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

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 P_proH 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.

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.

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.

To validate the functioning of our biobricks, a tasA overproducing strain was made under the control of the salt inducible P_proH 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.

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.

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.

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 function in the real world.

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.

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 no Blue Bio Energy, the use of GMOs in some cases, such as Blue Bio Energy, and the use of GMOs in a lot of everyday applications. This gave perspective about which underlying principles motivates stakeholders when forming an opinion on a new technology.

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.