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

Biofilm Module (LapA and expression system xylS2/Pm)

Analyze of the biofilm of the strains with the nahR-Psal expression system

The strains used in these experiments were generated by electroporation and transposition, as described in Protocols (Electroporation and transposition), and they are KT2442-TpMRB128 (nahR-Psal-lapG), KT2442-TpMRB133 (nahR-Psal-nasF-lapG), KT2442 lapG^--TpMRB128 and KT2442 lapG^--TpMRB133. We did serial dilution-based growth curves of these strains to test the activity of lapG under the nahR-Psal and nahR-Psal-nasF expression system in LB and LB+salicylate 2 mM, as described in Protocols (Dilution series-based growth curves). TpMRB128 is the part BBa_K1973006 and TpMRB133 is the part BBa_K1973007.

In the experimental design we have made 1-3 replays for each condition. We consider a condition as each combination of the strains, the presence of salicylate and the kind of expression system. We have taken 10 measurements corresponding to different dilutions in each condition. And we made 8 lectures of each dilution per biological replica. The data are represented like the average ± standard error. We have made a t-test check for the statistic analyze and we consider significant the values of p<0.05. It has been represented with: * for p<0.05, ** for p<0.01 and *** for p<0.001.

The growth curves test of the strains KT2442-TpMRB128 and KT2442 lapG^--TpMRB128 will reveal if the nahR-Psal expression system express correctly the lapG gene at the induction conditions with and without salicylate (Figure 4). After the experiments, we observed that both strains have a similar planktonic growth curves to the control and between them in LB and LB+salicylate. As regards the biofilm growth, KT2442 lapG^- produce 2-3 times more biofilm than the wild type and do not disperse the biofilm due to the fact that KT2442 lapG^- is a biofilm superproducer. The complementation of lapG in the lapG mutant under the induced expression system causes a delay of the biofilm formation, a reduction of the maximum amount of biofilm and the biofilm disperses. The expression of LapG in the wild type does not cause a big change in the behavior of the biofilm development, it only produces a little reduction of the amount of biofilm.

Figure 4. Serial dilution-based growth curves of the expression system nahR-Psal-lapG (20 hours). The image represents the growth curves of the strains wild type in LB (A), wild type in LB+salicylate 2 mM (B), lapG^- in LB (C) and lapG^- in LB+salicylate 2mM (D). We assay the strains wild type Tn7 Ø and lapG^- Tn7 Ø concurrently to compare the induction effect of the expression system. Each blot represent the average of 24 data.

We observe a possible biofilm dispersal when we induce the expression system in lapG mutant, but we can't be sure of that. So, we will repeat the experiment with a little modification, we will incubate the plates for 30 hours (Figure 5) instead of 20 hours to observe if the biofilm disperses at all or keeps high like the lapG mutant. We only did one replica of this experiment, so the results of the curves are not clear enough. But the biofilm growth tendency of the strain, when is induced by salicylate, shows that the expression of LapG protein promotes the biofilm dispersal like the wild type. The strains without inducing have grown in a different rate, although the tendency of both curves are similar. We suppose that their behavior is similar and the induction by salicylate does not affect to the biofilm development.

Figure 5. Serial dilution-based growth curves of the expression system nahR-Psal-nasF-lapG (30 hours). The image represents the growth curves of the strains lapG^- in LB (A) and lapG^- in LB+salicylate 2 mM (B). We assay the strain lapG^- Tn7 Ø concurrently to compare the induction effect of the expression system. Each blot represent the average of 8 data.

At the same way, we test the nahR-Psal-nasF expression system in the strains KT2442-TpMRB133 and KT2442 lapG^--TpMRB133 (Figure 6). In this case, the planktonic growth of the both strains keep a similar pattern to the control and between them. As same as before, the strain KT2442 generate 2-3 less time than the lapG mutant. As well as, the biofilm curve of induced KT2442 lapG^--TpMRB133 keeps the same tendency that the control strain, so this strain have the same biofilm generation in LB and LB+salicylate.

Figure 6. Serial dilution-based growth curves of the expression system nahR-Psal-nasF-lapG (20 hours). The image represents the growth curves of the strains wild type in LB (A), wild type in LB+salicylate 2mM (B), lapG^- in LB (C) and lapG^- in LB+salicylate 2 mM (D). We assay the strains wild type Tn7 Ø and lapG^- Tn7 Ø concurrently to compare the induction effect of the expression system. Each blot represent the average of 24 data.

Study of the activity of the mutant Pm promoters by a beta-galactosidase activity assay

First of all, we introduce the three variants of Pm (original Pm, Pm1, Pm2 and Pm3) with the fusion protein lacZ-gfpmut3 by triparental mating, as described in Protocols (Triparental mating), in the strains KT2442-TpMRB170 (nahR-Psal-xylS2) and KT2442-TpMRB159 (nahR-Psal-nasF-xylS2) that were generated by electroporation and transposition, as described in Protocols (Electroporation and transposition). We test the Pm promoters in LB and LB+salicylate 2 mM and our control is the plasmid with the fusion protein lacZ-gfpmut3 without promoter (pMRB1). We create this promoter by mutagenic PCR,as described in Protocols (Site-directed mutagenesis using overlap extension PCR), so we do not know the effect of this mutation in a promoter and it could convert in a constitutive or inducible by other molecule promoter, rise or reduce its basal or induce expression... TpMRB170 is the part BBa_K1973024, TpMRB159 is the part BBa_K1973021, Pm1 is the part BBa_K1973013, Pm2 is the part BBa_K1973014 and Pm3 is the part BBa_K1973015.

We test the 8 constructions using the beta-galactosidase activity assay in 2 different conditions (LB and LB+salicylate 2 mM) (Figure 7). We did 4 replicates of the strains with the transposon nahR-Psal-xylS2 (BBa_K1973024) and 2 replicates of the strains with the transposon nahR-Psal-nasF-xylS2 (BBa_K1973021). There is a significant difference between the expression of the promoters when the regulation module (expression of xylS2) is induced and when it is not induced. As well as, the strains with nahR-Psal-xylS2 have more expression when it is induced and when it is not induced in comparison with the strains with the attenuator.

Figure 7. Beta-galactosidase assay of Pm promoters. The image represents the beta-galactosidase activity of the different Pm promoters in the strains expressing xylS2 without nasF (A) and with the attenuator (B). We assay the strains KT2442-TpMRB170/pMRB1 and KT2442-TpMRB159/pMRB1 concurrently to compare the induction effect of the expression promoter.

The three variants of the Pm promoter work satisfactory and the Pm1 promoter (BBa_K1973013) is the variant that has the mayor expression in the system without the attenuator when it is induced. On the other hand, the Pm3 promoter (BBa_K1973915) is the variant that has the mayor expression in the system with the attenuator when it is induced. We have had problems with the expression of the original Pm promoter and it seems like this promoter does not work. So, we could not have made a comparison between the activity of the original Pm and its variants, but our mutant promoters work perfectly.