Difference between revisions of "Team:UCL/Fermentation"

 
(26 intermediate revisions by 3 users not shown)
Line 1: Line 1:
 
{{CSS_UCL6}}
 
{{CSS_UCL6}}
 
<html>
 
<html>
 +
<head>
 +
  <style>
 +
   
 +
#header2 {
  
 +
width: 100%;
 +
position: relative;
 +
margin: 0;
 +
padding: 0;
 +
top: 0;
 +
bottom: 0;
 +
margin: 0 auto;
 +
min-height: 100%;
 +
height: auto;
 +
vertical-align: middle;
 +
font-family: Raleway;
 +
letter-spacing: 1.1px; font-size: 13px;
 +
}
 +
 +
 +
 +
#header2:before
 +
{
 +
content: "";
 +
    background-image: url('https://static.igem.org/mediawiki/2015/b/bb/UCL_2015_Scaling_up_background.jpeg');
 +
  background-repeat: no-repeat;
 +
  background-attachment: fixed !important;
 +
  -webkit-background-size: cover;
 +
  -moz-background-size: cover;
 +
  -o-background-size: cover;
 +
  background-size: cover;
 +
-webkit-box-sizing: border-box;
 +
-moz-box-sizing: border-box;
 +
box-sizing: border-box;
 +
-webkit-mask-image: -webkit-gradient(linear, 0 0, 0 1200, from(rgba(0,0,0,1)), to(rgba(0,0,0,0.2)));
 +
    position: absolute;
 +
    top: 0px;
 +
    left: 0px;
 +
margin: 0;
 +
padding: 0;
 +
    width: 100%;
 +
    height: 100%;
 +
pointer-events: none;
 +
overflow: hidden;
 +
display: block;
 +
border: none;}
 +
 +
#mobilebackground {
 +
  background-image: url('https://static.igem.org/mediawiki/2015/a/a6/UCL_MG_1643_2.jpg');
 +
-webkit-mask-image: -webkit-gradient(linear, 0 0, 0 300, from(rgba(0,0,0,1)), to(rgba(0,0,0,0.2)));
 +
 +
    top: 0;
 +
    right: 0;
 +
    left: 0;
 +
    background-position: 0 0 0 0;
 +
position: fixed;
 +
    background-repeat: no-repeat;
 +
  -webkit-background-size: cover;
 +
  -moz-background-size: cover;
 +
  -o-background-size: cover;
 +
  background-size: cover;
 +
-webkit-box-sizing: border-box;
 +
-moz-box-sizing: border-box;
 +
box-sizing: border-box;
 +
display: none;
 +
width: 100%;
 +
height: 100%;
 +
 +
  }
 +
 +
 +
   
 +
.mainmenu {background-color: #fff !important;}
 +
.menu-items a {color: #493C54;}
 +
.buttonblack {background-color: #22343C; border-color: #22343C; }
 +
a.buttonblack { color: #fff !important; font-size: 12px;}
 +
.buttonblack:hover {border-color: #22343C; color: #22343C !important;}
 +
 +
 +
 +
 +
 +
#wrapper {width: 80%; height: 80%; margin: 0 auto; position: relative; top: 80px; opacity: 1; line-height: 2;}
 +
 +
#submenu {width: 100%; margin-top: 20px; text-align: center; position: relative;}
 +
 +
#text {margin-top: 50px; z-index: 9999;}
 +
#text h2 {font-size: 25px; text-decoration: none; border-bottom: 0px; display: block; background-color: #c97062; color: #fff; font-weight: bold; border-radius: 4px; padding: 10px; opacity: 0.5; }
 +
 +
.blockk:hover h2 {opacity: 0.9 !important; transition-property: opacity;
 +
    transition-duration: 1s;
 +
    transition-timing-function: ease; }
 +
 +
#text h4 {text-decoration: none; font-size: 14px; font-weight: normal !important; border-bottom: 0px; display: block;  padding-left: 5px; padding-top: 5px; padding-bottom: 5px; line-height: 2; text-align: justify; color: #272a2b}
 +
 +
  </style>
 +
  </head>
 +
 
 +
   
 
<body>
 
<body>
−
<!--
+
 
 
<div id="mobilebackground"></div>
 
<div id="mobilebackground"></div>
 
<div id="header2">
 
<div id="header2">
Line 10: Line 108:
  
 
<div class="bigtitle">
 
<div class="bigtitle">
−
<div class="titlecircle;"><span class="title2">Scaling Up</span> <p style="margin-top: -100px; line-height: 1.7;font-family:Raleway;">
+
<div class="titlecircle"><span class="title2">Scaling Up</span> <p style="margin-top: -100px; line-height: 1.7;font-family:Raleway;">
 
   </p>
 
   </p>
 
   </div>
 
   </div>
Line 17: Line 115:
 
<div id="text">
 
<div id="text">
 
<div class="blockk">
 
<div class="blockk">
−
<div id="text">
+
  <h2><span id="background" style="padding-top:150px;">Why bother going big?</span></h2>
−
<h3>Why bother going big?</h3>
+
  <br/>
 +
<h4>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.</h4>
 +
<h4>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 <a href="https://2015.igem.org/Team:UCL/Entrepreneurship"><b>entrepreneurial effort</b></a></h4>.
 +
<br/>
 +
<h4>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?
 +
  </h4>
 +
  <br/>
  
−
<p>After creating our biobricks and expressed them in bacteria 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.</p>
+
  <h2><span id="background" style="padding-top:150px;">Using a bioreactor</span></h2>
−
<p>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 <a href="https://2015.igem.org/Team:UCL/Entrepreneurship">entrepreneurial effort</a>.</p>
+
 
<br/>
 
<br/>
−
<p>The problem we faced was that we couldn't 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?
+
  <h4>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.</h4>
 +
 
 
<br/>
 
<br/>
 +
<h2><span id="background" style="padding-top:150px;">Procedure</span></h2>
 +
<br/>
 +
  <h4>
 +
<ol>
 +
<li>For the fermentation in the bioreactor we used DH5alpha cells transformed with a plasmid containing an expression cassette for our novel biobrick TPH1 downstream of an IPTG promoter.</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 the growth of the bacteria cultures.</li>
 +
<li>We then inoculated the first liquid culture into 250ml shakeflasks using glycerol stocks and gradually scaled up the volume, see picture</li>
 +
<img src="https://static.igem.org/mediawiki/2015/f/f6/Scale_up.png" style="width:40%;">
 +
<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>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.
 +
<a href="http://www.sciencedirect.com/science/article/pii/S0891584912017960" target="_blank">[1]</a></li>
 +
</ol>
 +
  </h4>
 +
<br/>
 +
  <h2><span id="background" style="padding-top:150px;">Measurement results</span></h2>
 +
<br/>
 +
  <h4 style="font-size:20px">pH shock</h4>
 +
<br/>
 +
<img src="https://static.igem.org/mediawiki/2015/7/7e/PH_shock.PNG" alt="Cell growth and pH shock" style="width:40%;">
 +
<br/>
 +
<h4>Immediately after reducing pH from 6.95 to 5 the culture did not seems to slow down in growth much. The OD measurement following the sudden increase showed a smaller cell density which is likely to be linked to decreased growth.</h4>
 +
<h4>The OD in the measurement taken an hour after returning the pH of the medium back to 6.96 was higher than immediately after the shock.</h4>
  
−
<h3>Using a bioreactor</h3>
+
<br/>
−
<p>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.
+
<h3>DO shock</h3>
−
Secondly, we could show that a large scale production of our probiotic bacteria is possible.</p>
+
<br/>
  
−
</div>
+
<img src="https://static.igem.org/mediawiki/2015/6/6b/DO-shock.PNG" alt="Cell growth and DO shock" style="width;30%;">
−
  </div>
+
−
  </div>
+
−
  </div>  -->
+
  
−
<div id="mobilebackground"></div>
+
<br/>
−
<div id="header2">
+
<h4>The reduction in oxygen concentration was carried out after measuring an increase in OD after the pH induced shock. The cell growth was smaller than during exponential phase after the reduction of oxygen. However, growth still continued at a slower rate after.</h4>
 +
<br/>
  
−
<div id="wrapper">
+
  <h2><span id="background" style="padding-top:150px;">Discussion</span></h2>
−
 
+
<br/>
−
<div class="bigtitle">
+
<h4> The experiment showed that we could successfully grow engineered probiotics at large scale which would allow us to produce engineered bacteria in commercially relevant amounts. The cultures survived the shock of pH, similar to certain transitions in the gut and maintained growing after. Even the complete absence of oxygen flow for half an hour did not kill the culture although diminishing growth.</h4>
−
  <div class="titlecircle"><span class="title2" style="line-height:80px;">Scaling Up</span><hr style="height:2px; border:none; color:#22343; background-color:#000;"><p style="margin-top: 5%; line-height: 1.7;font-family:Raleway;"><br><span class="arrow"> <a data-scroll data-options='{ "easing": "linear" }' href="#submenu" >&#x25BC; </a></span>
+
  
−
</p> 
+
<div style="height:50px"></div>
−
 
+
−
</div></div>
+
−
<div id="submenu">
+
 
</div>
 
</div>
−
 
+
  </div>
−
 
+
   </div>
−
<div id="text">
+
−
   <div class="blockk">
+
−
<h7><span id="background" style="font-family:font-family: 'Lato', sans-serif;;">Background</span> </h7>
+
−
<hr style="height:1px; border:none; color:#22343; background-color:#000;">
+
−
    <br>
+
−
    <h4> After creating our biobricks and expressed them in bacteria 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.
+
−
    </h4>
+
−
<br>
+
−
    <br>
+
 
   </div>
 
   </div>
  
−
<div class="blockk">
 
−
<h7 style="font-size:200%;"><span style="margin-top: -100px; font-family:font-family: 'Lato', sans-serif;font-weight:100;">Infamous Insidious <!--<u> --><b style="color:#22343;">Inflammation</b><!--</u>--></span> </h7>
 
−
<hr style="height:1px; border:none; color:#22343; background-color:#000;">
 
−
  <br>
 
−
 
 
−
<h4> Clinical depression is likely caused by a chronic low grade-response to inflammation
 
−
 
−
<span class="content"><span class="tooltip" style="z-value:1000000;"><a href="http://www.biomedcentral.com/1741-7015/11/200" target="_blank"><b>[Link]</b></a> M. Berk et al. So depression is an inflammatory disease, but where does the inflammation come from? in European Journal for Medical Research, 2013 </span><b>[1]</b></span>.
 
  
−
Although the pathway from inflammation to depression is complex and not fully understood it has been shown that the immune response is often accompanied by symptoms such as oxidative and nitrosative stress in the gut.
 
−
</h4>
 
 
</body>   
 
</body>   
  
 
</html>
 
</html>

Latest revision as of 19:52, 16 November 2015

'

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. For the fermentation in the bioreactor we used DH5alpha cells transformed with a plasmid containing an expression cassette for our novel biobrick TPH1 downstream of an IPTG promoter.
  2. 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 the growth of the bacteria cultures.
  3. We then inoculated the first liquid culture into 250ml shakeflasks using glycerol stocks and gradually scaled up the volume, see picture
  4. 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.
  5. 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.
  6. 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]


Measurement results


pH shock


Cell growth and pH shock

Immediately after reducing pH from 6.95 to 5 the culture did not seems to slow down in growth much. The OD measurement following the sudden increase showed a smaller cell density which is likely to be linked to decreased growth.

The OD in the measurement taken an hour after returning the pH of the medium back to 6.96 was higher than immediately after the shock.


DO shock


Cell growth and DO shock

The reduction in oxygen concentration was carried out after measuring an increase in OD after the pH induced shock. The cell growth was smaller than during exponential phase after the reduction of oxygen. However, growth still continued at a slower rate after.


Discussion


The experiment showed that we could successfully grow engineered probiotics at large scale which would allow us to produce engineered bacteria in commercially relevant amounts. The cultures survived the shock of pH, similar to certain transitions in the gut and maintained growing after. Even the complete absence of oxygen flow for half an hour did not kill the culture although diminishing growth.