g. enterohaemorrhagic Escherichia coli (EHEC),
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| − | 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>
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| − | [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.
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| − | 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> | | </ol> |
| | </div> | | </div> |
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