Team:Valencia/Project2

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<h1>pH-stat: Culture of <i>Synechocystis sp.</i> PCC 6803 </h1>
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<h2>Objectives of  the culture</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  in order  to  activate  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.
 +
 
 +
<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>
 +
<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>
 +
 
 +
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>
 +
 
 +
<h2>Growing cyanobacteria under laboratory conditions</h2>
 +
 
 +
<p>The materials used were: </p>
 +
 
 +
<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>
 +
<li>Commercial COMPO Fertilizer</li>
 +
<li>Distilled, tap and Type II analytical water</li>
 +
<li>Cardboard boxes</li>
 +
<li>Household aluminium foil</li></ul>
 +
 
 +
<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>
 +
 
 +
<h3><b>Study of growth parameters</b></h3>
 +
<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>
 +
 
 +
 
 +
<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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 +
 
 +
<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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<center><img src="https://static.igem.org/mediawiki/2011/a/a8/Valencia_Grafica_750_exp1.gif" width="420"/></center>
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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:
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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>
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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>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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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.  
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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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Results and discussion
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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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The  other conditions remained  constant in both cases, as are detailed in the following table:
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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_Synecho_Tabla_1.jpg|center]]
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<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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<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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<center><img src="https://static.igem.org/mediawiki/2011/8/83/Valencia_Grafica_750_exp2.gif"width="420"/></center>
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[[Image:Valencia_Graficas1_Synecho.gif|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>For technical reasons, a power cut  for  an entire weekend,  on our arrival the following Monday, the cultures  showed  a transparent colour  which had nothing to do with their usual blue-green algae colour  ( cyanobacteria).So, we got rid of the remains and started a  new  culture. This time  we chose the least contaminated water  which still  showed a good margin of survival, 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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[[Image:Valencia_Graficas2_Synecho.gif|center]]
+
<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>
 +
<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>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>
+
<center><img src="https://static.igem.org/mediawiki/2011/4/45/Valencia_PHvariation.png" width="600"/></center>
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<h2><b>La evolución temporal del pH en el cultivo</b></h2>  
+
<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>Se hicieron experimentos para comprobar la variación temporal del pH con los últimos cultivos establecidos, pues son los que mayor concentración hemos obtenido y en los cuales se observa con el microscopio que la mayoría de células están en estado reproductivo. Se impone en el cultivo un nuevo fotoperiodo de 4h/4h, y medimos el pH de éstos 11 veces, 1 cada hora. Obtubimos:</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>Las variaciones totales del pH son menores de lo esperado en nuestro cultivo, hecho que puede ser debido a que la concentración total de Synechocystis en nuestro cultivo sea inferior a la concentración obtenida con otro medio, como por ejemplo el BG11, medio recomendado para el cultivo de cianobacterias (Portilla, A. et. al., 2009).</p>
+
<center><img src="https://static.igem.org/mediawiki/2011/1/12/Valencia_pHSheffield.png" width="600"/></center>
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<p>Tenemos conocimiento gracias a un grupo colaborador de que las variaciones de pH pueden incrementarse del orden de 1,5 grados en nueve días (Paula Tamagnini, fom IBMC, Porto. Personal comunication. http://www.ibmc.up.pt/index.php?id=447#ibmc). Estas variaciones pueden ser suficientes para activar o inactivar las colicinas.</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>
-
<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>
+
<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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