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.

Figure 2. (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).

This could be due to the hydrophobic properties from BslA. The tasA and bslA+ and the double tasA bslA+mutant showed comparable phenotypes with both single mutants when grown without NaCl. When grown on NaCl, the phenotype of the double tasA bslA+ mutant strain shows a combination of both phenotypes from the single mutants. The strong dense biofilm from the tasA single mutant and the hydrophobic properties from the bslA+ single mutant.

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.

Validation of tasA and the P_proH inducible promoter

Bacillus subtilis is capable of coping with fluctuating salt concentrations. One of the genes involved is proH, this is an 1-pyrroline-5-caboxylate reductase. A study has shown that this gene is under control of the salt inducible proH promoter (Brills, J et al. 2011). The salt inducible promoter can be activated by a range of NaCl concentrations, respectively ranging from 0,1 M NaCl up to 1 M NaCl.

We have cloned this promoter out of the genome of Bacillus subtilis and combined it with tasA (BBa_K1597002) (amyloid-like fibers in the biofilm matrix). To confirm that the salt inducible promoter functions, our construct was placed in B. subtilis NCIB 3610 ΔcomI with the use of BBa_K823023 . B. subtilis with salt inducible PproH promoter and tasA construct (tasA+) and wild type were grown on Msgg media with and without 0,5M salt.

After 24 hours 50 mM thioflavin S (which is an amyloid fiber staining) was added to the biofilms (Figure 3). The biofilms were photographed with white light and fluorescence (430 nm excitation, 100 ms exposure) after 15 min incubation.

With the induction of NaCl, the strain containing the tasA overproduction cassette tasA+ shows more amyloid fibers are present after 24 hours. This is not the case for the wild type. This indicates that the salt promoter is indeed activated with NaCl, causing an overexpression of tasA.

Figure 4. (A) comI strain grown on Msgg without salt. (B) tasA+mutant grown on Msgg without salt.(C) comI strain grown on Msgg without salt.(D) tasA+mutant grown on Msgg with salt.

Another method to validate our salt promoter was with the characterization of phenotypes with different NaCl concentration. These results can be found in thetasA and bslA overproduction <a href="https://static.igem.org/mediawiki/parts/7/76/TasA_and_bslA_single_and_double_mutant.pdf">document</a>.

Another method is to measure TasA protein with thioflavin S over time in both wildtype and in the tasA overexpression strain. Both strains were grown on SSM media (supplemented with 10 µM thioflavin S) with different salt concentrations, ranging from 0M NaCl up to 1 M NaCl. Over time the fluorescence (em: 430 nm, ex: 485 nm, Gain50) and the OD (600 nm) were measured using a plate reader. The plates were incubated at 37 °C.

Strains grown on a NaCl concentration above 0,5M showed little growth. Therefore these results are not shown here. A bar chart was plotted after 1 hour and after 3 hours for both   the control strain and the tasA+ strain. After one hour the tasA+ strain shows a higher fluorescence than the  control (Figure 5A). After 3 hours the tasA+ strain shows, with an induction of 0,1M; 0,2M and 0,3M NaCl, a significant increase than the control(Figure 5B). This confirms that both the salt-inducible (<a href="http://parts.igem.org/Part:BBa_K1597000">BBa_K1597000</a>) and the tasA biobrick function in B. subtilis

Figure 5. (A) The difference in fluorescent emission between the control strain and the tasA+ strain one hour after NaCl addition. (B) The difference in fluorescent emission between the control strain and the tasA+ strain three hours after NaCl addition.

Ion-selectivity

To test the ion selectivity, the volts generated by the membrane was measured and the ion-selectivity can be calculated using the Nernst equation.

E is the mV generated. Alpha is the ion-selectivity, z is the charge of the ion passing the membrane. R the gas constant, T the temperature.

With a salt solution of 30 g sodium chloride and demi water with 1 g/l sodium chloride the maximum potential is 86 mV. Only the monovalent ion, so ions with a charge or 1 have been used in our setup. By comparing the measurements with the maximum potential the ion-selectivity can be calculated.

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.

Figure 6. Energy potential of different B.subitilis strains.