Team:UPO-Sevilla/Experiments

EXPERIMENTS

Glycerol Module

Bacterial growth measurement

This physiological assay was performed as described in Protocols (Bacterial growth measurement). By measuring bacterial growth in a medium with glycerol as the only carbon source, we can explain the glycerol consumption of the different strains.

Growth of bacteria expressing glpF (with the BBa_K1973027 part inserted in the genome) was compared to that of the control, bacteria with an “empty” mini-Tn7, a Tn7 with no added information, in different conditions. It was expected that bacteria expressing glpF in presence of an activator would reach higher growth rates than the control.

The growth media prepared for this assay were minimal media with different carbon sources and different concentrations: 25 mM succinate, 25 mM glycerol, 5 mM glycerol, 1 mM glycerol and 0,2 mM glycerol. Each medium was prepared twice, and we added 20 μl of salycilate 1M (a final concentration of 2 mM) to one of each kind. Salycilate acts as an activator of the expression system. Minimal medium components are the following (for a 100 ml stock): 10 ml NaCl-P 10X, 1 ml NH₄Cl 100 g/L, 1 ml vitamins solution 100X, 0,2 ml microelements solution 500X, the carbon source and water until reaching the final volume.

By performing this assay for the first time, we saw that both bacteria, that expressing glpF and the control, grew in a similar manner when using succinate as a sole carbon source. However, in no case exponential phase was reached when using glycerol. Then, we put the same microplate with the same bacteria in the fluorimeter to keep growing for another 23 hours. After that time, we saw bacteria had started growing in media with glycerol as a sole carbon source (Fig. 1).

We can stand up that modified bacteria reach a higher absorbance than control in a minimal media with glycerol 5 mM (data not shown). However, growth curves were not reliable as there was not a correlation between glycerol concentration and growth. Because of this, and because of the prolonged lag phase, we repeated the experiment.

The delay is due to a system developed in P. putida KT2442 that implies a prolonged lag phase when metabolizing glycerol. According to Escapa et al. (2013), this can be avoided by the addition of growth precursors, such as octanoic acid 1 mM. This acid, apart from improving the growth, would increase the useful molecule production.

We then repeated the experiment adding octanoic acid 1 mM as a growth precursor. In this case, we observed lag phase was substantially reduced, and bacteria reached exponential phase when growing in glycerol during the 23 hours the experiment lasts (Fig. 1). Results show bacteria expressing glpF reach a higher absorbance than control in media with glycerol (Fig. 2).

Figure 1. Growth graphs of experiments 1 and 2. Here we show growth curves in minimal media with salycilate and carbon source indicated in the legend. We did not add octanoic acid in experiment 1, where lag phase lasts between 35 and 36 hours. We did add octanoate in experiment 2, where lag phase is reduced to less than 10 hours.

Experiment 2 results show genetically modified bacteria expressing glpF grow much more than wild type in media with glycerol. This behavior is observed in all media with glycerol at different concentrations of this substrate (Fig. 2). This way, we conclude that the expression of the gene encoding for the glycerol transporter of the inner membrane is enough to increase consumption of this molecule, as modeling studies predicted.

In both experiments 2 and 3 (data not shown), we observe that wild type growth decreases as glycerol concentration increases. However, bacteria expressing glpF grow more as glycerol concentration increases (Fig. 2). GlpF seems to avoid the “damaging” effect of glycerol in bacterial growth. This means an advantage on an industrial scale, as modified bacteria would consume a higher concentration of glycerol than wild type, that is, more glycerol in less time.

Moreover, these results show growth in media with salycilate don’t differ significantly from that in media without it. This means basal expression of glpF is sufficient to increase growth in presence of glycerol.

Figure 2. Graphs showing bacterial growth in media with glycerol as a sole carbon source. Genetically modified bacteria reach a higher absorbance than wild type in both media with and without salycilate. Salycilate effect seems to not be significant for the increase in glycerol consumption.

Biofilm curves in glycerol

We also performed a second physiological assay involving the formation of biofilms of P. putida KT2442 expressing glpF. We wanted to study the effects of expressing this gene on the formation of a biofilm in a medium with glycerol as a sole carbon source. To that end we made a dilution series-based growth curves as indicated on Protocols (Dilution series-based growth curves), comparing our strain containing the BBa_K1973027 part and the wild type.

By measuring absorbance, we studied planktonic growth and biofilm of both strains in minimal media with glycerol 25 mM as a sole carbon source (composition indicated in Bacterial growth measurement) with and without salycilate.

We represented all data in graphs and studied the behavior of these bacteria. We can see that bacteria expressing glpF under the nahR-Psal expression system reach higher growth rates than wild type, as we saw in the previous assay (Bacterial growth measurement) (Fig. 3). This way, we have demonstrated this behavior through two different assays. Regarding biofilm formation, we observe wild type bacteria seem to reach higher absorbance levels at some points and at the end of the assay. However, both strains show a similar conduct (Fig. 3).

Figure 3. Graph showing planktonic growth and biofilm of modified bacteria expressing glpF and wild type in minimal media with glycerol as sole carbon source and salycilate. We can observe planktonic growth of modified bacteria reach higher absorbance levels than wild type.

Same results are obtained in media without salycilate (data not shown). As indicated in the previous assay, salycilate addition is not necessary to improve glycerol assimilation.

This assay shows us biofilm formation is similar in modified bacteria and wild type when growing in minimal media with glycerol. While planktonic growth is improved in modified bacteria, biofilm formation does not show significant differences.

Tables

Table 1. Oligonucleotides used in the mutagenic PCR of glpF. Here we represent the name of the oligonucleotides, their sequences and the number of nucleotides of each one. The nucleotides that are written in small letter do not hybridize with the gene sequence (prefix, suffix and mutations).

Table 2. All plasmids generated and needed for this module.

Table 3. Oligonucleotides used for sequencing the gene in pSB1K3

Table 4. Oligonucleotides used for verifying the insertion of the constructions in the genome of P. putida KT2442