Difference between revisions of "Template:Team:Groningen/CONTENT/MEASUREMENT/Measurement"

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 is pumped using two dropping funnels. These funnels can be loaded with salt solutions of different concentrations.

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.

\( E = \frac{RT}{zF} \ln \frac{[\text{ion outside cell}]}{[\text{ion inside cell}]} = 2.3026 \frac{RT}{zF} \log \frac{[\text{ion outside cell}]}{[\text{ion inside cell}]} \)

E is the is the potential in mV. F is Faradays constant, z is the charge of the ion passing the membrane, R the gas constant and T the temperature.

In our experiments salt and fresh water concentrations consisted of 30 g/L NaCl and 1 g/L NaCl in demiwater, resulting in a maximum potential of 86 mV. In Figure 6, the experimental values measured for biofilms of Bacillus are shown for various strains.

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.

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 of 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.

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.

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