Template:Team:Groningen/CONTENT/PROJECT/Results

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 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.

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 P_proH inducible promoter

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.

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 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.