Difference between revisions of "Template:Team:Groningen/CONTENT/PROJECT/Results"

 
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<div class="content module">
 
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<div class="subtitle">Overview of achievements</div>
  
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<div class="subtitle">
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<div class="text">Developed several genetic constructs which resulted in distinct biofilm phenotypes.</div>
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Overview
+
<div class="text">Biobricked the salt inducible promoter to control several biofilm genes.</div>
−
</div>
+
<div class="text">Validated both the salt inducible <i>P<sub>proH</sub></i> promoter (BBa_K1597000) and <i>tasA</i> (BBa_K1597002).</div>
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<div class="text">  
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<div class="text">A new shuttle vector (BBa_K1597001) was created for integration in the <i>thrC</i> locus in <i>B. subtilis</i>.</div>
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Characterized biofilm properties of B. subtilis. Natto, 3610 ComI, 168, JH642 and PY79
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<div class="text">Used combinations of these constructs to create a biofilm which is ion-selective.</div>
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</div>
+
<div class="text">Demonstrated ion selectivity of <i>B. subtilis</i> biofilm with our prototype in the real world.</div>
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<div class="text">  
+
<div class="text">y-PGA was modeled as a cation exchange membrane using Molecular Dynamics.</div>
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Demonstrated ion-selectivity of the B. subtilis biofilm and improved it with our biobricks.
+
<div class="text">Created future scenarios about our project and the use of GMOs.</div>
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</div>
+
<div class="text">Developed a card game to teach about synthetic biology in a fun way.</div>
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<div class="text">  
+
−
Modeled y-PGA as a Cation Exchange membrane using Molecular Dynamics
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</div>
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−
<div class="text">  
+
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Designed drip and continuous flow method to grow more robust biofilms.
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</div>
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<div class="text">  
+
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Created easy cheap test to measure rigidity of biofilms.  
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</div>
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<div class="text">  
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Created new backbone(K1597001) for integration into ThrC locus in B. subtilis and validated it.</div>
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−
<div class="text">  
+
  
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Constructs created for TasA, BslA, Abrb KO, SlrR, y-PGA and ProH
+
<div class="subtitle">Phenotypical differences after biobrick introduction in <i>B. subtilis</i></div>
−
</div>
+
<div class="text">The introduction of the developed biobricks in <i>B. subtilis</i> changes the behaviour of the bacterium in the biofilm state, resulting in observable differences in phenotype after growing for 24 hours.</div>
−
<div class="text">  
+
 
−
Created biobricks to increase the robustness.
+
<div class="subtitle">Created salt inducible promoter to control several biofilm genes</div>
−
</div>
+
<div class="text">To increase the the robustness and ion selectivity of the biofilm, several biobricks were developed, where an overproduction of biofilm involved genes were introduced to <i>B. subtilis</i>. Multiple genes were put under the control of the salt inducible promoter, in order to regulate their expression.</div>
−
<div class="text">  
+
 
−
Validated ThrC(K1597001), TasA(K1597002) and ProH (K1597000)
+
<div class="subtitle">Validation of <i>tasA</i> and the <i>P<sub>proH</sub></i> inducible promoter</div>
−
</div>
+
<div class="text">To validate the functioning of our biobricks, a <i>tasA</i> overproducing strain was made under the control of the salt inducible <i>P<sub>proH</sub></i> promoter. After induction, by adding NaCl in two different experiments, indeed an increase in TasA and change in phenotype of the biofilm was registered, thereby validating the functionality of the salt promoter.</div>
−
<div class="text">  
+
 
−
Created future scenario’s about GMO’s and sustainability and a cardgame for education
+
<div class="subtitle">A new shuttle vector</div>
−
</div>
+
<div class="text">An extra integration locus is useful especially when making a multiple mutant. Therefore a shuttle vector was designed and created (BBa_K1597001). The backbone is capable of reproducing in <i>E. coli</i> and integrating in the <i>thrC</i> locus from <i>B. subtilis</i>.</div>
−
<div class="text">  
+
 
 +
<div class="subtitle">Ion selectivity</div>
 +
<div class="text">To test the ion selectivity, the potential over the membrane was measured and compared to the theoretical maximum, which was calculated using the Nernst equation. A measured potential of 22 mV for a theoretical maximum of 86 mV resulted in an apparent ion selectivity of 0.26 for a mixed strain of <i>B. subtilis</i>.</div>
 +
 
 +
<div class="subtitle">Demonstration of working prototype</div>
 +
<div class="text">The ultimate test was if our prototype could work with actual sea and lake water. The potential over our biofilm overexpressing <i>tasA</i>, <i>bslA</i>, <i>slrR</i> and <i>ΔabrB</i> was measured with using water from the Waddenzee and the Ijselmeer. This resulted in measuring the highest potential so far, indicating that our prototype can indeed function in the real world.</div>
 +
 
 +
<div class="subtitle">Ion selectivity of y-PGA</div>
 +
<div class="text">To show how the ion selectivity of the biofilm could be enhanced, a molecular dynamics model was used. The model showed that a y-PGA membrane allows for selective diffusion of sodium atoms from salt to fresh water, and can in this way function as a cation exchange membrane. Overproduction of y-PGA in <i>B. subtilis</i> would contribute to a negative charge of the biofilm, increasing its ion selectivity.</div>
 +
 
 +
<div class="subtitle">Future scenarios for the use of GMOs</div>
 +
<div class="text">Before applying a new technology in society, it is vital that research has been done into the opinion of the public. Therefore three future scenarios were created, describing either the situation with no use of GMOs, the restricted use of GMOs, and the use of GMOs in a lot of everyday applications. Feedback on these scenarios provided insight into which underlying principles stakeholders are most determining in forming an opinion on a new technology.</div>
 +
 
 +
<div class="subtitle">Card game, the fun way of learning synthetic biology</div>
 +
<div class="text">The synthetic biology cardgame was designed and created with the aim of teaching about synbio and iGEM in a playful way. More than a hundred high school students have played it and all the feedback was used to create a game that is fun and educational. The teachers have been supportive as well and want to use the card game to explain synthetic biology at school.</div>
  
−
<div class="subtitle">
 
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Bacillus Subtilis & Growth Method
 
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</div>
 
−
<div class="text">
 
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<i>Drip flow experiment/ directed evolution</i>
 
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</div>
 
−
<div class="text">
 
−
To increase the robustness of the biofilm in a natural way the drip and continuous flow experiment had been performed. A biofilm was put in an angle of 15 degrees and a constant flow of medium was flowing over it. Only the cells with a high enough robustness were sticking to the carrier material. Unfortunately only one biofilm at a time was grown in this set up and it was requiring lots of growth medium. Several hundred plates were needed for our experiments so the drip and continuous flow experiments were stopped after a while to save time and money. The choice afterward was between submerged and at the air surface
 
 
</div>
 
</div>

Latest revision as of 13:43, 31 October 2015

Overview of achievements
Developed several genetic constructs which resulted in distinct biofilm phenotypes.
Biobricked the salt inducible promoter to control several biofilm genes.
Validated both the salt inducible PproH promoter (BBa_K1597000) and tasA (BBa_K1597002).
A new shuttle vector (BBa_K1597001) was created for integration in the thrC locus in B. subtilis.
Used combinations of these constructs to create a biofilm which is ion-selective.
Demonstrated ion selectivity of B. subtilis biofilm with our prototype in the real world.
y-PGA was modeled as a cation exchange membrane using Molecular Dynamics.
Created future scenarios about our project and the use of GMOs.
Developed a card game to teach about synthetic biology in a fun way.
Phenotypical differences after biobrick introduction in B. subtilis
The introduction of the developed biobricks in B. subtilis changes the behaviour of the bacterium in the biofilm state, resulting in observable differences in phenotype after growing for 24 hours.
Created salt inducible promoter to control several biofilm genes
To increase the the robustness and ion selectivity of the biofilm, several biobricks were developed, where an overproduction of biofilm involved genes were introduced to B. subtilis. Multiple genes were put under the control of the salt inducible promoter, in order to regulate their expression.
Validation of tasA and the PproH inducible promoter
To validate the functioning of our biobricks, a tasA overproducing strain was made under the control of the salt inducible PproH promoter. After induction, by adding NaCl in two different experiments, indeed an increase in TasA and change in phenotype of the biofilm was registered, thereby validating the functionality of the salt promoter.
A new shuttle vector
An extra integration locus is useful especially when making a multiple mutant. Therefore a shuttle vector was designed and created (BBa_K1597001). The backbone is capable of reproducing in E. coli and integrating in the thrC locus from B. subtilis.
Ion selectivity
To test the ion selectivity, the potential over the membrane was measured and compared to the theoretical maximum, which was calculated using the Nernst equation. A measured potential of 22 mV for a theoretical maximum of 86 mV resulted in an apparent ion selectivity of 0.26 for a mixed strain of B. subtilis.
Demonstration of working prototype
The ultimate test was if our prototype could work with actual sea and lake water. The potential over our biofilm overexpressing tasA, bslA, slrR and ΔabrB was measured with using water from the Waddenzee and the Ijselmeer. This resulted in measuring the highest potential so far, indicating that our prototype can indeed function in the real world.
Ion selectivity of y-PGA
To show how the ion selectivity of the biofilm could be enhanced, a molecular dynamics model was used. The model showed that a y-PGA membrane allows for selective diffusion of sodium atoms from salt to fresh water, and can in this way function as a cation exchange membrane. Overproduction of y-PGA in B. subtilis would contribute to a negative charge of the biofilm, increasing its ion selectivity.
Future scenarios for the use of GMOs
Before applying a new technology in society, it is vital that research has been done into the opinion of the public. Therefore three future scenarios were created, describing either the situation with no use of GMOs, the restricted use of GMOs, and the use of GMOs in a lot of everyday applications. Feedback on these scenarios provided insight into which underlying principles stakeholders are most determining in forming an opinion on a new technology.
Card game, the fun way of learning synthetic biology
The synthetic biology cardgame was designed and created with the aim of teaching about synbio and iGEM in a playful way. More than a hundred high school students have played it and all the feedback was used to create a game that is fun and educational. The teachers have been supportive as well and want to use the card game to explain synthetic biology at school.