Difference between revisions of "Team:Munich/Demonstrate"

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                     <div class="display-2 mb-0">
 
                     <div class="display-2 mb-0">
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                     Demostrate
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                     Demonstrate
 
                     </div>
 
                     </div>
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         <h4>......</h4>
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         <h4></h4>
  
 
</div>
 
</div>
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<main class="post-content">
 
<main class="post-content">
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<h2>Plaque Assay of MS3 phage over 8 hours</h2>
+
<div class="row mt-5">
 +
<div class="col-12">
 +
   
 +
    <p>Phactory revolutionizes the production of therapeutic phages independently from their bacterial hosts. Each step of our phage production line was optimized individually to meet Good Manufacturing Practice and guarantee all safety requirements for therapeutic phages. Specifically engineered E. coli strains are the basis for the preparation of our home made cell extract to minimize remaining endotoxins and to maximize phage assembling quality. The simplicity of Phactory enables phage assembly by simply adding high-purity phage genomic DNA to the cell extract. This opens flexibel and individualized medical treatment of patients. Highest phage purity and high titers are ensured by further filtration and purification steps. Furthermore Phactory involves the packaging of our phages in alginate capsules for protection from gastric acid after oral administration and intestinal delivery.</p>
 +
</div>
 +
</div>
 +
   
 +
<h2>Safety Requirements for Therapeutic Phages</h2>
 +
<div class="row align-items-center">
 +
<div class="col-6 col-md-3">
 +
    <img src="https://static.igem.org/mediawiki/2018/8/80/T--Munich--Measurement_1.png" class="img-fluid rounded mr-3" alt="A generic square placeholder image with rounded corners in a figure." style="width:50px;"> <span style="color: rgba(0, 158, 198,1); font-weight: bold;">Identification</span>
 +
</div>
 +
<div class="col-6 col-md-6">
 +
<span>Complete Genome</span>
 +
</div>
 +
</div>
 +
<div class="row align-items-center">
 +
<div class="col-6 col-md-3">
 +
    <img src="https://static.igem.org/mediawiki/2018/2/29/T--Munich--Measurement_2.png" class="img-fluid rounded mr-3" alt="A generic square placeholder image with rounded corners in a figure." style="width:50px;"> <span style="color: rgba(0, 158, 198,1); font-weight: bold;">Contamination</span>
 +
</div>
 +
<div class="col-6 col-md-6">
 +
<span>Microorganisms DNA Prophages
 +
</span>
 +
</div>
 +
</div>
 +
<div class="row align-items-center">
 +
<div class="col-6 col-md-3">
 +
    <img src="https://static.igem.org/mediawiki/2018/0/0b/T--Munich--Measurement_3.png" class="img-fluid rounded mr-3" alt="A generic square placeholder image with rounded corners in a figure." style="width:50px;"> <span style="color: rgba(0, 158, 198,1); font-weight: bold;">Toxicity</span>
 +
</div>
 +
<div class="col-6 col-md-6">
 +
<span>5 Endotoxin units / kg bm</span>
 +
</div>
 +
</div>
 +
<div class="row align-items-center">
 +
<div class="col-6 col-md-3">
 +
    <img src="https://static.igem.org/mediawiki/2018/0/09/T--Munich--Measurement_4.png" class="img-fluid rounded mr-3" alt="A generic square placeholder image with rounded corners in a figure." style="width:50px;"> <span style="color: rgba(0, 158, 198,1); font-weight: bold;">pH</span>
 +
</div>
 +
<div class="col-6 col-md-6">
 +
<span>6,5 – 7,5</span>
 +
</div>
 +
</div>
 +
<div class="row align-items-center">
 +
<div class="col-6 col-md-3">
 +
    <img src="https://static.igem.org/mediawiki/2018/3/32/T--Munich--Measurement_5.png" class="img-fluid rounded mr-3" alt="A generic square placeholder image with rounded corners in a figure." style="width:50px;"> <span style="color: rgba(0, 158, 198,1); font-weight: bold;">Purity</span>
 +
</div>
 +
<div class="col-6 col-md-6">
 +
<span>Absence of visible</span>
 +
</div>
 +
</div>
 +
<div class="row align-items-center">
 +
<div class="col-6 col-md-3">
 +
    <img src="https://static.igem.org/mediawiki/2018/8/84/T--Munich--Measurement_6.png" class="img-fluid rounded mr-3" alt="A generic square placeholder image with rounded corners in a figure." style="width:50px;"> <span style="color: rgba(0, 158, 198,1); font-weight: bold;">Preservation</span>
 +
</div>
 +
<div class="col-6 col-md-6">
 +
<span>2-8°C</span>
 +
</div>
 +
</div>
 +
   
 +
   
 +
   
 +
<h2>High Quality Transcription-Translation Machinery </h2>
 
<div class="row">
 
<div class="row">
−
<div class="col-12 px-0 rounded">
+
<div class="col-12">
−
<video autoplay loop poster="https://static.igem.org/mediawiki/2018/a/ae/T--Munich--timelapsePic.png" style="width:100%;">
+
   
−
<source src="https://static.igem.org/mediawiki/2018/4/4a/T--Munich--timelapse.mp4">
+
    <p>The preparation of home made cell extract for the production of therapeutics is very time intensive. For this reason we designed an msbB mutant strain to get steps for the elimination of endotoxin Lipid A off the protocol.</p>
−
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+
−
</video>
+
 
</div>
 
</div>
 
</div>
 
</div>
−
 
+
   
 +
<div class="row align-items-center">
 +
<div class="col-12 col-md-6">
 +
<div class="h4">
 +
Low Endotoxin amount<img src="https://static.igem.org/mediawiki/2018/0/0b/T--Munich--Measurement_3.png" class="img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." style="width:50px;"></div>
 +
    <p>A LAL test was used to measure endotoxin values. We achieved a 49 fold lower Lipid A value in our home made cell extract compared to the commercialized myTX-TL.</p>
 +
<div class="h4">
 +
Physiological pH Range<img src="https://static.igem.org/mediawiki/2018/0/09/T--Munich--Measurement_4.png" class="img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." style="width:50px;"></div>
 +
    <p>We have approved, that the assembled phage solution has a physiological pH to ensure the requirement of the correct pH range for therapeutic phages.</p>
 +
</div>
 +
<div class="col-12 col-md-6">
 +
<figure class="figure">
 +
    <img src="https://static.igem.org/mediawiki/2018/5/53/T--Munich--Results_Wl2_LAL.png" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." >
 +
    <figcaption class="figure-caption">Endotoxin content in different cell-extracts determined by LAL-Test. Error bars indicate standard deviation of the measured plateau values. Error bars indicate SD.</figcaption>
 +
    </figure>
 +
</div>
 +
</div>
 +
   
 
<div class="row">
 
<div class="row">
−
<div class="col-12 px-0 rounded">
+
<div class="col-12">  
−
<video controls poster="https://static.igem.org/mediawiki/2018/2/2a/T--Munich--Clinical_trialll.png" style="width:100%;">
+
    <p>Furthermore, we investigated different steps of the protocol to optimize cell extract quality concerning the following:</p>
−
<source src="https://static.igem.org/mediawiki/2018/b/b3/T--Munich--Phactory_Clinical_Trial_V2_lq.mov">
+
−
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</video>
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</div>
 
</div>
 
</div>
 
</div>
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+
   
−
     <div class="row">
+
      
−
    <div class="col-12 text-center">
+
<div class="row align-items-center">
−
     <figure class="figure" >
+
<div class="col-12 col-md-6">
−
     <img src="https://static.igem.org/mediawiki/2018/f/fc/T--Munich--Demonstrate_3S_Phage_3D_video_TEM-_fertig.gif" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure.">
+
<div class="h4">
 +
Transcription analyzis via Malachite Green Aptamer</div>
 +
     <p>We compared the fluorescence time trace of a Malachite Green binding aptamer in different cell extract samples. The fluorescence time traces decline after 30-50min indicating, that RNA degradation starts to prevail over transcription. Differences in the observed kinetics can be explained by variations in cell extract composition.</p>
 +
</div>
 +
<div class="col-12 col-md-6">
 +
<figure class="figure">
 +
     <img src="https://static.igem.org/mediawiki/2018/f/f3/T--Munich--MG_aptamer.png" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." >
 +
    <figcaption class="figure-caption"></figcaption>
 
     </figure>
 
     </figure>
−
     </div>
+
</div>
−
     </div>
+
</div>
−
+
      
−
<h2>Project safety</h2>     
+
<div class="row align-items-center">
 +
<div class="col-12 col-md-6">
 +
<div class="h4">
 +
Translation analyzis via fluorescence assay with mTurquoise</div>
 +
     <p>Translation efficiency is five times higher than in commercialized TX/TL.</p>
 +
</div>
 +
<div class="col-12 col-md-6">
 +
<figure class="figure">
 +
    <img src="https://static.igem.org/mediawiki/2018/7/7c/T--munich--Interlab_AbsorbanceOD600.pdf" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." >
 +
    <figcaption class="figure-caption">Absorbance (OD = 600 nm) mean values at the start and end of the experiment for all devices.</figcaption>
 +
    </figure>
 +
</div>
 +
</div>
 +
   
 +
<h2>Preservation of Cell Extract</h2>     
 +
    <div class="row align-items-center">
 +
<div class="col-12 col-md-6">
 +
<div class="h4">
 +
Storage and shipping of cell extract by lyophilization<img src="https://static.igem.org/mediawiki/2018/8/84/T--Munich--Measurement_6.png" class="img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." style="width:50px;"></div>
 +
    <p>To make our cell extract accessible to everyone everywhere, we seeled to ensure long term storage and shipping at room temperature. Therefore we created lyophilized cell extract. The results show, that the tested samples retained 70% and 90% of expression quality respectively after lyophilization. </p>
 +
    <p>Furthermore, we tested the functionality of phages assembled in our home made cell extract after lyophilization and found functional phages afterwards.</p>
 +
</div>
 +
<div class="col-12 col-md-6">
 +
<figure class="figure">
 +
    <img src="https://static.igem.org/mediawiki/2018/4/49/T--Munich--Results-WL2_LYO_vs_FRESH.png" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." >
 +
    <figcaption class="figure-caption">Our two tested samples of cell extract retained 70 and 90 % of expression quality respectively after lyophilization.</figcaption>
 +
    </figure>
 +
</div>
 +
</div>
 +
   
 +
<h2>Phage Assembly</h2>
 
<div class="row">
 
<div class="row">
 
<div class="col-12">
 
<div class="col-12">
 +
    <p>The simplicity of Phactory offers the possibility of producing phages simply by adding the specific phage DNA to the TX-TL machinery. Moreover, Phactory yields excellent titers of assembled phages by far higher as required for therapeutic use.</p>
 +
</div>
 +
</div>
 
      
 
      
−
    <p>The careless use of antibiotics is leading mankind into a dangerous direction. The WHO calculated that 900,000 resistant infections occure in hospitals annually (<a href="#phareferences">1</a>), killing 63,000 patients (<a href="#phareferences">2</a>). Also, each year antibiotic resistance results in 8 million more hospitalization days adding more than $20 billion to US healthcare costs (<a href="#phareferences">3</a>). Moreover, the use of antibiotics is expected to reduce the microbiome diversity, which in turn has been related to malnutrition and several types of other diseases (<a href="#phareferences">4</a>). Therefore, it is essential to investigate other forms of therapy for bacterial infections. Over the last years phage therapy has been re-discovered, because of the promising results seen in the Eastern Europe (<a href="#phareferences">5</a>). Now the western countries have started to focus on this field of research with studies like „Phage4Cure“. Our cell free assembly platform increases safety and supports the field in the following ways:</p>
+
<div class="row align-items-center">
 +
<div class="col-12 col-md-6">
 +
<div class="h4">
 +
High Titer</div>
 +
    <p>Phactory makes high yield of phages of up to 10<sup>12</sup> PFU/mL in a small reaction volume of 9 µL possible</p>
 
</div>
 
</div>
−
+
<div class="col-12 col-md-6">
−
<div class="col-12 col-md-6">  
+
<figure class="figure">
−
    <h3>Overcoming the antibiotic crisis by phages</h3>
+
    <img src="https://static.igem.org/mediawiki/2018/7/76/T--Munich--Results--WL3_Titer_difference.png" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." >
−
     <p>Phages have been proven to work on pathogens, which are resistant to antibiotics (<a href="#phareferences">6</a>). Furthermore, by the production of therapeutic phages the treatment repertoire is increased not endangering and curing patients, to which conventional treatment has no effect (<a href="#phareferences">7</a>).</p>  
+
     <figcaption class="figure-caption"> Overview of the successfully assembled phages. MS2 (RNA-phage), T4 (DNA phage), T5 (DNA phage), T7 (DNA phage), CLB-P2 (clinically relevant), CLB-P2 (clinically relevant), GEC-3S P2 (clinically relevant).</figcaption>
 +
    </figure>
 
</div>
 
</div>
 +
</div>
 +
   
 +
<div class="row align-items-center">
 
<div class="col-12 col-md-6">
 
<div class="col-12 col-md-6">
−
    <h3>Time saving production of phages can save a patient’s life</h3>
+
<div class="h4">
−
     <p>We have shown that we can assemble a phage of our choice in 3h, whereas the native isolation of the same phage takes 6h. Referring to the treatment of a patient this time difference can be lifesaving.</p>  
+
High Purity<img src="https://static.igem.org/mediawiki/2018/3/32/T--Munich--Measurement_5.png" class="img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." style="width:50px;"></div>
 +
     <p>The abscence of impurities in the phage solutions produced in Phactory was certified by Transmission Electron Microscopy (TEM) imaging.</p>
 
</div>
 
</div>
 
<div class="col-12 col-md-6">
 
<div class="col-12 col-md-6">
−
    <h3>Phages are more specific than antibiotics:</h3>
+
<figure class="figure">
−
     <p>Antibiotics target abundant characteristics of bacteria. Thus, they can be used against several bacteria (<a href="#phareferences">8</a>), however also impacting the human microbiome (<a href="#phareferences">9</a>). Whereas phages exhibit a high specificity for restricted subpopulations making the treatment with phages saver (<a href="#phareferences">10</a>).</p>  
+
     <img src="https://static.igem.org/mediawiki/2018/1/15/T--Munich--CLB_P3_human_practice.jpeg" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." >
 +
    <figcaption class="figure-caption">TEM (Transmission Electron Microscopy) of the CLB-P3 phage</figcaption>
 +
    </figure>
 
</div>
 
</div>
−
<div class="col-12 col-md-6">   
 
−
    <h3>Long term use of phages is saver than antibiotics</h3> 
 
−
    <p>Like bacterial pathogens develop with the human body, phages adapt to the development of bacteria (<a href="#phareferences">11</a>). The resistance behavior of bacteria against phages has been studied. The research of several groups indicates, that we can avoid causing an “phage crisis” like we have an “antibiotics crisis” (<a href="#phareferences">12</a>).</p>
 
−
</div>
 
−
<div class="col-12 col-md-6"> 
 
−
    <h3>Evade contamination by other phages during purification</h3> 
 
−
    <p>The biggest problem for native or environment derived phages is, that during isolation pro-phages or other phages can be isolated, too. Because we use only the DNA of phages for our assembly, there is no possibility that we unintentional generate a mix of phages. This assumption was also verified by us with nanopore sequencing.</p>
 
 
</div>
 
</div>
 +
   
 +
     
 +
   
 +
<h2>Sequ-Into</h2>
 +
   
 +
<div class="row align-items-center">
 
<div class="col-12 col-md-6">
 
<div class="col-12 col-md-6">
−
    <h3>Pathogen free production of therapeutic phages</h3>
+
<div class="h4">
−
     <p>The major advantage of our method is, that we have proven to produce clinically relevant phages for life threatening pathogens. Those pathogens are not needed for the production in our TX-TL system in a laboratory. This finding was shown with the production of anti- enterohemorrhagic E. coli (EHEC) phages in our S1 laboratory. Whereas the EHEC bacteria is an organism of the biosafety level 3 like Salmonella Typhi oder Yersinia pestis (<a href="#phareferences">13</a>).</p>  
+
Phage Assembly from High quality Phage DNA <img src="https://static.igem.org/mediawiki/2018/8/80/T--Munich--Measurement_1.png" class="img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." style="width:50px;"><img src="https://static.igem.org/mediawiki/2018/2/29/T--Munich--Measurement_2.png" class="img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." style="width:50px;"></div>
 +
     <p>For Phactory we intend to only use sequenced phage DNA to ensure the production of the volitional phage. We optimized the protocol for phage DNA purification to eliminate the possibility of generating a mix of phages unintentionally. Our software Sequ-Into was used for accurate quantification of DNA contamination levels. With our optimized purification methods we achieved a final DNA purity of 96 %. In comparison to that common phage DNA purification methods only achieve a purity of 65 %.</p>
 
</div>
 
</div>
 
<div class="col-12 col-md-6">
 
<div class="col-12 col-md-6">
−
    <h3>Phages for the industrial sector</h3> 
+
<figure class="figure">
−
     <p>The application of phages is not only restricted for a clinical practice. It has also been evaluated for the agricaltural sector as a replacement of antibiotics. The administration was easy, successful and no side effects were seen (<a href="#phareferences">14</a>). </p>  
+
     <img src="https://static.igem.org/mediawiki/2018/9/92/T--Munich--Contamination.png" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." >
 +
    <figcaption class="figure-caption"></figcaption>
 +
    </figure>
 
</div>
 
</div>
 +
</div>
 +
   
 +
   
 +
<h2>Purification</h2>
 +
<div class="row">
 +
<div class="col-12">
 +
    <p>To fulfill the strict requirements for therapeutically used phages further purification is required to produce a high quality product.</p>
 +
</div>
 +
</div>
 +
   
 +
<div class="row align-items-center">
 
<div class="col-12 col-md-6">
 
<div class="col-12 col-md-6">
−
    <h3>Overall effect on public health</h3> 
+
<div class="h4">
−
    <p>As clearly presented in the previous sections phage therapy is a safe and promising treatment option, reducing site effects by the high specificity of phages (<a href="#phareferences">15</a>). As shown by us the phage production is easy, stable, economic and safe making it an ideal treatment in industrialized and but also development countries. One of the biggest problem as seen in our survey is the public awareness concerning the use of antibiotics and the knowledge about other treatment options. This indicates, that the people have to be further educated about the risks of needless use of antibiotics. Moreover, the practical application must be further supported and tested like we did it during iGEM.</p>  
+
Ultra-Filtration for phage purification <img src="https://static.igem.org/mediawiki/2018/2/29/T--Munich--Measurement_2.png" class="img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." style="width:50px;"></div>
 +
    <p>By ultra-filtration phages got successfully separated from submolecular content.</p>
 
</div>
 
</div>
−
</div>  
+
<div class="col-12 col-md-6">
−
 
+
<figure class="figure">
−
     
+
    <img src="https://static.igem.org/mediawiki/2018/8/88/T--Munich--Purification.jpg" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." >
−
<h2>SWOT Analysis</h2>  
+
    <figcaption class="figure-caption"></figcaption>
 +
    </figure>
 +
</div>
 +
</div>
 +
   
 +
<h2>Packaging</h2>
 
<div class="row">
 
<div class="row">
 
<div class="col-12">
 
<div class="col-12">
 +
    <p>Phactory yields phages with toxicity levels that allow oral administration to the patient. However, oral delivery requires protection of the phages from rapid degradation in the acidic gastric juice. Therefore, we encapsulated our phages in alginate. This protects our phages successfully until release into the gastric system. We were able to prove functionality of encapsulated and released phages. </p>
 +
</div>
 +
</div>
 
      
 
      
−
    <p>Because many parts of Phactory seem innovative and new, we are trying to commercialize some aspects. The first step was writing a patent, which is currently pending. Moreover, we have reached out to the “UnternehmerTUM” an institution, which is specialized in consulting scientists from the university for establishing a company and applying for the corresponding grants. </p>
+
<div class="row align-items-center">
−
    <p>The first step before writing the patent and seeking out for consulting was evaluating our project by the “SWOT” analysis, which is used by many companies. SWOT stands for “"Strengths Weaknesses Opportunities and Threats" and is necessary for realizing an objective by taking risks into consideration. Based on the internal analysis (strength/weakness) you optimize your market position. The external analysis (opportunities/threats) gives a cost benefit ratio of the project.</p>
+
<div class="col-12 col-md-4">
−
       
+
<figure class="figure">
−
     <h3>Internal analysis:</h3>  
+
    <img src="https://static.igem.org/mediawiki/2018/9/9c/T--Munich--Results_Droplets_zstack-400_8bit.gif" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." >
−
     <h4>Strength:</h4>
+
     <figcaption class="figure-caption">Animated Zoom Through Z-Stack</figcaption>
−
    <p>We have proven that our system works (applicability)</p>  
+
     </figure>
−
     <p>Until now pathogen bacteria were necessary to produce phages, not anymore (safety)</p>  
+
</div>
−
     <p>The production pipeline is profitable (economy)</p>
+
<div class="col-12 col-md-4">
−
    <h4>Weakness:</h4>
+
<figure class="figure">
−
     <p>Availability and Quality of Phage-DNA</p>  
+
    <img src="https://static.igem.org/mediawiki/2018/4/4f/T--Munich--Results_Joe_SGF_Phages.png" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." >
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     <p>Dependence on the quality of our cell-free system</p>
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     <figcaption class="figure-caption">growth fermenter vs. shaking flask side by side with FI mtq2</figcaption>
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<figure class="figure">
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     <img src="https://static.igem.org/mediawiki/2018/a/a2/T--Munich--Results_Droplets_phages.png" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure." >
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    <figcaption class="figure-caption">growh fermenter vs. shaking flask side by side with FI mtq2</figcaption>
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     <h3>External analysis:</h3>
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    <h4>Opportunities:</h4>
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<h2>The Product</h2>
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    <p>There is a high demand on alternatives to antibiotics due to resistant bacteria Phages have been approved in parts of the EU (Belgiukm, France)</p>  
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<div class="row">
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    <h4>Threats:</h4> 
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<div class="col-12">
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     <p>Phactory is open source due to iGEM and therefore easy to copy</p>
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     <p>Phactory presents a complete production line from the optimized transcription-translation machinery as a platform for phage assembly to the packaging for final treatment. After all, our phages show excellent activity.</p>
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    <p>If enough new antibiotics will be developed, this treatment will be favored</p>
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<h3>Plaque Assay of T7 phage over 6 hours</h3>
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<p style="color: red;">Notes: [Bild vor der UnternehmerTUM (muss noch hochgeladen werden/ Whiteboard mit Kalkulationen “Franzi/Kilian”)]</p>
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<div id="phareferences" class="row">
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<h2>References</h2>
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<h2>Clinically Relevant Bacteriophages</h2><div class="row">
 
<div class="col-12">
 
<div class="col-12">
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<ol>
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    <p>It was of great importance for us to demonstrate the medical relevance of bacteriophages manufactured with Phactory. We received the SEC-3S bacteriophage from the Queen Astrid Hospital as well as the P2 and P3 bacteriophages from the group of Laurent Debarbieux at the Pasteur Institute in Paris. These bacteriophages are specific for the E.Coli strain O104:H4, also known as EHEC, which caused 53 deaths during an outbreak in Germany in 2011. As indicated in <a href="https://2018.igem.org/Team:Munich/Human_Practices#clinically">the microscopy images</a>, we successfully assembled these medically relevant bacteriophages in our cell-free system.
−
<li><a href="">Chan, Margaret. "Antimicrobial resistance in the European Union and the world." World Health Organization (2012).</a></li>
+
We conducted a LAL-test and compared the results with magistral bacteriophage preparation regulations. According to these results, our bacteriophages are orally applicable. In the future we will work on reaching even higher titers with Phactory, so that we can apply more extensive purification protocols to allow intravenous application of bacteriophage therapy.</p>
−
<li><a href="">Chan, M. "Combat drug resistance: no action today means no cure tomorrow." World Health Day 6 (2011).</a></li>
+
−
<li><a href="">Hughes, James M. "Preserving the lifesaving power of antimicrobial agents." Jama 305.10 (2011): 1027-1028.</a></li>
+
−
<li><a href="">Lozupone, C. A., Stombaugh, J. I., Gordon, J. I., Jansson, J. K., Knight, R., Diversity, stability and resilience of the human gut microbiota. Nature 2012, 489, 220–230.</a></li>
+
−
<li><a href="">Expert round table on acceptance and re‐implementation of bacteriophage therapy. "Silk route to the acceptance and re‐implementation of bacteriophage therapy." Biotechnology Journal 11.5 (2016): 595-600.</a></li>
+
−
<li><a href="">Lin, Derek M., Britt Koskella, and Henry C. Lin. "Phage therapy: an alternative to antibiotics in the age of multi-drug resistance." World journal of gastrointestinal pharmacology and therapeutics8.3 (2017): 162.</a></li>
+
−
<li><a href="">Slopek, Stefan, et al. "Results of bacteriophage treatment of suppurative bacterial infections. VI. Analysis of treatment of suppurative staphylococcal infections." Archivum immunologiae et therapiae experimentalis 33.2 (1985): 261-273.</a></li>
+
−
<li><a href="">Fridkin, Scott K., and Robert P. Gaynes. "Antimicrobial resistance in intensive care units." Clinics in Chest Medicine 20.2 (1999): 303-316.</a></li>
+
−
<li><a href="">MCCURDY, ROBERT S., and ERWIN NETER. "Effects of penicillin and broad-spectrum antibiotics on the emergence of a gram-negative bacillary flora in the upper respiratory tract of infants." Pediatrics 9.5 (1952): 572-576.</a></li>
+
−
<li><a href="">Frisch, Arthur W., and Philip Levine. "Specificity of the Multiplication of Bacteriophage." The Journal of Immunology 30.1 (1936): 89-108.</a></li>
+
−
<li><a href="">Lenski, Richard E., and Bruce R. Levin. "Constraints on the coevolution of bacteria and virulent phage: a model, some experiments, and predictions for natural communities." The American Naturalist 125.4 (1985): 585-602.</a></li>
+
−
<li><a href="">Örmälä, Anni-Maria, and Matti Jalasvuori. "Phage therapy: should bacterial resistance to phages be a concern, even in the long run?." Bacteriophage 3.1 (2013): e24219.</a></li>
+
−
<li><a href="">Chosewood, L. Casey. Biosafety in microbiological and biomedical laboratories. Diane Publishing, 2007.</a></li>
+
−
<li><a href="">Colom, Joan, et al. "Microencapsulation with alginate/CaCO 3: A strategy for improved phage therapy." Scientific reports 7 (2017): 41441.</a></li>
+
−
<li><a href="">Golkar, Zhabiz, Omar Bagasra, and Donald Gene Pace. "Bacteriophage therapy: a potential solution for the antibiotic resistance crisis." The Journal of Infection in Developing Countries 8.02 (2014): 129-136.</a></li>
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<h3>Clinical Trial</h3>
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</main>
 
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Revision as of 01:10, 18 October 2018

Phactory

Demonstrate

Phactory revolutionizes the production of therapeutic phages independently from their bacterial hosts. Each step of our phage production line was optimized individually to meet Good Manufacturing Practice and guarantee all safety requirements for therapeutic phages. Specifically engineered E. coli strains are the basis for the preparation of our home made cell extract to minimize remaining endotoxins and to maximize phage assembling quality. The simplicity of Phactory enables phage assembly by simply adding high-purity phage genomic DNA to the cell extract. This opens flexibel and individualized medical treatment of patients. Highest phage purity and high titers are ensured by further filtration and purification steps. Furthermore Phactory involves the packaging of our phages in alginate capsules for protection from gastric acid after oral administration and intestinal delivery.

Safety Requirements for Therapeutic Phages

A generic square placeholder image with rounded corners in a figure. Identification
Complete Genome
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Microorganisms DNA Prophages
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5 Endotoxin units / kg bm
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6,5 – 7,5
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Absence of visible
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2-8°C

High Quality Transcription-Translation Machinery

The preparation of home made cell extract for the production of therapeutics is very time intensive. For this reason we designed an msbB mutant strain to get steps for the elimination of endotoxin Lipid A off the protocol.

Low Endotoxin amountA generic square placeholder image with rounded corners in a figure.

A LAL test was used to measure endotoxin values. We achieved a 49 fold lower Lipid A value in our home made cell extract compared to the commercialized myTX-TL.

Physiological pH RangeA generic square placeholder image with rounded corners in a figure.

We have approved, that the assembled phage solution has a physiological pH to ensure the requirement of the correct pH range for therapeutic phages.

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Endotoxin content in different cell-extracts determined by LAL-Test. Error bars indicate standard deviation of the measured plateau values. Error bars indicate SD.

Furthermore, we investigated different steps of the protocol to optimize cell extract quality concerning the following:

Transcription analyzis via Malachite Green Aptamer

We compared the fluorescence time trace of a Malachite Green binding aptamer in different cell extract samples. The fluorescence time traces decline after 30-50min indicating, that RNA degradation starts to prevail over transcription. Differences in the observed kinetics can be explained by variations in cell extract composition.

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Translation analyzis via fluorescence assay with mTurquoise

Translation efficiency is five times higher than in commercialized TX/TL.

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Absorbance (OD = 600 nm) mean values at the start and end of the experiment for all devices.

Preservation of Cell Extract

Storage and shipping of cell extract by lyophilizationA generic square placeholder image with rounded corners in a figure.

To make our cell extract accessible to everyone everywhere, we seeled to ensure long term storage and shipping at room temperature. Therefore we created lyophilized cell extract. The results show, that the tested samples retained 70% and 90% of expression quality respectively after lyophilization.

Furthermore, we tested the functionality of phages assembled in our home made cell extract after lyophilization and found functional phages afterwards.

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Our two tested samples of cell extract retained 70 and 90 % of expression quality respectively after lyophilization.

Phage Assembly

The simplicity of Phactory offers the possibility of producing phages simply by adding the specific phage DNA to the TX-TL machinery. Moreover, Phactory yields excellent titers of assembled phages by far higher as required for therapeutic use.

High Titer

Phactory makes high yield of phages of up to 1012 PFU/mL in a small reaction volume of 9 µL possible

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Overview of the successfully assembled phages. MS2 (RNA-phage), T4 (DNA phage), T5 (DNA phage), T7 (DNA phage), CLB-P2 (clinically relevant), CLB-P2 (clinically relevant), GEC-3S P2 (clinically relevant).
High PurityA generic square placeholder image with rounded corners in a figure.

The abscence of impurities in the phage solutions produced in Phactory was certified by Transmission Electron Microscopy (TEM) imaging.

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TEM (Transmission Electron Microscopy) of the CLB-P3 phage

Sequ-Into

Phage Assembly from High quality Phage DNA A generic square placeholder image with rounded corners in a figure.A generic square placeholder image with rounded corners in a figure.

For Phactory we intend to only use sequenced phage DNA to ensure the production of the volitional phage. We optimized the protocol for phage DNA purification to eliminate the possibility of generating a mix of phages unintentionally. Our software Sequ-Into was used for accurate quantification of DNA contamination levels. With our optimized purification methods we achieved a final DNA purity of 96 %. In comparison to that common phage DNA purification methods only achieve a purity of 65 %.

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Purification

To fulfill the strict requirements for therapeutically used phages further purification is required to produce a high quality product.

Ultra-Filtration for phage purification A generic square placeholder image with rounded corners in a figure.

By ultra-filtration phages got successfully separated from submolecular content.

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Packaging

Phactory yields phages with toxicity levels that allow oral administration to the patient. However, oral delivery requires protection of the phages from rapid degradation in the acidic gastric juice. Therefore, we encapsulated our phages in alginate. This protects our phages successfully until release into the gastric system. We were able to prove functionality of encapsulated and released phages.

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Animated Zoom Through Z-Stack
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growth fermenter vs. shaking flask side by side with FI mtq2
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growh fermenter vs. shaking flask side by side with FI mtq2

The Product

Phactory presents a complete production line from the optimized transcription-translation machinery as a platform for phage assembly to the packaging for final treatment. After all, our phages show excellent activity.

Plaque Assay of T7 phage over 6 hours

Clinically Relevant Bacteriophages

It was of great importance for us to demonstrate the medical relevance of bacteriophages manufactured with Phactory. We received the SEC-3S bacteriophage from the Queen Astrid Hospital as well as the P2 and P3 bacteriophages from the group of Laurent Debarbieux at the Pasteur Institute in Paris. These bacteriophages are specific for the E.Coli strain O104:H4, also known as EHEC, which caused 53 deaths during an outbreak in Germany in 2011. As indicated in the microscopy images, we successfully assembled these medically relevant bacteriophages in our cell-free system. We conducted a LAL-test and compared the results with magistral bacteriophage preparation regulations. According to these results, our bacteriophages are orally applicable. In the future we will work on reaching even higher titers with Phactory, so that we can apply more extensive purification protocols to allow intravenous application of bacteriophage therapy.

Clinical Trial