Difference between revisions of "Team:Amsterdam/Project/Phy param/Ecoli"

The problem that we are addressing is urgent, and we need a feasible solution fast! We think E. coli is the ideal candidate for this, as it can grow on many of the compounds that Synechocystis has been engineered to secrete [1], and is the workhorse of synthetic biology: most bio-bricks are developed for and tested in E. coli. Thus, in theory E. coli is the ideal chemoheterotroph for our consortium. However, if we want to create meaningful models that can help us to rationally design our consortium, we need to measure how E. coli will behave in conditions that would be used for co-culturing with cyanobacteria. To predict growth behaviour in co-culture, we need to know if E. coli can grow under these conditions, how fast it can grow on carbon compounds that Synechocystis could be engineered to secrete, and at what rates it will consume these substrates. Furthermore, we would like to measure these parameters not only for wild-type E. coli, but also for an arginine overproducing strain, that could be used in a consortia with an arginine auxotrophic Synechocystis.

E. coli is usually grown in rich media such as LB-medium at 37°C, providing enough nutrients to ensure fast growth. In our consortium, E. coli will be grown at conditions which are ideal for Synechocystis to ensure enough production of carbon compounds on which E. coli can feed. Cyanobacteria are usually grown at 30°C in BG-11 medium, a minimal medium containing some minerals and sodium nitrate as a source for nitrogen. However, E. coli grows slower at this temperature, is not able to grow without a carbon source such as glucose, and it can’t use nitrate as a source for nitrogen. Therefore, the BG-11 needs to be supplemented with a carbon source and an extra nitrogen source such as ammonium chloride. In our consortia, the carbon source would be provided by the cyanobacteria, but here the carbon compound was added artificially to mimic that scenario.

To obtain enough viable cells for experiments, E. coli was grown overnight in LB medium in shaking (200 rpm) flasks at a temperature of 30°C. Cells were spinned down, washed twice in BG-11 and used to inoculate an overnight culture in BG-11 supplemented with 18mM ammonium chloride and 20-40 mM of either glucose, glycerol, lactate or acetate. This culture was (after spinning down and washing twice in BG-11) used for experiments.

One important parameter for predicting how E. coli will behave in co-culture with a carbon compound generating Synechocystis is the biomass yield of E. coli, i.e. the amount of biomass that E. coli can make with a certain amount of substrate. To measure this, biomass formation needs to be monitored at different concentrations of the substrate. In figure 1 the growth curves are shown for E. coli strain K-12 MG1655 growing in BG-11 supplemented with 5 mM ammonium chloride and 0 - 5 mM of glucose, glycerol, lactate or acetate. The maximum OD600 reached during 2 days of monitoring growth under these conditions is shown in figure 2. Yields are listed in table 1. In figure 3, growth of E. coli is shown in BG-11 media supplemented with 40 mM acetate and 0 - 5 mM ammonium chloride. The maximum OD600 reached during this experiment is plotted in figure 4. The figures show that E. coli is unable to grow on BG-11 with only nitrate, and a linear increase of the amount of biomass formed with increasing ammonium chloride concentration, with a yield of 0.27 OD/mM ammonium. carbon source growth rate (h-1) uptake rate (mM/h/OD) acetate 0.031 ± 0.002 1.7 glucose 0.289 ± 0.014 1.4 glycerol 0.203 ± 0.030 1.8 lactate 0.144 ± 0.016 2.8

Two other important parameters that are needed to rationally design our consortia are the growth rate of E. coli and the rate at which it consumes the carbon source that Synechocystis would produce. Furthermore, we would like to know how these parameters differ for an arginine overproducing strain of E. coli (the arginine strain was kindly provided by Prof. Dr. D. Chalier from Brussels Free University [2]). In figure 5 (top plot) OD600 of three E. coli cultures grown in BG-11 supplemented with 5mM ammonia and 3 mM of glucose, glycerol, lactate or acetate are shown for the first 9 hours of the experiment. The growth rate was estimated by finding the best fit of an exponential curve to 3 consecutive time points, and are listed in table 2. The plot on the bottom (figure 5) shows the concentration of each carbon source as measured by HPLC. In figure 6 the same data is plotted for E. coli strain K-12 MG1655 grown on acetate, but also the “growth on” and “consumption of” acetate by E. coli strain K12 p4x and p4xb2. These are a wildtype (p4x) E. coli strain and a derivative strain (p4xb2) that overproduces arginine. Unfortunately, arginine could not be measured. Growth rates and uptake rates of the three strains are listed in table 3. strain growth rate (h-1) uptake rate (mM/h/OD) MG1655 0.031 ± 0.002 1.7 P4X 0.064 ± 0.06 0.008 P4XB2 0.013 ± 0.0004 1.6