Team:Valencia/Engineering

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        <ul>
 
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        <li><a href="#Engineerin">Engineering</a></li>
 
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        <li><a href="#Bioreactor">Bioreactor</a></li>
 
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        <li><a href="#Advance co-culture">Advance co-culture</a></li>
 
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<br><br>
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<h2><b>Simple Batch Bioreactors</b></h2><br><br>
 +
<h3>CO<sub>2</sub> Pump</h3>
 +
<br>
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<div id="Titulos">
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To enhance a faster growth rate in our Synechococcus broth culture and taking advantage of the bacterium being photosynthetic, we decided to make a device with a CO2 pump in order to make the transformations quickly.
<br><br>
<br><br>
-
Bioreactor
 
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</div>
 
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<div id="HomeCenterCenter">
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<u>Materials:</u>
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<br><br>
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text.
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<ul style="list-style-type: square">
 +
<li>A bottle of water (1.5 litres) + a cap so the bottle remains hermetically sealed.</li>
 +
<li>PVC tubes.</li>
 +
<li>Rapid fixation glue.</li>
 +
<li>30g of sugar.</li>
 +
<li>One spoon of yeast (Saccharomyces cerevisae).</li>
 +
<li>Water.</li>
 +
<li>Methylene blue.</li>
 +
</ul>
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<div id="Titulos">
 
<br><br>
<br><br>
-
Advanced continuous coculture system
+
<center><a href:"http://2012.igem.org/wiki/images/6/6b/Air_Pump_Cris.jpg"><img src="http://2012.igem.org/wiki/images/6/6b/Air_Pump_Cris.jpg" widht="420" height="380"></a></center>
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</div>
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<center><b>Figure 1:</b> Diagram of the Co<sub>2</sub> pump.</center>
<br><br>
<br><br>
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<div id="HomeCenterCenter">
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<u>Construction:</u>
-
<p align="justify">
+
-
Our furthest goal is to build a continuous culture system with spatial and temporal decoupling from the photosynthetic and the bioluminescent module, totally autonomous.
+
<br><br>
<br><br>
-
<h3><u>Design:</u></h3>
+
 
 +
First of all, we bored holes to the bottle and sealed the edges. Then we cut the tubes so they fit in the holes properly. Fill in one third of the botle approximately with water at 100ºC. Then add up the sugar. After that, shake the bottle so the sugar dissolves in the water. Add the spoon of yeast and shake again.
 +
<br><br>
 +
Fresh yeast is a living organism, a micro fungus able to perform fermentation: in absence of oxygen converts the sugar into alcohol and liberates CO<sub>2</sub>.
 +
<br><br>
 +
At last, pour 1 litre of Methylene blue into another bottle, used as a contamination control device, in order to purify the air that got into our culture. And finally we connect the air pumps to keep a turbulence regine inside the bioreactor.
 +
 
 +
<br><br>
 +
<center><a href:"http://2012.igem.org/wiki/images/3/3f/Air_Pump1_Cris.jpg"><img src="http://2012.igem.org/wiki/images/3/3f/Air_Pump1_Cris.jpg" width="420" height="380"></a></center>
 +
<center><b>Figure 2:</b>Photograph of the actual CO<sub>2</sub> pump we build.</center>
 +
<br><br>
 +
 
 +
The results of our experiment were not the expected, having proved that our cyanobacteria grew up faster if we provided an extra input of CO<sub>2</sub>,bu the culture didn’t develop well due to contamination problems. We do not know if the yeast caused the problem or the bottle just wasn’t properly sealed.<br><br>
 +
 
 +
<h3>Air Pump</h3>
<br>
<br>
 +
This <i>Synechococcus</i> bioreactor was built in a 2l Kitasatos flask with all openings thoroughly sealed with parafilm. Through this controlled interfaces we bored in sterile input and output tubing. The input tube opening falls below the liquid level and is in charge of bubbling air pumped from the environment to remove CO<sub>2</sub> used in photosynthesis. It differs from the previous bioreactor in this aspect, as it focuses on removing environmental CO<sub>2</sub> instead of boosting the culture with high carbonation.<br>
 +
To avoid the contamination problems that happened at the previous design, we attached a clarification filter of 0.45micron pore size to retain the microorganisms from the incoming air flow.<br><br>
 +
<center><a href="http://2012.igem.org/wiki/images/a/a5/Imagenairpump.JPG"><img src="http://2012.igem.org/wiki/images/a/a5/Imagenairpump.JPG" width="450" height="400"></a></center>
 +
<center><b>Figure 3:</b> <i>Synechococcus</i> culture growing with the air pump</center>
 +
<br><br>
 +
To enhance better gas diffusion from the bubbled air, we built a bubble-diffusor stone rod at the end of the input tube, see below.<br><br>
 +
<center><a href="http://2012.igem.org/wiki/images/4/43/Piedraaaa.png"><img src="http://2012.igem.org/wiki/images/4/43/Piedraaaa.png"width="410" height="420"></a></center>
 +
<center><b>Figure 4:</b> Bubble-diffusor stone rod working.</center>
 +
<br>
 +
<br><br>
 +
<h3>Simple biphasic coculture bioreactor:</h3>
 +
<center><br><img align="center" src="http://2012.igem.org/wiki/images/2/20/IMG_0399.JPG" width="220" higth="170" border="10"></a></center>
 +
<br><br>
 +
We designed and built a simple biphasic coculture bioreactor prototype, easy to transport and operate, though not stable for long-term functioning. The construction is composed of 2 flasks of 10ml connected through a 0.45micron pore membrane from a clarification syringe filter. All joints were sealed with parafilm. The bioreactor is airtight,  so oxygen and carbon dioxide levels would deplete with growing biomass if the proportions of each culture (autotrophic/heterotrophic) are not in metabolic equilibrium.
 +
<br><br><br>
 +
<h2><b>Advanced continuous coculture system</b></h2>
 +
<br><br>
 +
Our furthest goal is to build a continuous culture system with spatial and temporal decoupling from the photosynthetic and the bioluminescent module, totally autonomous.
 +
<br><br>
 +
<h3>Design:</h3>
 +
<br><br>
Set 2 separate culture modules, a flat wide one for <i>S. elongatus cscB</i> as a solar module and a smaller compact one for <i>A. fischeri</i> as a biobulb. Prepare an open system so that there can be gas exchange from the cultures with the atmosphere (to let the system at as a CO<sub>2</sub> sink), but with a Pasteurian design opening to avoid contamination from deposition.
Set 2 separate culture modules, a flat wide one for <i>S. elongatus cscB</i> as a solar module and a smaller compact one for <i>A. fischeri</i> as a biobulb. Prepare an open system so that there can be gas exchange from the cultures with the atmosphere (to let the system at as a CO<sub>2</sub> sink), but with a Pasteurian design opening to avoid contamination from deposition.
 +
<br><br>
<br><br>
Set tubing connecting both cultures, protecting each end with a syringe filter 0.45 microns of pore size. The fluid medium dynamics is borne by a pair of peristaltic reversible-flow pumps, which switch flow direction every 30-60 seconds to avoid collapsing any side of the membranes with jammed cells. The membranes allow the exchange of gases, ions, water, sucrose and AHL, but not cells, so that populations do not mix. This is fundamental to guarantee the light-efficiency of each compartment, without having cyanobacteria shading light emission from <i>A. fischeri</i>, and permitting a spatial decoupling of the photosynthetic module and the biolamp.
Set tubing connecting both cultures, protecting each end with a syringe filter 0.45 microns of pore size. The fluid medium dynamics is borne by a pair of peristaltic reversible-flow pumps, which switch flow direction every 30-60 seconds to avoid collapsing any side of the membranes with jammed cells. The membranes allow the exchange of gases, ions, water, sucrose and AHL, but not cells, so that populations do not mix. This is fundamental to guarantee the light-efficiency of each compartment, without having cyanobacteria shading light emission from <i>A. fischeri</i>, and permitting a spatial decoupling of the photosynthetic module and the biolamp.
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All electric devices would be powered by a small solar panel, to preserve to the last detail the energetic autonomy of the system.
All electric devices would be powered by a small solar panel, to preserve to the last detail the energetic autonomy of the system.
<br><br>
<br><br>
-
Routine analyses of the system would be carried out on sucrose, oxygen and AHL levels in the liquid medium. Luminescence would be measured at night to acquire an experimental Light/Time curve.
+
Routine analyses of the system would be carried out on sucrose, oxygen and AHL levels in the liquid medium. Luminescence would be measured at night to acquire an experimental Light/Time curve.<br><br>
 +
<center>
 +
<img src="http://2012.igem.org/wiki/images/8/8b/Botella.jpg" width="120" heigth="100"> <img src="http://2012.igem.org/wiki/images/b/bd/IMG_0414.JPG" width="200" height="200">
 +
<br>
 +
</center>
 +
 
 +
In our dry lab we tried different pumping and membrane systems which would ensure fast fluid exchange between the individual culture volumes without fast saturation of the membrane with bacterial mass. We ended up building a balanced flow circulation setting with relatively large-size pore membrane (0.45micron cellulose clarification filter modules) which still had a high saturation rate, near 1 minute. This gives us an estimate of the required rate of flow direction reversal we have to program in the microcontroller.
 +
 
<br><br>
<br><br>
[Future design development: Achieve a knock-out/directed mutagenesis of the luxI autoinducer genes in <i>A. fischeri</i> to unable isolated autoinduction. This would render the system totally dependent on the AHL production of the transformed <i>S. elongatus</i>. In such situation, turning valves of different membrane pore size at the cell-stopping sections would result extremely useful, to manually switch on/off the diffusion of AHL from the <i>Synechococcus</i> culture to the biolamp. This enables an element of human control to turn on/off the lights over the normal day/night fashion].
[Future design development: Achieve a knock-out/directed mutagenesis of the luxI autoinducer genes in <i>A. fischeri</i> to unable isolated autoinduction. This would render the system totally dependent on the AHL production of the transformed <i>S. elongatus</i>. In such situation, turning valves of different membrane pore size at the cell-stopping sections would result extremely useful, to manually switch on/off the diffusion of AHL from the <i>Synechococcus</i> culture to the biolamp. This enables an element of human control to turn on/off the lights over the normal day/night fashion].
Line 114: Line 102:
<img src="http://2012.igem.org/wiki/images/5/58/Advanced_coculture_vlc_BBFJFJBFJBFJ.JPG">
<img src="http://2012.igem.org/wiki/images/5/58/Advanced_coculture_vlc_BBFJFJBFJBFJ.JPG">
</center>
</center>
 +
<center>
 +
<b>Figure a:</b> Advanced continuous cocultive device, showing in both culture compartments<br> with optical cell density monitors (green diodes), reversible-flow pumps at th sides,<br> bacteriological (Fb) and activated carbon (Fc) filtering and medium recycle system <br>at the middle. Evaporation loss automatic replenisher at the top (H<sub>2</sub>O).
 +
</center>
 +
<br><br>
 +
<h2><b>Measuring sucrose of the cscB</b></h2><br>
 +
The cscB Synechococcus strain is capable to export sucrose at a concentration of 36mg/lh (Ducat et al. 2012). In order to confirm this in the cscB strain we were growing in the lab we designed two different methods:<br><br>
 +
<ol>
 +
<li>Measuring glucose with Fehling reactive.</li>
 +
<li>Measuring glucose with a standard glucometer.</li>
 +
</ol>
<br><br>
<br><br>
-
<h3><u>Results:</u></h3><br>
+
For the transformation of saccharose into glucose an invertase enzyme was needed which we couldn´t obtain. We will try to get it before the Jamboree to confirm the data of the Ducat et al. 2012 paper.
-
Construction of inter-flask pumping system with 0.45micron pore membranes.<br>
+
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Latest revision as of 03:42, 27 September 2012



Engineering



Simple Batch Bioreactors



CO2 Pump


To enhance a faster growth rate in our Synechococcus broth culture and taking advantage of the bacterium being photosynthetic, we decided to make a device with a CO2 pump in order to make the transformations quickly.

Materials:

  • A bottle of water (1.5 litres) + a cap so the bottle remains hermetically sealed.
  • PVC tubes.
  • Rapid fixation glue.
  • 30g of sugar.
  • One spoon of yeast (Saccharomyces cerevisae).
  • Water.
  • Methylene blue.


Figure 1: Diagram of the Co2 pump.


Construction:

First of all, we bored holes to the bottle and sealed the edges. Then we cut the tubes so they fit in the holes properly. Fill in one third of the botle approximately with water at 100ºC. Then add up the sugar. After that, shake the bottle so the sugar dissolves in the water. Add the spoon of yeast and shake again.

Fresh yeast is a living organism, a micro fungus able to perform fermentation: in absence of oxygen converts the sugar into alcohol and liberates CO2.

At last, pour 1 litre of Methylene blue into another bottle, used as a contamination control device, in order to purify the air that got into our culture. And finally we connect the air pumps to keep a turbulence regine inside the bioreactor.

Figure 2:Photograph of the actual CO2 pump we build.


The results of our experiment were not the expected, having proved that our cyanobacteria grew up faster if we provided an extra input of CO2,bu the culture didn’t develop well due to contamination problems. We do not know if the yeast caused the problem or the bottle just wasn’t properly sealed.

Air Pump


This Synechococcus bioreactor was built in a 2l Kitasatos flask with all openings thoroughly sealed with parafilm. Through this controlled interfaces we bored in sterile input and output tubing. The input tube opening falls below the liquid level and is in charge of bubbling air pumped from the environment to remove CO2 used in photosynthesis. It differs from the previous bioreactor in this aspect, as it focuses on removing environmental CO2 instead of boosting the culture with high carbonation.
To avoid the contamination problems that happened at the previous design, we attached a clarification filter of 0.45micron pore size to retain the microorganisms from the incoming air flow.

Figure 3: Synechococcus culture growing with the air pump


To enhance better gas diffusion from the bubbled air, we built a bubble-diffusor stone rod at the end of the input tube, see below.

Figure 4: Bubble-diffusor stone rod working.



Simple biphasic coculture bioreactor:




We designed and built a simple biphasic coculture bioreactor prototype, easy to transport and operate, though not stable for long-term functioning. The construction is composed of 2 flasks of 10ml connected through a 0.45micron pore membrane from a clarification syringe filter. All joints were sealed with parafilm. The bioreactor is airtight, so oxygen and carbon dioxide levels would deplete with growing biomass if the proportions of each culture (autotrophic/heterotrophic) are not in metabolic equilibrium.


Advanced continuous coculture system



Our furthest goal is to build a continuous culture system with spatial and temporal decoupling from the photosynthetic and the bioluminescent module, totally autonomous.

Design:



Set 2 separate culture modules, a flat wide one for S. elongatus cscB as a solar module and a smaller compact one for A. fischeri as a biobulb. Prepare an open system so that there can be gas exchange from the cultures with the atmosphere (to let the system at as a CO2 sink), but with a Pasteurian design opening to avoid contamination from deposition.

Set tubing connecting both cultures, protecting each end with a syringe filter 0.45 microns of pore size. The fluid medium dynamics is borne by a pair of peristaltic reversible-flow pumps, which switch flow direction every 30-60 seconds to avoid collapsing any side of the membranes with jammed cells. The membranes allow the exchange of gases, ions, water, sucrose and AHL, but not cells, so that populations do not mix. This is fundamental to guarantee the light-efficiency of each compartment, without having cyanobacteria shading light emission from A. fischeri, and permitting a spatial decoupling of the photosynthetic module and the biolamp.

Cell density would be regulated by output tubes from each culture which would recycle broth after passing it through a filter/skimmer. This process would be regulated by a turbidimeter (in theory) or by sampling-and-testing, at least in the first experimental prototype.

Water loss due to evaporation would be solved by a water-level-wise lever opening a valve which would let in some distilled water from a small deposit, when the volume of the liquid falls below certain threshold.

A microcontroller would be used as hardware to coordinate pump flow switch, input signals from turbidimeters and control of the cell density control system.

All electric devices would be powered by a small solar panel, to preserve to the last detail the energetic autonomy of the system.

Routine analyses of the system would be carried out on sucrose, oxygen and AHL levels in the liquid medium. Luminescence would be measured at night to acquire an experimental Light/Time curve.


In our dry lab we tried different pumping and membrane systems which would ensure fast fluid exchange between the individual culture volumes without fast saturation of the membrane with bacterial mass. We ended up building a balanced flow circulation setting with relatively large-size pore membrane (0.45micron cellulose clarification filter modules) which still had a high saturation rate, near 1 minute. This gives us an estimate of the required rate of flow direction reversal we have to program in the microcontroller.

[Future design development: Achieve a knock-out/directed mutagenesis of the luxI autoinducer genes in A. fischeri to unable isolated autoinduction. This would render the system totally dependent on the AHL production of the transformed S. elongatus. In such situation, turning valves of different membrane pore size at the cell-stopping sections would result extremely useful, to manually switch on/off the diffusion of AHL from the Synechococcus culture to the biolamp. This enables an element of human control to turn on/off the lights over the normal day/night fashion].

Figure a: Advanced continuous cocultive device, showing in both culture compartments
with optical cell density monitors (green diodes), reversible-flow pumps at th sides,
bacteriological (Fb) and activated carbon (Fc) filtering and medium recycle system
at the middle. Evaporation loss automatic replenisher at the top (H2O).


Measuring sucrose of the cscB


The cscB Synechococcus strain is capable to export sucrose at a concentration of 36mg/lh (Ducat et al. 2012). In order to confirm this in the cscB strain we were growing in the lab we designed two different methods:

  1. Measuring glucose with Fehling reactive.
  2. Measuring glucose with a standard glucometer.


For the transformation of saccharose into glucose an invertase enzyme was needed which we couldn´t obtain. We will try to get it before the Jamboree to confirm the data of the Ducat et al. 2012 paper.