Difference between revisions of "Team:Amsterdam/Project/emulsions"

Stable Romances tend to evolve when there is a perfect understanding between the individuals. Whether a match is a perfect fit (or not!) can be tested when partners are forced to interact for instance, when people move in together. So imagine applying the same principle to consortia were we deduced that an underlying property of all consortia is that ultimately they will display a so-called “high-yield strategy”. By High-yield strategy, it is meant that a more efficient use of natural resources leading to a greater number of individuals will be observed. This is even more so the case when instability is a serious threat. ,

What if there was a way to select these high-yield strategy consortia and actually make them interact better? How would one do this? Well… just like with people, this is possible to test by making microbes move in together.

We decided to tackle this problem by looking at studies done using emulsion based techniques selecting for high-yield strategies (Bachmann et al.). In this paper, the authors discuss the tradeoff that occurs between growth rate and growth yield in microbes. They demonstrate that compartmentalizing the availability of public goods while serial propagating leads to the selection of organisms with increased yield strategy, i.e. able to generate ever higher cell numbers (biomass) from the same portion of substrate. We apply the same logic to consortia by developing a dedicated emulsion-based protocol.

The basic principle is that by randomly combining different individuals of each side of the partnership in isolated compartments (micro-droplets), perfect matches will lead to increased numbers of offspring of both photo- and chemotroph. If propagated in time, ultimately this will enrich the mixture in both photo- and chemotrophs that do well when placed in a consortium while purging the population of the ones that do not.

This method can be used to select “naturally” occurring consortia, or to evolve the synthetic consortia that have been rationally engineered. For example, chemotrophs that use more efficiently the carbon source provided by the phototroph can be co-selected at the same time that the population is enriched in phototrophs that, not only grow efficiently on the required nutrient produced by the chemotroph, but also are more capable of fixing CO2 and “willing” to release the C-source. After this process, biobricks of choice can be added to the chemotroph in order to produce a compound with the knowledge that the chemotroph consumes the produced carbon more efficiently – and hence (if all things alike) is potentially able to make the product of interest also more efficiently.

Emulsions were created using 700μl HFE 7500 mixed with 0.2% picosurf as a surfactant. This was mixed with 300μl of medium (BG11 enriched with TES buffer, Bicarbonate and 5mM ammonium chloride) with the expectation to create 4.6x106 emulsion with an average size of 50μm (Figure 1). Incubation was performed under high light conditions at 30°C. The breaking of the emulsion was performed using breaking solution (1H,1H,2H,2H-perfluorooctanol), It is important to note that the oil in this solution was heavier than the water thus the medium phase remains on top and is easily extractable after adding 700μl of medium. Emulsion integrity is determined using a microscope and viewing average size of the emulsion.

Cell counts were determined using a Coulter Counter. Cells suspended in electrolyte are separated via a microchannel compartment. The cell is drawn in causing a brief change in electrical impedance, which is recorded by the machine. In our case, one could clearly distinguish between Synechocystis and E.coli cells due to the fact that E.coli is on average 1μm and Synechocystis about 2-3μm

Another method used for counting was Fluorescence-activated cell sorting (FACS). This method allowed for cell identification based upon fluorescence and specific light scattering techniques. We could clearly differentiate between Synechocystis, which presents fluorescence at the far red spectrum in contrast to E.coli that shows no autofluorescence.

As shown by Bachmann et al., the emulsion technique has already been used with Lactococcus lactis, a chemotroph. We wanted to test if Synechocystis, a photoautotroph, could grow inside the emulsion droplets. Problems we could have encountered include 1) nature of the medium which is different from lactis, 2) whether CO2 is possible of entering into the medium and 3) whether Synechocystis received enough light.

Our results demonstrate that Synechocystis is in fact able to grow inside the emulsions and we are able to break the emulsion droplets open to extract higher number of cells than the initial concentration. However, after day 7, Synechocystis only undergoes 2 doublings which is slow given that it’s usual generation time is between 12-17 hours. We believe that by optimizing the growth conditions more doublings could be achieved. One possible modification would be to grow Synechocystis inside an incubator where each sample freely receives the same amount of light with little shading from other samples. The use of a Tube roller could improve the amount of light received by the emulsions, however initial attempts at this showed that after a few days emulsion integrity was compromised.

To establish a working emulsion method, we had to prove that we would not lose a lot of cells by creating and breaking the emulsions(link protocol). This is important because we want to have enough cells to serially propagate. First, recovery rate experiments were carried out on each organism separately. The first experiment was done at different pH levels to determine the effect of pH on recovery rates as Synechocystis makes the medium basic whereas E. coli makes it acidic. This was done in BG11 without addition of TES buffer. Data showed that E.coli has a much larger recovery rate than Synechocystis but in contrast it has large variance due to pH (Figure 9). Synechocystis shows a smaller recovery rate but is affected much less by pH change.