Template:Team:Groningen/CONTENT/MEASUREMENT/Measurement

Open circuit voltage

Our project requires a convenient way to measure ion selectivity and biofilm robustness. Although ion selectivity and robustness are difficult to measure directly, the so-called open circuit potential provides a convenient indicator of both ion selectivity and robustness.

Consider a perfect cation exchange membrane which allows all cations to pass but blocks all anions. If such a membrane is used to separate two compartments containing water with different NaCl concentrations, selective diffusion of ions takes place. Specifically, positively charged sodium ions diffuse across the membrane in the towards the lower salt concentration.

Hence, a net positive charge is transported across the membrane. Thus, one compartment gains a net positive charge while the other gains a net negative charge. This difference in charge between the compartments creates an electric field, which counteracts the diffusion of sodium across the membrane. The famous Nernst equation predicts that all diffusion stops when a potential difference of 86 mV is reached.

Since a potential difference is only observed if selective diffusion takes place and, moreover, the potential at which the net charge flow is zero is dependent on the selectivity of the membrane, measurements of the open circuit potential can be used to calculate the (apparent) selectivity of the membrane.

The core of our setup consists of a flow cell originally designed as a microbial fuel cell. This cell consists of two compartments through which water flows are directed using two dropping funnels. These funnels can be loaded salt solutions of different concentrations.

Our measurement device consists of this microbial fuel cell in which the fresh water compartment is separated from the salt water compartment by a membrane.

The potential between the two compartments was measured using two commercial Ag/AgCl reference electrodes connected to a PREMA 5000 multimeter. The measurements for non-GMO membranes were repeated using a high quality potentiostat <model no?> to check if the multimeter was not drawing too much current.

Growing biofilms

The word biofilm is used to denote a pleidae of different extracellular structures created by bacteria. The willingness of bacteria to stick together on a surface using secreted extracellular materials is usually considered the defining property of a biofilm, though it is hard to define a clear boundary between closely packed colonies and a low density biofilm. Biofilms are usually grouped according to the type of surface they inhabit, depicted in table 1.

Solid-liquid

Thin and uniform

Solid-air

Thicker and wrinkly, heterogeneous

Liquid-air

Floating pellicles

The effect of the surface on the biofilm phenotype

Since (by definition) liquid-air pellicles cannot be grown on a solid strength-providing carrier material, they are uninteresting for our project. The solid-air type is the most well-known and can be grown on a simple agar plate. This is convenient, but our setup is nothing like an agar plate, and it is not clear that a biofilm grown on a solid-air interface can cope with the water flow in our setup. The solid-liquid type has the advantage of living at the right kind of surface, but is difficult to grow.

The effect of genetic constructs on biofilm phenotype

To see the difference in robustness phenotypes were characterized. To test if the developed biobricks (biofilm involved genes) had an effect in the bacterium, phenotypical studies were performed. The biobricks that have been created were used to create new B. Subtilis NCBI 3610 ΔcomI strains, mutant strains. With these biobricks four single mutant strains (respectively abrB knockout, slrR+, tasA+ and bslA+) and two double mutant strains (respectively abrB knockout with slrR+ and tasA+ with bslA+) were created.

The abrB knockout results in an overexpression of matrix genes according to the studies. These genes are involved in different aspects of the biofilm forming. With the help from Molecular Genetics from the University of Groningen, an abrB knockout strain was obtained. After growing this mutant for 24 hours on Msgg(Figure 2B), the biofilm was compared to the the B. subtilis comI strain(Figure 2A). The abrB knockout strain showed a smaller and thicker biofilm compared to the control. This phenotype is probably the result of much matrix proteins, keeping the cells together.

Figure2. (A) B. subtilis comI control grown on Msgg (B) abrB knockout mutant grown on Msgg. (C) slrR mutant grown on Msgg. (D) abrB knockout slrR double mutant grown on Msgg.

Another mutant strain is the overproduction of slrR (a transcriptional regulator) through expressing slrR under a strong promoter and RBS. The slrR gene has an important role in biofilm production; regulating the biofilm formation through regulating genes such as the eps operon. B. Subtilis with an overexpression of slrR has shown a bigger biofilm than the wild type control. This phenotype could be due to the overexpression of the eps operon, leading to many extracellular polysaccharide substances. Resulting in a larger biofilm. The combination of the abrB knockout and slrR+ mutant resulted in a comparable biofilm to the control. The process behind the this interaction is not known, however comparing these phenotypes clearly show an interaction.

Both bslA and tasA were constructed under the salt-inducible PProH-promoter. Therefore the phenotyping was done on Msgg with NaCl and Msgg without NaCl. tasA, bslA+ and the double tasA bslA+ mutant strain showed a bigger biofilm than the control. This could be the result of that there is no clear distinction between the phenotype and the wildtype. However, these mutant strains on Msgg with NaCl shows a complete different strain than without NaCl. Where tasA+ mutant grown without larger biofilm, the tasA mutant grown on Msgg with NaCl (Figure 3D) has shown a smaller and more dense biofilm. Which is the direct result from the amyloid-like fibers from the tasA gene. The bslA+ mutant (Figure 3B) grown on Msgg without NaCl showed comparable phenotypes with the tasA mutant without NaCl. Ice crystal like structure were visible with salt induction(Figure 3E).

Figure 3. (A) tasA mutant grown on Msgg without NaCl. (B)bslA+ mutant grown on Msgg without NaCl.(C) tasAbslA+ double mutant grown on Msgg without NaCl.(D) tasA mutant grown on Msgg with NaCl. (E) bslA+ mutant grown on Msgg with NaCl.(F) tasA bslA+ double mutant grown on Msgg with NaCl.