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

Revision as of 11:36, 19 October 2015

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.

Figure1. (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.

@@ Line 65: / Line 65: @@
 The effect of genetic constructs on biofilm phenotype
 </div>
 <div class="text">
 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 <i>B. Subtilis</i> NCBI 3610 Δ<i>comI</i> strains, mutant strains. With these biobricks four single mutant strains (respectively <i>abrB</i> knockout, <i>slrR+</i>, <i>tasA+</i> and <i>bslA+</i>) and two double mutant strains  (respectively <i>abrB</i> knockout with <i>slrR+</i> and <i>tasA+</i> with <i>bslA+</i>) were created.
 </div>
 <div class="text">
-The <i>abrB</i> 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 <i>abrB</i> knockout strain was obtained. After growing this mutant for 24 hours on Msgg(Figure 1B), the biofilm was compared to the the <i>B. subtilis comI</i> strain(Figure A). The <i>abrB</i> 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.
+The <i>abrB</i> 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 <i>abrB</i> knockout strain was obtained. After growing this mutant for 24 hours on Msgg(Figure 2B), the biofilm was compared to the the <i>B. subtilis comI</i> strain(Figure 2A). The <i>abrB</i> 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.
 </div>
+{{Template:Team:Groningen/TEMPLATES/OBJECT/FIGURE
+	|type=small
+	|imgversion=/f/f3
+	|image=example
+	|caption=Figure1. (A) <i>B. subtilis comI</i> control grown on Msgg (B) <i>abrB</i> knockout mutant grown on Msgg. (C) <i>slrR</i> mutant grown on Msgg. (D) <i>abrB</i> knockout <i>slrR</i> double mutant grown on Msgg.
+}}