Difference between revisions of "Team:Stuttgart/Collaborations"
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| − | + | <div class="container has-text-centered"> | |
| − | + | <h1 class="title"> | |
| − | + | Collaboration | |
| − | + | </h1> | |
| − | + | </div> | |
</div> | </div> | ||
| − | </section> | + | </section> |
| − | + | <div style="padding: 1rem 0;" class="container"> | |
| − | <div style="padding: 1rem 0;" class="container"> | + | |
<div class="columns is-centered"> | <div class="columns is-centered"> | ||
| − | + | <div class="column is-three-quarters"> | |
| − | + | <h2 class="title is-4">Validation of our new algae based PhyCoVi medium</h2> | |
| − | + | <p> | |
| − | + | Besides our own experiments with the algae extract, we wanted to have a cross-validation from other iGEM teams | |
| − | + | and their organisms. Through this, we can make sure, that everybody can work with our medium and also improve | |
| − | + | the product itself, if problems occur. Kindly, the iGEM teams of Hamburg, Tübingen, TU Darmstadt, | |
| − | + | Freiburg, and TU Dresden offered their help. Therefore, we prepared 15 ml tubes with two variations of | |
| − | + | PhyCoVi and a LB control dry medium and sent them to the teams, respectively. Additionally, they received a | |
| − | + | SOP for their growth curve including an Excel sheet to plot their results directly. Before we discuss their | |
| − | + | results individually, it can be said, that we are happy with every result and already improved the product | |
| − | + | concerning the issues that occurred for some teams. | |
| + | </p> | ||
| + | </div> | ||
</div> | </div> | ||
| − | </div> | + | </div> |
| − | + | ||
| − | <div class="container" style="padding-top: 3rem;"> | + | <div class="container" style="padding-top: 3rem;"> |
<div class="columns"> | <div class="columns"> | ||
| − | + | <div class="column"> | |
| − | + | <div> | |
| − | + | <h2 class="title is-5">Hamburg (<i>E. coli</i> DH5-α)</h2> | |
| − | + | <canvas id="team-hamburg"></canvas> | |
| − | + | <small | |
| − | + | >Figure 1: Growth curve of E. coli DH5-α created by the iGEM Team Hamburg. PhyCoVi medium (blue), | |
| − | + | PhyCoVi medium with additional soy peptone (green) and High salt LB medium were tested (black). PhyCoVi | |
| − | + | medium contained 10 g/L additional NaCl, High Salt LB medium contained 20 g/L NaCl.</small | |
| − | + | > | |
| − | + | <br /> | |
| − | + | <p> | |
| + | As seen in Fig. 1, <i>E. coli</i> was able to grow in all three media. The PhyCoVi medium including | ||
| + | additional soy peptone and the high salt LB medium show similar growth curves after 45 minutes. The | ||
| + | PhyCoVi medium without supplements shows a very long lag phase, growth occurs after 135 minutes. The | ||
| + | DH5-α strain is very robust and optimized for optimal transformation <a href="#link-1"><sup>1</sup></a | ||
| + | >. These optimizations also seem to have a positive effect on high salt concentrations. | ||
| + | </p> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="column"> | |
| − | + | <div> | |
| − | + | <h2 class="title is-5">Tübingen (<i>E. coli</i> Nissle 1917)</h2> | |
| − | + | <canvas id="team-tuebingen"></canvas> | |
| − | + | <small | |
| − | + | >Figure 2: Growth curve of E. coli Nissle 1917 created by the iGEM Team Tübingen. PhyCoVi medium | |
| − | + | (blue), PhyCoVi medium with additional soy peptone (green) and High salt LB medium were tested (black). | |
| − | + | PhyCoVi medium contained 10 g/L additional NaCl, High Salt LB medium contained 20 g/L NaCl.</small | |
| − | + | > | |
| − | + | <br /> | |
| + | <p> | ||
| + | Fig. 2 shows the growth curves by Team Tübingen. Probably due to algae lipid residues in the PhyCoVi | ||
| + | medium samples, OD(600) measurements reveal negative values. But still, growth of <i>E. coli</i> Nissle 1917 | ||
| + | can be observed in the PhyCoVi medium supplemented with soy peptone. As described | ||
| + | <a href="#link-2"><sup>2</sup></a | ||
| + | >, this wildtype strain has a low tolerance for high salt medium, therefore the reduced growth is expected. | ||
| + | </p> | ||
</div> | </div> | ||
| + | </div> | ||
</div> | </div> | ||
<div class="columns"> | <div class="columns"> | ||
| − | + | <div class="column"> | |
| − | + | <div> | |
| − | + | <h2 class="title is-5">TU Dresden (<i>E. coli</i> GB05)</h2> | |
| − | + | <canvas id="team-dresden"></canvas> | |
| − | + | <small | |
| − | + | >Figure 3: Growth curve of E. coli GB05 created by the iGEM Team TU Dresden. PhyCoVi medium (blue), PhyCoVi | |
| − | + | medium with additional soy peptone (green) and High salt LB medium were tested (black). PhyCoVi medium | |
| − | + | contained 10 g/L additional NaCl, High Salt LB medium contained 20 g/L NaCl.</small | |
| − | + | > | |
| − | + | <br /> | |
| − | + | <p> | |
| + | The results of the iGEM Team TU Dresden shows very low growth for all three media, possibly caused by the | ||
| + | high salt concentrations (Fig. 3). After a long adaptation phase, <i>E. coli</i> GB05 was able to grow in | ||
| + | high salt LB medium. Growth in PhyCoVi medium supplemented with soy peptone shows high fluctuations, | ||
| + | probably due to the already discussed problem with insoluble medium compounds. The used strain is able to | ||
| + | take up linear DNA fragments but seems not to be able to cope with high salt media | ||
| + | <a href="#link-3"><sup>3</sup></a | ||
| + | >. | ||
| + | </p> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="column"> | |
| − | + | <div> | |
| − | + | <h2 class="title is-5">TU Darmstadt (<i>E. coli</i> BL21 (DE3))</h2> | |
| − | + | <canvas id="team-darmstadt"></canvas> | |
| − | + | <small | |
| − | + | >Figure 4: Growth curve of E. coli BL21 (DE3) created by the iGEM TU Darmstadt. PhyCoVi medium (blue), | |
| − | + | PhyCoVi medium with additional soy peptone (green) and High salt LB medium were tested (black). PhyCoVi | |
| − | + | medium contained 10 g/L additional NaCl, High Salt LB medium contained 20 g/L NaCl.</small | |
| − | + | > | |
| + | <br /> | ||
| + | <p> | ||
| + | While <i>E. coli</i> BL21 (DE3) in High Salt LB medium reaches high OD values, growth in both algae-based | ||
| + | media is very slow with long adaptation phases (Fig. 4). Also, the OD(600) values exhibit high standard | ||
| + | deviations. As already discussed, this can be explained by algea remnants, such as lipids and cell wall | ||
| + | remnants, which had not been removed properly. The strain is one of the most frequent used | ||
| + | <i>E. coli</i> strains and it grows best on low salt medium <a href="#link-4"><sup>4</sup></a | ||
| + | >. | ||
| + | </p> | ||
</div> | </div> | ||
| + | </div> | ||
</div> | </div> | ||
<div class="columns"> | <div class="columns"> | ||
| − | + | <div class="column"> | |
| − | + | <div> | |
| − | + | <h2 class="title is-5">Freiburg (<i>E. coli</i> DH5-α)</h2> | |
| − | + | <canvas id="team-freiburg"></canvas> | |
| − | + | <small | |
| − | + | >Figure 5: Growth curve of E. coli DH5-α created by the iGEM Team Freiburg. PhyCoVi medium (blue), | |
| − | + | PhyCoVi medium with additional soy peptone (green) and High salt LB medium were tested (black). PhyCoVi | |
| − | + | medium contained 10 g/L additional NaCl, High Salt LB medium contained 20 g/L NaCl.</small | |
| − | + | > | |
| − | + | <br /> | |
| − | + | <p> | |
| − | + | This measurement was conducted in a 96-well plate reader. The quality of both algae-based media are impaired | |
| − | + | by algea lipid and cell wall remnants and therefore show negative growth rates. But thank you, Team | |
| − | + | Freiburg, for conducting the experiment over such a long time course. <br /><br /> | |
| − | + | </p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</div> | </div> | ||
| + | </div> | ||
| + | <div class="column"> | ||
| + | <h2 class="title is-5">Conclusion</h2> | ||
| + | <p> | ||
| + | In conclusion, the collaboration was a full success! It showed that different strains are able to grow in our | ||
| + | developed PhyCoVi medium. After evaluating the collaboration results, we increased our clean-up efforts during | ||
| + | the production of our medium. Algea lipids and cell wall aggregates were removed by including an additional | ||
| + | autoclavation step followed by centrifugation of the medium. | ||
| + | </p> | ||
| + | <figure class="image"> | ||
| + | <img | ||
| + | style="width: 350px; margin: 0 auto;" | ||
| + | src="https://2019.igem.org/wiki/images/thumb/8/89/T--Stuttgart--Collab_Fig7.png/800px-T--Stuttgart--Collab_Fig7.png" | ||
| + | /> | ||
| + | </figure> | ||
| + | <small | ||
| + | >Figure 6: PhyCoVi powder and medium before additional steps for clean-up is displayed on the left side. On | ||
| + | the right side the PhyCoVi powder and medium after additional steps for clean-up can be seen.</small | ||
| + | > | ||
| + | <br /> | ||
| + | <p> | ||
| + | The result of this improvement can be seen in | ||
| + | <a | ||
| + | target="_blank" | ||
| + | href="https://2019.igem.org/wiki/images/thumb/8/89/T--Stuttgart--Collab_Fig7.png/800px-T--Stuttgart--Collab_Fig7.png" | ||
| + | >Fig. 6.</a | ||
| + | > | ||
| + | The left part shows the old version of our medium, which exhibits a higher turbidity as the advanced medium | ||
| + | seen in the right part of | ||
| + | <a | ||
| + | target="_blank" | ||
| + | href="https://2019.igem.org/wiki/images/thumb/8/89/T--Stuttgart--Collab_Fig7.png/800px-T--Stuttgart--Collab_Fig7.png" | ||
| + | >Fig. 6.</a | ||
| + | > | ||
| + | </p> | ||
| + | </div> | ||
</div> | </div> | ||
<div class="columns"> | <div class="columns"> | ||
| − | + | <div class="column"> | |
| − | + | <div class="notification"> | |
| − | + | <h3 class="title is-5">References</h3> | |
| − | + | <ol> | |
| − | + | <li id="link-1"> | |
| − | + | Douglas Hanahan, Joel Jessee, Fredric R. Bloom. Plasmid transformation of Escherichia coli and other | |
| − | + | bacteria, Methods in Enzymology, Academic Press, Volume 204, 1991, Pages 63-113, | |
| − | + | https://doi.org/10.1016/0076-6879(91)04006-A | |
| − | + | </li> | |
| − | + | <li id="link-2"> | |
| − | + | Bury S, Soundararajan M, Bharti R, von Bünau R, Förstner KU, Oelschlaeger TA. The Probiotic | |
| − | + | Escherichia coli Strain Nissle 1917 Combats Lambdoid Bacteriophages stx and λ. Front Microbiol. | |
| − | + | 2018 May 29;9:929. doi: 10.3389/fmicb.2018.00929 | |
| − | + | </li> | |
| − | + | <li id="link-3"> | |
| − | + | Jun Fu, Madeleine Teucher, Konstantinos Anastassiadis, William Skarnes, A. Francis Stewart, Chapter Eight | |
| − | + | - A Recombineering Pipeline to Make Conditional Targeting Constructs, Editor(s): Paul M. Wassarman, | |
| − | + | Philippe M. Soriano, Methods in Enzymology, Academic Press, Volume 477, 2010, Pages 125-144, | |
| − | + | https://doi.org/10.1016/S0076-6879(10)77008-7. | |
| − | + | </li> | |
| − | + | <li id="link-4"> | |
| − | + | New England Biolabs FAQ: What are the solutions/recipes (C2527): | |
| − | + | https://international.neb.com/faqs/2012/02/22/what-are-the-solutions-recipes-c2527, 2019 New England | |
| − | + | Biolabs, last visited 10.15.2019 | |
| − | + | </li> | |
| − | + | </ol> | |
| − | + | ||
</div> | </div> | ||
| + | </div> | ||
</div> | </div> | ||
| − | </div> | + | </div> |
| − | <script> | + | <script> |
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| − | + | 535: { plus: 0.008498366, minus: 0.00849836 } | |
| − | + | } | |
| − | + | }, | |
| − | + | { | |
| − | + | label: 'PhyCoVi+Soy Peptone', | |
| − | + | backgroundColor: window.chartColors.green, | |
| − | + | borderColor: window.chartColors.green, | |
| − | + | fill: false, | |
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| − | + | 1.363333333, | |
| − | + | 1.373333333 | |
| − | + | ], | |
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| − | + | 45: { plus: 0.004082483, minus: 0.004082483 }, | |
| − | + | 90: { plus: 0.012472191, minus: 0.012472191 }, | |
| − | + | 135: { plus: 0.010274023, minus: 0.010274023 }, | |
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| − | + | 315: { plus: 0.006236096, minus: 0.006236096 }, | |
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| − | + | }, | |
| − | + | { | |
| − | + | label: 'High Salt LB', | |
| − | + | backgroundColor: window.chartColors.black, | |
| − | + | borderColor: window.chartColors.black, | |
| − | + | fill: false, | |
| − | + | lineTension: 0, | |
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| − | + | ], | |
| − | + | errorBars: { | |
| − | + | 0: { plus: 0.008498366, minus: 0.008498366 }, | |
| − | + | 45: { plus: 0.014337209, minus: 0.014337209 }, | |
| − | + | 90: { plus: 0.008498366, minus: 0.008498366 }, | |
| − | + | 135: { plus: 0.014719601, minus: 0.014719601 }, | |
| − | + | 180: { plus: 0.012472191, minus: 0.012472191 }, | |
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| − | + | 360: { plus: 0.067986927, minus: 0.067986927 }, | |
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| − | + | 450: { plus: 0.051044643, minus: 0.051044643 }, | |
| − | + | 535: { plus: 0.045521668, minus: 0.045521668 } | |
| − | + | } | |
| − | + | } | |
| − | + | ] | |
| + | }, | ||
| + | options: { | ||
| + | responsive: true, | ||
| + | title: { | ||
| + | display: true, | ||
| + | text: 'iGEM Team TU Dresden E. coli GB05' | ||
}, | }, | ||
| − | + | scales: { | |
| − | + | xAxes: [ | |
| − | + | { | |
| + | scaleLabel: { | ||
display: true, | display: true, | ||
| − | + | labelString: 'Time [min]' | |
| − | + | } | |
| − | + | } | |
| − | + | ], | |
| − | + | yAxes: [ | |
| − | + | { | |
| − | + | scaleLabel: { | |
| − | + | display: true, | |
| − | + | labelString: 'OD600' | |
| − | + | } | |
| − | + | } | |
| − | + | ] | |
| − | + | }, | |
| − | + | plugins: { | |
| − | + | chartJsPluginErrorBars: { | |
| − | + | /** | |
| − | + | * stroke color, or array of colors | |
| − | + | * @default: derived from borderColor | |
| − | + | */ | |
| − | + | color: '#666', | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | /** | |
| − | + | * bar width in pixel as number or string or bar width in percent based on the barchart bars width (max 100%), or array of such definition | |
| − | + | * @default 10 | |
| − | + | */ | |
| − | + | width: 10 | '10px' | '60%', | |
| − | + | /** | |
| − | + | * lineWidth as number, or as string with pixel (px) ending | |
| − | + | */ | |
| − | + | lineWidth: 2 | '2px', | |
| − | + | /** | |
| − | + | * lineWidth as number, or as string with pixel (px) ending, or array of such definition | |
| − | + | * @default 2 | |
| − | + | */ | |
| − | + | lineWidth: 2 | '2px', | |
| − | + | /** | |
| − | + | * whether to interpet the plus/minus values, relative to the value itself (default) or absolute | |
| − | + | * @default false | |
| − | + | */ | |
| − | + | absoluteValues: false | |
| − | + | } | |
| − | + | ||
} | } | ||
| + | } | ||
}; | }; | ||
// FREIBURG | // FREIBURG | ||
var configFreiburg = { | var configFreiburg = { | ||
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| − | + | data: { | |
| − | + | labels: [0, 45, 90, 135, 180, 225, 270, 315, 360, 405, 450, 495, 540, 585, 630, 675, 720, 765, 810, 855], | |
| − | + | datasets: [ | |
| − | + | { | |
| − | + | label: 'PhyCoVi', | |
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| − | + | borderColor: window.chartColors.blue, | |
| − | + | fill: false, | |
| − | + | lineTension: 0, | |
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| − | + | 180: { plus: 0.002596295, minus: 0.002596295 }, | |
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| − | + | 855: { plus: 0.001690569, minus: 0.001690569 } | |
| − | + | } | |
| − | + | }, | |
| − | + | { | |
| − | + | label: 'High Salt LB', | |
| − | + | backgroundColor: window.chartColors.black, | |
| − | + | borderColor: window.chartColors.black, | |
| − | + | fill: false, | |
| − | + | lineTension: 0, | |
| − | + | data: [ | |
| − | + | 0.041666667, | |
| − | + | 0.04911111, | |
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| − | + | 0.291, | |
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| − | + | 0.370777777, | |
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| − | + | 0.367555553, | |
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| − | + | errorBars: { | |
| − | + | 0: { plus: 0.00095258, minus: 0.00095258 }, | |
| − | + | 45: { plus: 0.000643103, minus: 0.000643103 }, | |
| − | + | 90: { plus: 0.000549974, minus: 0.000549974 }, | |
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| − | + | 270: { plus: 0.00244949, minus: 0.00244949 }, | |
| − | + | 315: { plus: 0.003767143, minus: 0.003767143 }, | |
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| − | + | 450: { plus: 0.008495097, minus: 0.008495097 }, | |
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| − | + | 630: { plus: 0.009464359, minus: 0.009464359 }, | |
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| − | + | 810: { plus: 0.012412906, minus: 0.012412906 }, | |
| − | + | 855: { plus: 0.013718286, minus: 0.013718286 } | |
| − | + | } | |
| − | + | } | |
| − | + | ] | |
| − | + | }, | |
| − | + | options: { | |
| − | + | responsive: true, | |
| − | + | title: { | |
| − | + | display: true, | |
| − | + | text: 'iGEM Team Freiburg E. coli C321.ΔA' | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
}, | }, | ||
| − | + | scales: { | |
| − | + | xAxes: [ | |
| − | + | { | |
| + | scaleLabel: { | ||
display: true, | display: true, | ||
| − | + | labelString: 'Time [min]' | |
| − | + | } | |
| − | + | } | |
| − | + | ], | |
| − | + | yAxes: [ | |
| − | + | { | |
| − | + | scaleLabel: { | |
| − | + | display: true, | |
| − | + | labelString: 'OD600' | |
| − | + | } | |
| − | + | } | |
| − | + | ] | |
| − | + | }, | |
| − | + | plugins: { | |
| − | + | chartJsPluginErrorBars: { | |
| − | + | /** | |
| − | + | * stroke color, or array of colors | |
| − | + | * @default: derived from borderColor | |
| − | + | */ | |
| − | + | color: '#666', | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | /** | |
| − | + | * bar width in pixel as number or string or bar width in percent based on the barchart bars width (max 100%), or array of such definition | |
| − | + | * @default 10 | |
| − | + | */ | |
| − | + | width: 10 | '10px' | '60%', | |
| − | + | /** | |
| − | + | * lineWidth as number, or as string with pixel (px) ending | |
| − | + | */ | |
| − | + | lineWidth: 2 | '2px', | |
| − | + | /** | |
| − | + | * lineWidth as number, or as string with pixel (px) ending, or array of such definition | |
| − | + | * @default 2 | |
| − | + | */ | |
| − | + | lineWidth: 2 | '2px', | |
| − | + | /** | |
| − | + | * whether to interpet the plus/minus values, relative to the value itself (default) or absolute | |
| − | + | * @default false | |
| − | + | */ | |
| − | + | absoluteValues: false | |
| − | + | } | |
| − | + | ||
} | } | ||
| + | } | ||
}; | }; | ||
// Tuebingen | // Tuebingen | ||
var configTuebingen = { | var configTuebingen = { | ||
| − | + | type: 'line', | |
| − | + | data: { | |
| − | + | labels: [0, 45, 90, 135, 180, 225, 270, 315, 360], | |
| − | + | datasets: [ | |
| − | + | { | |
| − | + | label: 'PhyCoVi', | |
| − | + | backgroundColor: window.chartColors.blue, | |
| − | + | borderColor: window.chartColors.blue, | |
| − | + | fill: false, | |
| − | + | lineTension: 0, | |
| − | + | data: [-0.56, -0.506666667, -0.296666667, -0.47, -0.07, -0.363333333, -0.443333333, -0.623333333, -0.43], | |
| − | + | errorBars: { | |
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| − | + | 45: { plus: 0.080138769, minus: 0.080138769 }, | |
| − | + | 90: { plus: 0.046963343, minus: 0.046963343 }, | |
| − | + | 135: { plus: 0.12980755, minus: 0.12980755 }, | |
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| − | + | 225: { plus: 0.013123346, minus: 0.013123346 }, | |
| − | + | 270: { plus: 0.015456031, minus: 0.015456031 }, | |
| − | + | 315: { plus: 0.132056385, minus: 0.132056385 }, | |
| − | + | 360: { plus: 0.06794605, minus: 0.067946057 } | |
| − | + | } | |
| − | + | }, | |
| − | + | { | |
| − | + | label: 'PhyCoVi+Soy Peptone', | |
| − | + | backgroundColor: window.chartColors.green, | |
| − | + | borderColor: window.chartColors.green, | |
| − | + | fill: false, | |
| − | + | lineTension: 0, | |
| − | + | data: [-0.133333333, 0.18, 0.606666667, 0.68, 0.56, 1.253333333, 1.306666667, 1.14, 1.393333333], | |
| − | + | errorBars: { | |
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| − | + | 45: { plus: 0.016329932, minus: 0.016329932 }, | |
| − | + | 90: { plus: 0.086055538, minus: 0.086055538 }, | |
| − | + | 135: { plus: 0.159216833, minus: 0.159216833 }, | |
| − | + | 180: { plus: 0.044158804, minus: 0.044158804 }, | |
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| − | + | 270: { plus: 0.176839538, minus: 0.176839538 }, | |
| − | + | 315: { plus: 0.195746435, minus: 0.195746435 }, | |
| − | + | 360: { plus: 0.217191672, minus: 0.21719167 } | |
| − | + | } | |
| − | + | }, | |
| − | + | { | |
| − | + | label: 'High Salt LB', | |
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| − | + | ], | |
| − | + | errorBars: { | |
| − | + | 0: { plus: 0.012472191, minus: 0.012472191 }, | |
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Revision as of 20:38, 21 October 2019
Collaboration
Validation of our new algae based PhyCoVi medium
Besides our own experiments with the algae extract, we wanted to have a cross-validation from other iGEM teams and their organisms. Through this, we can make sure, that everybody can work with our medium and also improve the product itself, if problems occur. Kindly, the iGEM teams of Hamburg, Tübingen, TU Darmstadt, Freiburg, and TU Dresden offered their help. Therefore, we prepared 15 ml tubes with two variations of PhyCoVi and a LB control dry medium and sent them to the teams, respectively. Additionally, they received a SOP for their growth curve including an Excel sheet to plot their results directly. Before we discuss their results individually, it can be said, that we are happy with every result and already improved the product concerning the issues that occurred for some teams.
Hamburg (E. coli DH5-α)
Figure 1: Growth curve of E. coli DH5-α created by the iGEM Team Hamburg. PhyCoVi medium (blue), PhyCoVi medium with additional soy peptone (green) and High salt LB medium were tested (black). PhyCoVi medium contained 10 g/L additional NaCl, High Salt LB medium contained 20 g/L NaCl.As seen in Fig. 1, E. coli was able to grow in all three media. The PhyCoVi medium including additional soy peptone and the high salt LB medium show similar growth curves after 45 minutes. The PhyCoVi medium without supplements shows a very long lag phase, growth occurs after 135 minutes. The DH5-α strain is very robust and optimized for optimal transformation 1. These optimizations also seem to have a positive effect on high salt concentrations.
Tübingen (E. coli Nissle 1917)
Figure 2: Growth curve of E. coli Nissle 1917 created by the iGEM Team Tübingen. PhyCoVi medium (blue), PhyCoVi medium with additional soy peptone (green) and High salt LB medium were tested (black). PhyCoVi medium contained 10 g/L additional NaCl, High Salt LB medium contained 20 g/L NaCl.Fig. 2 shows the growth curves by Team Tübingen. Probably due to algae lipid residues in the PhyCoVi medium samples, OD(600) measurements reveal negative values. But still, growth of E. coli Nissle 1917 can be observed in the PhyCoVi medium supplemented with soy peptone. As described 2, this wildtype strain has a low tolerance for high salt medium, therefore the reduced growth is expected.
TU Dresden (E. coli GB05)
Figure 3: Growth curve of E. coli GB05 created by the iGEM Team TU Dresden. PhyCoVi medium (blue), PhyCoVi medium with additional soy peptone (green) and High salt LB medium were tested (black). PhyCoVi medium contained 10 g/L additional NaCl, High Salt LB medium contained 20 g/L NaCl.The results of the iGEM Team TU Dresden shows very low growth for all three media, possibly caused by the high salt concentrations (Fig. 3). After a long adaptation phase, E. coli GB05 was able to grow in high salt LB medium. Growth in PhyCoVi medium supplemented with soy peptone shows high fluctuations, probably due to the already discussed problem with insoluble medium compounds. The used strain is able to take up linear DNA fragments but seems not to be able to cope with high salt media 3.
TU Darmstadt (E. coli BL21 (DE3))
Figure 4: Growth curve of E. coli BL21 (DE3) created by the iGEM TU Darmstadt. PhyCoVi medium (blue), PhyCoVi medium with additional soy peptone (green) and High salt LB medium were tested (black). PhyCoVi medium contained 10 g/L additional NaCl, High Salt LB medium contained 20 g/L NaCl.While E. coli BL21 (DE3) in High Salt LB medium reaches high OD values, growth in both algae-based media is very slow with long adaptation phases (Fig. 4). Also, the OD(600) values exhibit high standard deviations. As already discussed, this can be explained by algea remnants, such as lipids and cell wall remnants, which had not been removed properly. The strain is one of the most frequent used E. coli strains and it grows best on low salt medium 4.
Freiburg (E. coli DH5-α)
Figure 5: Growth curve of E. coli DH5-α created by the iGEM Team Freiburg. PhyCoVi medium (blue), PhyCoVi medium with additional soy peptone (green) and High salt LB medium were tested (black). PhyCoVi medium contained 10 g/L additional NaCl, High Salt LB medium contained 20 g/L NaCl.
This measurement was conducted in a 96-well plate reader. The quality of both algae-based media are impaired
by algea lipid and cell wall remnants and therefore show negative growth rates. But thank you, Team
Freiburg, for conducting the experiment over such a long time course.
Conclusion
In conclusion, the collaboration was a full success! It showed that different strains are able to grow in our developed PhyCoVi medium. After evaluating the collaboration results, we increased our clean-up efforts during the production of our medium. Algea lipids and cell wall aggregates were removed by including an additional autoclavation step followed by centrifugation of the medium.
The result of this improvement can be seen in Fig. 6. The left part shows the old version of our medium, which exhibits a higher turbidity as the advanced medium seen in the right part of Fig. 6.
References
- Douglas Hanahan, Joel Jessee, Fredric R. Bloom. Plasmid transformation of Escherichia coli and other bacteria, Methods in Enzymology, Academic Press, Volume 204, 1991, Pages 63-113, https://doi.org/10.1016/0076-6879(91)04006-A
- Bury S, Soundararajan M, Bharti R, von Bünau R, Förstner KU, Oelschlaeger TA. The Probiotic Escherichia coli Strain Nissle 1917 Combats Lambdoid Bacteriophages stx and λ. Front Microbiol. 2018 May 29;9:929. doi: 10.3389/fmicb.2018.00929
- Jun Fu, Madeleine Teucher, Konstantinos Anastassiadis, William Skarnes, A. Francis Stewart, Chapter Eight - A Recombineering Pipeline to Make Conditional Targeting Constructs, Editor(s): Paul M. Wassarman, Philippe M. Soriano, Methods in Enzymology, Academic Press, Volume 477, 2010, Pages 125-144, https://doi.org/10.1016/S0076-6879(10)77008-7.
- New England Biolabs FAQ: What are the solutions/recipes (C2527): https://international.neb.com/faqs/2012/02/22/what-are-the-solutions-recipes-c2527, 2019 New England Biolabs, last visited 10.15.2019