Difference between revisions of "Team:UCL/Fermentation"

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<li>Before incubating liquid cultures for the fermentation we carried out measurements in LB (Luria Broth) and TB (terrific broth) to establish what medium is ideal for bacterial growth.</li>
 
<li>Before incubating liquid cultures for the fermentation we carried out measurements in LB (Luria Broth) and TB (terrific broth) to establish what medium is ideal for bacterial growth.</li>
 
<li>We then inoculated the first liquid culture into 250ml shakeflasks using glycerol stocks and gradually scaled up the volume, see picture</li>
 
<li>We then inoculated the first liquid culture into 250ml shakeflasks using glycerol stocks and gradually scaled up the volume, see picture</li>
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<img src="https://static.igem.org/mediawiki/2015/f/f6/Scale_up.png" style="width:100%;">
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<img src="https://static.igem.org/mediawiki/2015/f/f6/Scale_up.png" style="width:20%;">
 
<li>We set up the initial parameters for the fermentation, including pH, DO, temperature and rotational frequency. To view the exact set-up of the fermentor, go to <a href="https://2015.igem.org/Team:UCL/Protocols"><b>Protocols</b></a>.</li>
 
<li>We set up the initial parameters for the fermentation, including pH, DO, temperature and rotational frequency. To view the exact set-up of the fermentor, go to <a href="https://2015.igem.org/Team:UCL/Protocols"><b>Protocols</b></a>.</li>
 
<li>Over a period of 15 hours we measured the OD600 of the culture to determine the growth rate. After six hours we induced a pH shock from pH 6.95 to 5 within 45 minutes and afterwards a sudden increase back to base within 10 minutes. Especially the sudden increase was meant to simulate the transition from the stomach into the upper intestine.</li>
 
<li>Over a period of 15 hours we measured the OD600 of the culture to determine the growth rate. After six hours we induced a pH shock from pH 6.95 to 5 within 45 minutes and afterwards a sudden increase back to base within 10 minutes. Especially the sudden increase was meant to simulate the transition from the stomach into the upper intestine.</li>

Revision as of 01:32, 19 September 2015

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Scaling Up

Why bother going big?

After creating our biobricks and expressing them in probiotics we needed to test two essential assumptions of our project. The first was that our bacteria can survive a shock of pH and oxygen concentration which they would need to go through when traveling through the digestive system.

The other assumption was that we could create the right growth conditions for the bacteria to produce them in large amounts. The latter point would be necessary in order to mass-produce bacteria commercially which is essential for our entrepreneurial effort

.
The problem we faced was that we could not control these conditions precisely or without great effort, as a normal incubator allows only control of temperature and shaking frequency. How could we overcome these problems?


Using a bioreactor

We were able to successfully test our assumptions on a larger scale than usually possible in research labs by using a 7l bioreactor. This allowed us to simulate the conditions existing in the transition from one gut-compartment into the other, under controlled conditions. Secondly, we could show that a large scale production of our probiotic bacteria is possible.

Procedure

  1. Before incubating liquid cultures for the fermentation we carried out measurements in LB (Luria Broth) and TB (terrific broth) to establish what medium is ideal for bacterial growth.
  2. We then inoculated the first liquid culture into 250ml shakeflasks using glycerol stocks and gradually scaled up the volume, see picture
  3. We set up the initial parameters for the fermentation, including pH, DO, temperature and rotational frequency. To view the exact set-up of the fermentor, go to Protocols.
  4. Over a period of 15 hours we measured the OD600 of the culture to determine the growth rate. After six hours we induced a pH shock from pH 6.95 to 5 within 45 minutes and afterwards a sudden increase back to base within 10 minutes. Especially the sudden increase was meant to simulate the transition from the stomach into the upper intestine.
  5. Shortly after we induced a DO shock to see whether bacteria could recover from such a shock which is relevant as oxygen concentrations can vary strongly in the lumen of the gut. [1]