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

Line 1: Line 1:
 
<div class="content module">
 
<div class="content module">
 +
<div class="text">
 +
-Developed several genetic constructs which resulted in distinct biofilm phenotypes
 +
</div>
 +
<div class="text">
 +
-    Created 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">
  
−
<div class="subtitle">
+
-    Used combinations of these constructs to create a biofilm which is ion selective
−
Overview
+
 
</div>
 
</div>
−
<div class="text">  
+
<div class="text">
−
Characterized biofilm properties of B. subtilis. Natto, 3610 ComI, 168, JH642 and PY79
+
-    Demonstrated ion-selectivity of <i>B. subtilis</i> biofilm with our prototype in the real-world
 
</div>
 
</div>
−
<div class="text">  
+
<div class="text">
−
Demonstrated ion-selectivity of the B. subtilis biofilm and improved it with our biobricks.
+
-   y-PGA was modeled as a Cation Exchange membrane using Molecular Dynamics
 
</div>
 
</div>
−
<div class="text">  
+
<div class="text">
−
Modeled y-PGA as a Cation Exchange membrane using Molecular Dynamics
+
-   Created future scenarios about our project and the use of GMOs.
 
</div>
 
</div>
−
<div class="text">  
+
<div class="text">
−
Designed drip and continuous flow method to grow more robust biofilms.
+
-    A card game was developed to teach about synthetic biology in a fun way
 
</div>
 
</div>
−
<div class="text">  
+
 
−
Created easy cheap test to measure rigidity of biofilms.  
+
 +
 
 +
 
 +
 
 +
<div class="subtitle">Phenotypical differences after biobrick introduction in <i>B. subtilis</i></div>
 +
<div class="text">
 +
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>
 +
 
 +
<div class="subtitle">
 +
Created salt inducible promoter to control several biofilm genes
 
</div>
 
</div>
−
<div class="text">  
+
<div class="text">
−
Created new backbone(K1597001) for integration into ThrC locus in B. subtilis and validated it.</div>
+
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>
−
<div class="text">  
+
  
−
Constructs created for TasA, BslA, Abrb KO, SlrR, y-PGA and ProH
+
<div class="subtitle">
 +
Validation of <i>tasA</i>  and the <i>P<sub>proH</sub></i> inducible promoter
 
</div>
 
</div>
−
<div class="text">  
+
<div class="text">
−
Created biobricks to increase the robustness.
+
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>
 +
 
 +
 
 +
<div class="subtitle">
 +
A new shuttle vector
 
</div>
 
</div>
−
<div class="text">  
+
<div class="text">
−
Validated ThrC(K1597001), TasA(K1597002) and ProH (K1597000)
+
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>
 +
 
 +
<div class="subtitle">
 +
Ion selectivity in the lab
 
</div>
 
</div>
−
<div class="text">  
+
<div class="text">
−
Created future scenario’s about GMO’s and sustainability and a cardgame for education
+
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.
 
</div>
 
</div>
−
 
  
 
<div class="subtitle">
 
<div class="subtitle">
−
Bacillus Subtilis & Growth Method
+
Demonstration of working prototype
 
</div>
 
</div>
 +
 
<div class="text">
 
<div class="text">
−
<i>Drip flow experiment/ directed evolution</i>
+
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)
 +
</div>
 +
 
 +
<div class="subtitle">
 +
y-PGA
 
</div>
 
</div>
 
<div class="text">
 
<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
+
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.
 
</div>
 
</div>
 +
 +
<div class="subtitle">Future scenarios for Blue Bio Energy</div>
 +
<div class="text">
 +
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>
 +
 +
 +
<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 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>
 +
 +
 +
<div class="subtitle">Demonstration prototype</div>
 +
 +
<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>
 +
 +
<div class="text">
 +
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>

Revision as of 03:48, 19 September 2015

-Developed several genetic constructs which resulted in distinct biofilm phenotypes

- Created 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.

- A card game was developed 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. This resulted in different phenotypes after growing for 24 hours, where we can conclude that the developed biobricks have an impact on B. subtilis.

Created salt inducible promoter to control several biofilm genes

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 B. subtilis . Several of these genes were controlled under the developed salt inducible promoter. Introduction of these biobrick led to different phenotypes.

Validation of tasA and the PproH inducible promoter

To validate that our biobricks function as expected, a tasA overproducing strain was made under the control of the salt inducible PproH promoter. The use for salt inducible PproH 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???)


A new shuttle vector

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 E. coli and integrating in the thrC locus from B. subtilis.

Ion selectivity in the lab

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.

Demonstration of working prototype

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)

y-PGA

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 B. subtilis could make the biofilm ion-selective for sodium ions. This could increase the total amount of energy generated.

Future scenarios for Blue Bio Energy
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)


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 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.


Demonstration prototype
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. B. subtilis bslA achieved the highest energy potential with an impressive 18,5 mV. This is 21,5% of theoretically maximum. Another tested setup was B. subtilis with an overproduction of tasA,bslA, mixed with B. subtilis ΔabrB slrR. This mixture gave a 17,5 mV.
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 B. subtilis with an overproduction of tasA, bslA, mixed with B. subtilis 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 B. subtilis strains are capable of generating a energy potential over the membrane while simulating the actual conditions in the Netherlands.