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

 
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<div class="subtitle">Overview of achievements</div>
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-Developed several genetic constructs which resulted in distinct biofilm phenotypes
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-    Created salt inducible promoter to control several biofilm genes
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-    Validated both the salt inducible <i>P<sub>proH</sub></i> promoter (BBa_K1597000) and  <i>tasA</i> (BBa_K1597002)
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-    A new shuttle vector (BBa_K1597001) was created for integration in the <i>thrC</i> locus in <i>B. subtilis</i>
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-    Used combinations of these constructs to create a biofilm which is ion selective
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-    Demonstrated ion-selectivity of <i>B. subtilis</i> biofilm with our prototype in the real-world
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-    y-PGA was modeled as a Cation Exchange membrane using Molecular Dynamics
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-    Created future scenarios about our project and the use of GMOs.
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-    A card game was developed to teach about synthetic biology in a fun way
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<div class="text">Developed several genetic constructs which resulted in distinct biofilm phenotypes.</div>
 +
<div class="text">Biobricked the salt inducible promoter to control several biofilm genes.</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>
 +
<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>
 +
<div class="text">Used combinations of these constructs to create a biofilm which is ion-selective.</div>
 +
<div class="text">Demonstrated ion selectivity of <i>B. subtilis</i> biofilm with our prototype in the real world.</div>
 +
<div class="text">y-PGA was modeled as a cation exchange membrane using Molecular Dynamics.</div>
 +
<div class="text">Created future scenarios about our project and the use of GMOs.</div>
 +
<div class="text">Developed a card game to teach about synthetic biology in a fun way.</div>
  
 
<div class="subtitle">Phenotypical differences after biobrick introduction in <i>B. subtilis</i></div>
 
<div class="subtitle">Phenotypical differences after biobrick introduction in <i>B. subtilis</i></div>
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<div class="text">
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<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>
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The introduction of the developed biobricks in <i>B. subtilis</i>, changes the behaviour of the bacterium. This resulted in different phenotypes after growing for 24 hours, where we can conclude  that the developed biobricks have an impact on <i>B. subtilis</i>. </div>
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<div class="subtitle">
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<div class="subtitle">Created salt inducible promoter to control several biofilm genes</div>
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Created salt inducible promoter to control several biofilm genes
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<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>
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</div>
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<div class="text">
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To increase the the robustness and the ion selectivity of the biofilm, several biobricks were developed, where an overproduction of biofilm involved genes were introduced to <i> B. subtilis</i> . Several of these genes were controlled under the developed salt inducible promoter. Introduction of these biobrick led to different phenotypes.</div>
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<div class="subtitle">
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<div class="subtitle">Validation of <i>tasA</i> and the <i>P<sub>proH</sub></i> inducible promoter</div>
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Validation of <i>tasA</i> and the <i>P<sub>proH</sub></i> inducible promoter
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<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>
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To validate that our biobricks function as expected, a <i>tasA</i> overproducing strain was made under the control of the salt inducible <i>P<sub>proH</sub></i> promoter. The use for salt inducible <i>P<sub>proH</sub></i> promoter showed an increasement of TasA after induction with NaCl in two different experiments. Induction of salt also showed a different phenotype.(link naar measurement???)</div>
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<div class="subtitle">A new shuttle vector</div>
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<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>
  
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<div class="subtitle">
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<div class="subtitle">Ion selectivity</div>
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A new shuttle vector
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<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>
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</div>
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<div class="text">
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An extra integration locus is welcome 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>
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<div class="subtitle">
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<div class="subtitle">Demonstration of working prototype</div>
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Ion selectivity in the lab
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<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>
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</div>
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<div class="text">
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To test the ion selectivity, the volts generated by the membrane  was measured and the ion-selectivity can be calculated using the Nernst equation. Calculation showed that a theoretical maximum energy potential 86 mV is.
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</div>
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<div class="subtitle">
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<div class="subtitle">Ion selectivity of y-PGA</div>
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Demonstration of working prototype
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<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>
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<div class="text">
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<div class="subtitle">Future scenarios for the use of GMOs</div>
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The ultimate test was if our prototype could work with actual seawater and water from the lake. This test was performed and the results are significant. It was expected that the ion-selectivity would be lower hence real sea and lake water was used for this experiment. However the obtained yield was higher than previous measurements. This indicates that our prototype can function in the real world. (link naar measurement)
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<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>
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<div class="subtitle">
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<div class="subtitle">Card game, the fun way of learning synthetic biology</div>
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y-PGA
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<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>
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</div>
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<div class="text">
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To show how the ion-selectivity  of the biofilm could be enhanced, a molecular dynamic model was used. The molecular dynamics showed that y-PGA could function as a cation exchange membrane. This will contribute to a negative charge of the biofilm, where an overexpression of y-PGA in <i>B. subtilis</i> could make the biofilm ion-selective for sodium ions. This could increase the total amount of energy generated. 
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<div class="subtitle">Future scenarios for Blue Bio Energy</div>
 
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<div class="text">
 
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Before applying a new technology in society, it is vital that research has been done into the reaction of the public. Therefore 3 future scenarios were created, with none Blue Bio Energy, Blue Bio Energy and too much Blue Bio Energy. This gave perspective about which underlying principles motivates stakeholders when forming an opinion on a new technology. (link to Human Practices on the scenarios)</div>
 
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<div class="subtitle">
 
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Card game, the fun way of learning synthetic biology
 
 
</div>
 
</div>
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<div class="text">
 
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The synthetic biology cardgame was designed and created with the aim of teaching about synbio and iGEM in a playful way. More than hundred children 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>
 
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<div class="subtitle">Demonstration prototype</div>
 
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<div class="text"> To validate if our project could actually work a prototype was developed where the different strains could be tested on the chosen carrier. First, different strains were tested with two types of water; 30 g/L NaCl and 1g/L NaCl. This was done to recreate the effect of salt and fresh water. In the graph (Figure 1) you can see the different energy potentials from the tested strains, where a theoretical max of 86 mV could be achieved. <i>B. subtilis bslA</i>  achieved the highest energy potential with an impressive 18,5 mV. This is 21,5% of theoretically maximum. Another tested setup was <i>B. subtilis</i> with an overproduction of <i> tasA,bslA</i>, mixed with <i>B. subtilis ΔabrB slrR</i>. This mixture gave a 17,5 mV. </div>
 
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<div class="text">
 
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Although these test showed that an energy potential could be generated, a real-world prototype was not yet demonstrated. This could be achieved with the use of real seawater and water from a fresh lake(movie). Due to time limits only the <i>B. subtilis</i> with an overproduction of tasA, <i>bslA</i>, mixed with <i>B. subtilis</i> ΔabrB slrR was measured with these circumstances. This measurement resulted in a 21,5 mV energy potential, which is 25% of the theoretically maximum. In the movie the amount of mV is visible on the multimeter. The flow cell can also be seen (link naar flow cell), where there are two compartments, one with fresh and one with salt water. In between of these compartments a biofilm can be put in. Our engineered <i>B. subtilis</i> strains are capable of generating a energy potential over the membrane while simulating the actual conditions in the Netherlands. </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.