Difference between revisions of "Team:Munich/Results"

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Cultivation
 
Cultivation
 
</h3>
 
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<p>Cultivation is the first step in cell extract preparation. The original cell extract preparation protocol uses shaking flask cultivation for biomass production and states that cell harvest at OD 1,8-2,0 is strictly required to produce high-quality extract. To be able to transfer this to the bioreactor we first obtained growth data for both shaking flask and bioreactor cultivation.
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</p>
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<p>growh fermenter vs. shaking flask side by side with FI mtq2</p>
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<p>The growth curve from shaking flask cultivation showed us that harvest at OD 1,8-2 correlates to the mid-to late logarithmic growth phase. In bioreactor fermentation this correlates to OD between 4 and 6. We decided to test which of these ODs is best to harvest cells for cell extract preparation.
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</p>
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<p>
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This experiment gave 2 important results:
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</p>
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<p>
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<ul style="">
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<li>the optimal OD to harvest culture from the bioreactor is around 5. This gives the highest protein content and also the best expression.</li>
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<li>preparing cell extract from bioreactor cultivated cells under comparable conditions gives equal quality extract than cell extract from shaking flask cultivated cells.</li>
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</ul>
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</p>
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<p>
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In our small lab-scale bioreactor with 2 L cultivation volume we were able to obtain 20 g cell pellet. 2 L cultivation in shaking flasks only yields 4,5 g pellet.
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</p>
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<h3>
 
<h3>
 
Lysis
 
Lysis
 
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</h3>
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<p>
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Of the three commonly used methods of cell lysis, each has teir advantages and drawbacks. For cell extract preparation bead beating is most established, but the caveat there, is that it doesn’t leave room for upscaling, besides being tedious and time-consuming. Therefore, we decided early on to focus our optimization approach on Sonication as method for cell lysis.
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</p>
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<h3>
 
<h3>
 
Processing
 
Processing
 
</h3>
 
</h3>
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<p>
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The suggested steps in cell extract processing are:
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</p>
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<p>
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<ul style="">
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<li>run-off reaction</li>
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<li>dialysis</li>
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<li>storage at -80°C</li>
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</ul>
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</p>
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<p>
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We found that the run-off reaction as suggested in the Sun et.al paper is indeed beneficial and that dialysis is superior to diafiltration via centrifugal filters.
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</p>
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<h3>
 
Quality Control
 
Quality Control
 
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</h3>
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<h3>
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Storage
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</h3>
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<p>
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For long term storage of our cell-extract we decided to try lyophilization. After initial tests we found that:
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</p>
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<p>
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<ul style="">
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<li>cell extract quality is only preserved when cell-extract is mixed with the txtl- reaction buffer prior to lyophilization. </li>
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<li>the retention of quality does not depend on the size of lyophilized extract aliquots.</li>
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<li>We then tested the expression quality of several of our home-made cell extracts from fresh vs. lyophilized aliquots.</li>
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</ul>
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</p>
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<p>
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plot: lyophilisation
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</p>
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<p>
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Our two tested samples of cell extract retained 70 and 90 % of expression quality respectively after lyophilization.
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</p>
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<h2>
 
<h2>
 
Bacteriophage Assembly
 
Bacteriophage Assembly

Revision as of 11:22, 16 October 2018

Phactory

Results

First Blick of Phactory


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