Team:Valencia/Project2

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<h1><b>pH-stat: Culture of <i>Synechocystis sp.</i> PCC 6803</b></h1>
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<h2><b>Objectives of  the culture</b></h2>
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<b>General</b>
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  <p>By  introducing  a culture of Synechocystis sp cianobaceria. PCC 6803 we intent  to ensure that the pH changes resulting from growth and proliferation  function  as  a switch  to enable the denaturation of the colicins, so that in the artificial system created, as time passes, the colicins produced by Escherichia coli  will increase their concentration in their  habitat, being inactive until the pH reaches the optimum range of activation, which is when they ´ll get  activated and will produce cell lysis and kill the pathogens. </p>
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<b>Specific</b>
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To develop  what we have  stated above, we need to know:  
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• How to establish  the culture at the laboratory
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• The  temporal evolution  of  the pH in the culture.
 
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<h2><b>Establishing  the culture  under laboratory conditions.</b></h2>
 
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The  material and methods  needed are :
 
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• 18 Watts  fluorescent tubes of white light, special tubes for aquariums  that divide the spectrum mostly between the peaks of the visible  red and blue  light,  which stimulate the  photosynthesis.
 
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• Volumetric flasks, 100 ml and 200 ml
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• Commercial Fertilizer Brand COMPO
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• Distilled, tap and analytical  water (Type II)  
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<div class=col_center_top><b>pH-Stat: Controlling pH through photons</b></div><!-- fin clase col_left_top-->
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<p>As you can see on the following figure, we have effectively accomplished the control of pH through the autotrophic growth of <i>Synechocystis</i> sp. PCC 6803</p>. Thus opening the door to the construction of the CopH, the controller of pH.
 +
 
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<center><img src="https://static.igem.org/mediawiki/2011/4/45/Valencia_PHvariation.png" width="600"/></center>
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<b>Why is this important?</b>
 +
<p>Well, we wanted to use pH as a switch for the action of li <a href="https://2011.igem.org/Team:Valencia/Project1 " TARGET="_blank" title="Bacteriocins ">bacteriocins</a>.</p><br/><br/>
 +
 
 +
<b>How have we done this?</b>
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<p><i>Short answer</i>: cyanobacteria growing <b>only</b> on light, CO<sub>2</sub>, commercial fertilizer and H<sub>2</sub>O</p>
 +
<p><i>Long answer</i>: keep reading!<br/></p>
 +
 
 +
<h2>Objectives of the culture</h2>
 +
<p>By introducing a culture of cyanobaceria <i>Synechocystis</i> sp. PCC 6803 we intent to ensure that the pH changes resulting from growth and proliferation function as a switch to enable the denaturation of the colicins. The objective is that, as time goes by, the colicins produced by <i>Escherichia coli</i> will be produced but inactive until the pH reaches the optimum range of activation, thus getting activated and producing cell lysis and killing pathogens. </p>
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<p>In addition, our cultures, to make low-cost bioreactor were performed in a fed-batch type, in which the addition of nutrientres occurs periodically, so that the objetive of this is to maintain its exponential growth phase without reaching the limit maximum load.</p>
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In order to develop what we have stated above, we need to know:
 +
<ul>
 +
<li>How to establish the culture at the laboratory</li>
 +
<li>The temporal evolution of the pH in the culture</li>
 +
</ul>
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<p>The first experiment consisted of a series of six cultures in which environmental conditions varied so as to know the habitat preferences of the cyanobacterium. We distinguished  two different groups: </p>
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<h2>Growing cyanobacteria under laboratory conditions</h2>  
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#<p>The first, consisting of  C1, C2 and C3, which  have  tap , distilled and analytical  water respectively. It also uses a reflector box, which increases the irradiation of light on the culture, and a magnetic stirrer, which prevents the deposition of cells on the bottom.</p>  
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<p>The materials used were: </p>
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#<p>The  second group , which are C4, C5 and C6, without reflector  or  magnetic stirrer, but we  kept the water  in the same order as before.</p>  
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<ul>
 +
<li>18W fluorescent tubes of white light, special tubes for aquariums that divide the spectrum mostly between the peaks of the visible red and blue light, which stimulate the photosynthesis.</li>
 +
<li>100 ml and 200 ml flasks</li>
 +
<li>Air Pumps</li>
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<li>Commercial COMPO Fertilizer</li>
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<li>Distilled, tap and Type II analytical water</li>
 +
<li>Cardboard boxes</li>
 +
<li>Household aluminium foil</li></ul>
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<h3>Results and discussion</h3>  
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<p>We decided to perform fed-batch growth, in which the addition of nutrientres occurs periodically, so that we could maintain cyanobacterial exponential growth phase as long as possible without reaching the maximum load limit.<br/>
 +
We designed a low-cost photobioreactor using our lamps, cardboard boxes and aluminum foil in ordert to build a reflector box in which we made all our measurements</p>
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<p>The  other conditions remained  constant in both cases, as are detailed in the following table:</p>
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<h3><b>Study of growth parameters</b></h3>
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<p>Our first experiment consisted of a growth variables study so as to know the habitat preferences of the cyanobacterium. We made six cultures with different media conditions and we looked at its effects on growth. We distinguished two different groups: </p>
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[[Image:Valencia_Synecho_Tabla_1.jpg|center]]
 
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<p>The day after having inoculated the culture, the absorbency data were collected and  the cells / ml  counted with a Neubauer chamber. The absorbancy was measured at 440 nm and 750 nm as the first was the highest value according to the spectrophotometer after a sweep, and the second was taken  according to references (Burrows, EH, et. Al., 2009, JF Allen, 2008). In the account we distinguished  between simple cells, ie those which are not in the reproductive period, and those  which are.</p>
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<p> 1. First group, consisting of cultures C1, C2 and C3, which have tap, distilled and analytical water, respectively. It also uses a reflector box, which increases the irradiation of light on the culture, and a magnetic stirrer, which prevents the deposition of cells on the bottom.</p></li>
 +
<p> 2. The second one, made up of cultures C4, C5 and C6, without reflector box or magnetic stirrer.</p></li>
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<center><img src="https://static.igem.org/mediawiki/2011/8/81/Valencia_Synecho_Tabla_1.jpg" width="600"/></center>
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[[Image:Valencia_Grafica_750_exp1.gif|center]]
 
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<p>Cultures is seen as 1, 2, 4 and 5, tend to decrease in chlorophyll content, an indicator of the concentration of the substance diminishes over time in our culture, ie, they tend to die out. In contrast, cultures 3 and 6, not only survive, but also increases its concentration according to the evidence given by the spectrophotometer, showing a growth in them.</p>
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<p>The day after having inoculated the culture, absorbancy data were collected and cell concentration measured on a Neubauer chamber. The absorbancy was measured at 440 nm and 750 nm as the first was the highest absorbancy value after a spectrophotometer sweep and the second was taken according to references (Burrows, EH, et al., 2009 & JF Allen, 2008).</p>
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<p>The following two graphs show the number of cells <i> Synechocystis </i> sp., counted with the Neubauer chamber and a microscope. The first picture shows the cells in the reproductive or Siamese, which, as noted, tend to increase in number while they are not reproducing tend to decrease, as shown in the second graph.</p>
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<center><img src="https://static.igem.org/mediawiki/2011/a/a8/Valencia_Grafica_750_exp1.gif" width="420"/></center>
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[[Image:Valencia_Grafica_Growing_exp1.gif|center]]
 
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[[Image:Valencia_Grafica_Not_growing_exp1.gif|center]]
 
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<p>Cultures C1, C2, C4 and C5 tend to decrease in chlorophyll content, an indicator of the concentration of cells diminishes over time, i.e., they are dying. In contrast, cultures C3 and C6, not only survive, but also increase their concentration, thus they grow.</p>
 +
<p>We distinguished between simple cells, i.e. those which are in the reproductive period and those which are not. The following two graphs show the number of growing and non-growing <i>Synechocystis</i> cells, counted with the Neubauer chamber and a microscope. You can see </p>
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<center><img src="https://static.igem.org/mediawiki/2011/0/0b/Valencia_Circulos.png" width="400"/></center>
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<br><br>
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<p>As can be seen, except for crops 3 and 6, all others aren´t in optimal conditions for growth, so they tend to stay rather than to grow.</p>
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<p>The first graph shows the cells in the reproductive or Siamese state, which, as noted, tend to increase in number while the ones non-growing tend to decrease, as shown in the second graph.</p>
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<p>After this experiment we concluded after observing the differences between them, we got rid of the remains and assemble new crops with the intention of taking them to higher concentrations, we got rid of the remains and started a  new  culture. This time  we chose the least contaminated water whith better growth, analytical water, to which we added  a higher concentration of fertilizer to stimulate growth. Besides ,we decided to stop using the reflector  box as we had evidence that  such a bright light caused    photo-inhibition. The volumes and other conditions  kept constant. </p>
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<center><img src="https://static.igem.org/mediawiki/2011/1/10/Valencia_Grafica_Growing_exp1.gif" width="420"/><img src="https://static.igem.org/mediawiki/2011/f/f7/Valencia_Grafica_Not_growing_exp1.gif" width="420"/></center>
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[[Image:Valencia_Grafica_750_exp2.gif|center]]
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<p>As can be seen, cultures 3 and 6 grow pretty well, all other cultures are not under optimal conditions.</p>
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[[Image:Valencia_Grafica_Growing_exp2.gif|center]]
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[[Image:Valencia_Grafica_Not_growing_exp2.gif|center]]
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<p>Synechocistys  was  observed to be  viable/ feasible  under laboratory conditions. The problem we found is that it is very easy for the culture to get  contaminated , appearing unwanted cells pretty soon.</p>
+
<p>From that we concluded that major factors for cyanobacterial growth were type of water and the reflector box. With that on mind, we chose to grow cells under Type II analytical water, to which we added a higher concentration of fertilizer to stimulate growth. Besides, we decided to drop the use of the reflector box as we had evidences that s much bright light might cause photo-inhibition. The volumes and other conditions were the same as Table above.</p>
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<h2><b>The temporal evolution of the pH in the culture</b></h2>
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<center><img src="https://static.igem.org/mediawiki/2011/8/83/Valencia_Grafica_750_exp2.gif"width="420"/></center>
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<center><img src="https://static.igem.org/mediawiki/2011/b/bb/Valencia_Grafica_Growing_exp2.gif" width="420"/><img src="https://static.igem.org/mediawiki/2011/b/b9/Valencia_Grafica_Not_growing_exp2.gif" width="420"/></center>
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<p>Experiments were carried out to verify the temporal variation of pH with the latest cultures we had established, those with the greatest concentration, and in which we could observe with the microscope that most cells were in the reproductive status.  A new 4h/4h photoperiod culture imposed and we measured the pH 12 times, every hour.  What we obtained was this:</p>
 
-
[[Image:Valencia_Grafica_PH_variation.gif|center]]
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<p>We accomplished to grow <i>Synechocystis</i> sp. PCC6803 on a low-cost photobioreactor under laboratory conditions. The major problem we found is that this culture gets easily contaminated as it does not use antibiotics.</p>
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<p>In our culture, the total variations of pH were lower than expected ;a fact that might be due to the fact that  the total concentration of Synechocystis in our culture was below  the concentration obtained in another habitat, such as  the BG11, recommended habitat  for  the growth of cyanobacteria (Portilla, A. et. al., 2009).</p>
+
<h2>Temporal evolution of the pH in the culture</h2>
 +
<p>Experiments were carried out to verify the temporal variation of pH in growing cultures. A 4h/4h photoperiod culture imposed and we measured the pH 12 times, every hour. Resulting in this:</p>
-
<p>Thanks to a group of we knew that the variations in pH can increase more than 2 degrees between the dark and the light phase (Paula Tamagnini, fom IBMC, Porto. Personal comunication. Http://www.ibmc.up.pt/index. php? id = 447 # IBMC). These variations may be sufficient to activate or inactivate the colicins, and be used as a switch for our project. The results obtained by Paula Tamagnini, the IBMC, are shown below:</p>
+
<center><img src="https://static.igem.org/mediawiki/2011/4/45/Valencia_PHvariation.png" width="600"/></center>
 +
<p>This figure depicts a variation of up to a unit of pH. This might not be sufficient for colicin denaturation, but it's still significant, taking into account the low-cost growth device, the suboptimal growth media, the fed-batch growth mode and our inexperienced hands. We are confident that growing <i>Synechocystis</i> in chemostat mode and with  BG11 growth medium (far better for cyanobacterial growth, Portilla, A. et. al., 2009) we could have had much better results.</p>
-
<p>The culture show the results of which were in continuous culture, 6h/6h The photoperiod is used.</p>
+
<p>In fact, partner groups in Porto and Sheffield have accomplished, in a very similar experimental design, but in continuous cultures and 6h/6h photoperiod, to shift pH to up to three units, from pH 8 to pH 11 and back (<a href="http://www.ibmc.up.pt/index.php?id=447" TARGET="_blank" title="Prof. Paula Tamagnini">Prof. Paula Tamagnini</a>, fom IBMC, Porto and <a href="http://www.sheffield.ac.uk/cbe/people/staffprofiles/pwright/index" TARGET="_blank" title="Prof. Phillip Wright">Prof. Phillip Wright</a>, Sheffield University, personal communication). These variations may be sufficient to activate and inactivate bacteriocins, allowing to use pH as a switch. The results they obtained are shown here:</p>
-
<p>The pH values ​​in a measurement taken continuously for 295 hours, then observe the changes produced both between phases and the dark lighting and the general trend of increasing pH to more basic pH's. Between day and night variations is seen as reaching beyond two degrees, one strong enough variation to make it serve as a switch.</p>
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<center><img src="https://static.igem.org/mediawiki/2011/1/12/Valencia_pHSheffield.png" width="600"/></center>
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<p>The overall trend is growing to the basicity, but tend to stabilize the pH variations between day and night with the passage of time.</p>
+
<p>The overall trend is towards basicity, stabilizing pH variations among day and night with time.</p>
<h2><b>References</b></h2>
<h2><b>References</b></h2>
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<p>Burrows, E. H., <i>et. al.</i>, 2009. Optimization of pH and Nitrogen for Enhanced Hydrogen Production by <i>Synechocystis</i> sp. PCC 6803 via Statistical and Machine Learning Methods. Wiley InterScience. 25: 1009-1018</p>
<p>Burrows, E. H., <i>et. al.</i>, 2009. Optimization of pH and Nitrogen for Enhanced Hydrogen Production by <i>Synechocystis</i> sp. PCC 6803 via Statistical and Machine Learning Methods. Wiley InterScience. 25: 1009-1018</p>
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<p>Allen J.F., <i>et. al.</i>, 2008. Evaluation of Acid Stress Tolerance in <i>Synechocystis</i> sp. PCC 6803 Mutants Lacking Signal Transduction-Related Genes sigB, sigD, and rre15Photosynthesis. Energy from the Sun: 14th International Congress on Photosynthesis, 1519–1522.</p>
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<p>Allen J.F., <i>et. al.</i>, 2008. Evaluation of Acid Stress Tolerance in <i>Synechocystis</i> sp. PCC 6803 Mutants Lacking Signal Transduction-Related Genes sigB, sigD, and rre15. Photosynthesis. Energy from the Sun: 14th International Congress on Photosynthesis, 1519–1522.</p>
<p>Portilla, A. <i>et. al.</i>, 2009. Evaluación del rendimiento de producción de aceite en cuatro microalgas nativas de las provincias ecuatorianas de Orellana, Esmeraldas, Imbabura y Pichincha. http://www3.espe.edu.ec:8700/bitstream/21000/427/1/T-ESPE-029605.pdf</p>
<p>Portilla, A. <i>et. al.</i>, 2009. Evaluación del rendimiento de producción de aceite en cuatro microalgas nativas de las provincias ecuatorianas de Orellana, Esmeraldas, Imbabura y Pichincha. http://www3.espe.edu.ec:8700/bitstream/21000/427/1/T-ESPE-029605.pdf</p>
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Latest revision as of 03:33, 22 September 2011



pH-Stat: Controlling pH through photons

As you can see on the following figure, we have effectively accomplished the control of pH through the autotrophic growth of Synechocystis sp. PCC 6803

. Thus opening the door to the construction of the CopH, the controller of pH.
Why is this important?

Well, we wanted to use pH as a switch for the action of li bacteriocins.



How have we done this?

Short answer: cyanobacteria growing only on light, CO2, commercial fertilizer and H2O

Long answer: keep reading!

Objectives of the culture

By introducing a culture of cyanobaceria Synechocystis sp. PCC 6803 we intent to ensure that the pH changes resulting from growth and proliferation function as a switch to enable the denaturation of the colicins. The objective is that, as time goes by, the colicins produced by Escherichia coli will be produced but inactive until the pH reaches the optimum range of activation, thus getting activated and producing cell lysis and killing pathogens.

In order to develop what we have stated above, we need to know:
  • How to establish the culture at the laboratory
  • The temporal evolution of the pH in the culture

Growing cyanobacteria under laboratory conditions

The materials used were:

  • 18W fluorescent tubes of white light, special tubes for aquariums that divide the spectrum mostly between the peaks of the visible red and blue light, which stimulate the photosynthesis.
  • 100 ml and 200 ml flasks
  • Air Pumps
  • Commercial COMPO Fertilizer
  • Distilled, tap and Type II analytical water
  • Cardboard boxes
  • Household aluminium foil

We decided to perform fed-batch growth, in which the addition of nutrientres occurs periodically, so that we could maintain cyanobacterial exponential growth phase as long as possible without reaching the maximum load limit.
We designed a low-cost photobioreactor using our lamps, cardboard boxes and aluminum foil in ordert to build a reflector box in which we made all our measurements

Study of growth parameters

Our first experiment consisted of a growth variables study so as to know the habitat preferences of the cyanobacterium. We made six cultures with different media conditions and we looked at its effects on growth. We distinguished two different groups:

1. First group, consisting of cultures C1, C2 and C3, which have tap, distilled and analytical water, respectively. It also uses a reflector box, which increases the irradiation of light on the culture, and a magnetic stirrer, which prevents the deposition of cells on the bottom.

2. The second one, made up of cultures C4, C5 and C6, without reflector box or magnetic stirrer.

The day after having inoculated the culture, absorbancy data were collected and cell concentration measured on a Neubauer chamber. The absorbancy was measured at 440 nm and 750 nm as the first was the highest absorbancy value after a spectrophotometer sweep and the second was taken according to references (Burrows, EH, et al., 2009 & JF Allen, 2008).

Cultures C1, C2, C4 and C5 tend to decrease in chlorophyll content, an indicator of the concentration of cells diminishes over time, i.e., they are dying. In contrast, cultures C3 and C6, not only survive, but also increase their concentration, thus they grow.

We distinguished between simple cells, i.e. those which are in the reproductive period and those which are not. The following two graphs show the number of growing and non-growing Synechocystis cells, counted with the Neubauer chamber and a microscope. You can see



The first graph shows the cells in the reproductive or Siamese state, which, as noted, tend to increase in number while the ones non-growing tend to decrease, as shown in the second graph.

As can be seen, cultures 3 and 6 grow pretty well, all other cultures are not under optimal conditions.

From that we concluded that major factors for cyanobacterial growth were type of water and the reflector box. With that on mind, we chose to grow cells under Type II analytical water, to which we added a higher concentration of fertilizer to stimulate growth. Besides, we decided to drop the use of the reflector box as we had evidences that s much bright light might cause photo-inhibition. The volumes and other conditions were the same as Table above.

We accomplished to grow Synechocystis sp. PCC6803 on a low-cost photobioreactor under laboratory conditions. The major problem we found is that this culture gets easily contaminated as it does not use antibiotics.

Temporal evolution of the pH in the culture

Experiments were carried out to verify the temporal variation of pH in growing cultures. A 4h/4h photoperiod culture imposed and we measured the pH 12 times, every hour. Resulting in this:

This figure depicts a variation of up to a unit of pH. This might not be sufficient for colicin denaturation, but it's still significant, taking into account the low-cost growth device, the suboptimal growth media, the fed-batch growth mode and our inexperienced hands. We are confident that growing Synechocystis in chemostat mode and with BG11 growth medium (far better for cyanobacterial growth, Portilla, A. et. al., 2009) we could have had much better results.

In fact, partner groups in Porto and Sheffield have accomplished, in a very similar experimental design, but in continuous cultures and 6h/6h photoperiod, to shift pH to up to three units, from pH 8 to pH 11 and back (Prof. Paula Tamagnini, fom IBMC, Porto and Prof. Phillip Wright, Sheffield University, personal communication). These variations may be sufficient to activate and inactivate bacteriocins, allowing to use pH as a switch. The results they obtained are shown here:

The overall trend is towards basicity, stabilizing pH variations among day and night with time.

References

Burrows, E. H., et. al., 2009. Optimization of pH and Nitrogen for Enhanced Hydrogen Production by Synechocystis sp. PCC 6803 via Statistical and Machine Learning Methods. Wiley InterScience. 25: 1009-1018

Allen J.F., et. al., 2008. Evaluation of Acid Stress Tolerance in Synechocystis sp. PCC 6803 Mutants Lacking Signal Transduction-Related Genes sigB, sigD, and rre15. Photosynthesis. Energy from the Sun: 14th International Congress on Photosynthesis, 1519–1522.

Portilla, A. et. al., 2009. Evaluación del rendimiento de producción de aceite en cuatro microalgas nativas de las provincias ecuatorianas de Orellana, Esmeraldas, Imbabura y Pichincha. http://www3.espe.edu.ec:8700/bitstream/21000/427/1/T-ESPE-029605.pdf

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