-Developed several genetic constructs which resulted in distinct biofilm phenotypes
Template:Team:Groningen/CONTENT/PROJECT/Results
- 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.