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| | bank, and of having nothing to do: once or twice she had peeped into the | | bank, and of having nothing to do: once or twice she had peeped into the |
| | book her sister was reading, but it had no pictures or conversations in | | book her sister was reading, but it had no pictures or conversations in |
| − | it, 'and what is the use of a book,' thought Alice 'without pictures or<sup><a href="#phareferences">1</a></sup>There was nothing so VERY remarkable in that; nor did Alice think it so | + | it, 'and what is the use of a book,' thought Alice 'without pictures or |
| | + | Presently she began again. 'I wonder if I shall fall right THROUGH the |
| | + | earth! How funny it'll seem to come out among the people that walk with |
| | + | their heads downward! The Antipathies, I think--' (she was rather glad |
| | + | there WAS no one listening, this time, as it didn't sound at all the |
| | + | right word) '--but I shall have to ask them what the name of the country |
| | + | is, you know. Please, Ma'am, is this New Zealand or Australia?' (and |
| | + | she tried to curtsey as she spoke--fancy CURTSEYING as you're falling |
| | + | through the air! Do you think you could manage it?) 'And what an |
| | + | ignorant little girl she'll think me for asking! No, it'll never do to |
| | + | ask: perhaps I shall see it written up somewhere.'<sup><a href="#phareferences">1</a></sup>There was nothing so VERY remarkable in that; nor did Alice think it so |
| | VERY much out of the way to hear the Rabbit say to itself, 'Oh dear! | | VERY much out of the way to hear the Rabbit say to itself, 'Oh dear! |
| | Oh dear! I shall be late!' (when she thought it over afterwards, it | | Oh dear! I shall be late!' (when she thought it over afterwards, it |
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| | </div> | | </div> |
| | | | |
| − | <h3>Reducing Toxin Levels</h3>
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| | | | |
| − | <p>
| |
| − | In an attempt to push our purity towards enabling intravenous administration, we decided to perform fractionation in a pressure-driven size-exclusion filter system. Suitability of the phage product for intravenous application could then be assessed with an additional endotoxin test.
| |
| − | </p>
| |
| − | <p>
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| − | After these steps, our phages would be ready for animal testing, which we did not plan to attempt in the context of the iGEM competition
| |
| − | </p>
| |
| − |
| |
| − | </div>
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| − | </div>
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| − | <div class="row">
| |
| − | <div class="col-12">
| |
| − | <h3>Verifying the Function of the Phage Product</h3>
| |
| − |
| |
| − | <p>
| |
| − | To assess the antimicrobial action of our phage product, we combined two methods. We chose a plaque assay as a cheap and simple way to confirm the presence of functional phages and to quantify the phage titer. Reproduction and transcriptional capability of our phages however could be assessed by reverse transcriptase qPCR.
| |
| − | </p>
| |
| − | <h2>Encapsulation</h2>
| |
| − | <div class="row">
| |
| − | <div class="col-12">
| |
| − | <The goal of this module is to optimize the packaging of assembled phages to prepare them for
| |
| − | application to patients.>
| |
| − | <p>
| |
| − | The transport within the body often poses problems for the activity and integrity of phages, which can be addressed by packaging of the phages in a protective layer<sup><a href="#phareferences">5</a></sup>. For oral application of our phage product, sufficient stability for gastric passage has to be guaranteed. The highly acidic environment, as well as the presence of proteases composed the major challenges. Our packaging method thus needed to provide resistance to low pH. At the same time, phages needed to be released from their protection upon reaching intestinal fluid. These characteristics are provided by calcium-alginate microspheres<sup><a href= https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264180/>6</a></sup>. Lacking suitable encapsulation hardware, we decided to build our own nozzle to encapsulate the phages in monodisperse microcapsules.
| |
| − | </p>
| |
| − | <p>
| |
| − | The methods most suitable for quantification of size and assessment of monodispersity of the alginate capsules were brightfield and epifluorescence microscopy. To determine whether our encapsulation method fulfills our requirements of survival and release of phages in simulated gastric and intestinal fluid, respectively, we subsequently performed plaque assays.
| |
| − | </p>
| |
| − | </div>
| |
| − | </div>
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