Glycerol Module

DISCUSSION

Genetic modification performed in this module consists on the introduction of the glpF gene under an adjustable expression system. This gene codifies for an inner membrane porin that acts as a facilitator of glycerol transport to cytoplasm. This gene was obtained from the genome of P. aeruginosa PAO1, as this bacterium codifies for a protein with a higher maximum transport speed than that of the host bacterium. The genetic modification was performed with the help of the mini-Tn7 system, which was developed in a BioBrick format by members of the previous iGEM Team of our University in the year 2011¹. Mini-Tn7 system has a few advantages compared to other insertion systems. This system can adapt to a great variety of host bacteria, has a high efficiency and it permits insertion in a specific locus of the genome with a specific orientation, avoiding affecting other regions of the bacterial chromosome. Moreover, mini-Tn7 does not need to be continuously selected with antibiotics, as insertion is maintained for at least 100 generations in absence of antibiotics².

Functional assays show that bacteria have a prolonged lag phase when growing in presence of glycerol as sole carbon source in normal conditions. This phase can be reduced by adding a supplement, as can be octanoic acid. It constitutes a highly complex regulation system and role of octanoate is not still known. However, it could act by activating the global energetic system of the bacterium or by generating specific metabolites or cofactors for glycerol assimilation³.

P. putida populations growing in glycerol show a phenotypic variation due to a phenomenon called persistence. Because of this, a fraction of the population does not grow. Persistence protects against external factors, such as antibiotics, that affect growing bacteria, and it also permits the search for alternative carbon sources different from glycerol. This regulation is controlled by glpR gene, which codifies for a repressor of the glpF, glpK and glpD genes, involved in glycerol assimilation. The prolonged lag phase is due to the action of this repressor, as growth in glycerol is only possible when genes previously mentioned are expressed. This way, a mutant that does not express this repressor shows a reduction in the lag phase and a homogeneous growth of all population^3,4. GlpR repressor is controlled by glycerol-3-P (G3P) levels in such a way that a genetically modified bacterium that produces higher levels of this metabolite has a similar phenotype to ΔglpR mutant⁴. Therefore both suppression of the repressor or overexpression of G3P can help by increasing even more glycerol consumption.

As modeling studies predicted, glycerol transport from periplasm to cytoplasm supposes a bottleneck that has been avoided by glpF expression. We have demonstrated that the inner membrane porin synthesis permits a higher growth of the microorganism in presence of glycerol as a sole carbon source than that of the wild type.

Functional assays results also indicate that wild type growth is reduced as glycerol concentration increases. It is still not known the cause for this phenomenon, but it is thought to be due to a reduction of the water potential of the surroundings. This causes an osmotic stress that reduces cellular growth because it increases breathing rates and utilizes cell energy to keep turgidity⁵. Nevertheless, this “injurious” effect of glycerol is not observed in the case of bacteria expressing GlpF protein, which show a higher growth as glycerol concentration increases. This indicates expression of glpF somehow avoids this effect, probably by favoring its introduction and assimilation and helping the reduction of the water potential of the surroundings.

Regarding biofilm formation, we cannot see meaningful differences between modified bacteria and wild type when growing in minimal media with glycerol. This indicates glpF expression does not affect biofilm formation, but does improve bacterial growth.

References

1. http://parts.igem.org/Genome_Integration
2. Choi, K.-H., & Schweizer, H. P. (2006). mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nature Protocols, 1(1), 153–161. http://doi.org/10.1038/nprot.2006.24
3. Escapa, I. F., del Cerro, C., García, J. L., & Prieto, M. A. (2013). The role of GlpR repressor in Pseudomonas putida KT2440 growth and PHA production from glycerol. Environmental Microbiology, 15(1), 93–110. http://doi.org/10.1111/j.1462-2920.2012.02790.x
4. Schweizer, H. P., & Po, C. (1996). Regulation of glycerol metabolism in Pseudomonas aeruginosa: Characterization of the glpR repressor gene. Journal of Bacteriology, 178(17), 5215–5221.
5. Lambertsen, L., Sternberg, C., & Molin, S. (2004). Mini-Tn7 transposons for sitespecific tagging of bacteria with fluorescent proteins. Environmental Microbiology, 6(7), 726–732. http://doi.org/10.1111/j.1462-2920.2004.00605.x

THEY DIDN’T GO WELL…

We started trying to clone oprB gene, which codifies for a transporter of the outer membrane. It was hard to complete the mutagenic PCR (really hard!!!) and we did a first attempt to clone it into pSB1K3 plasmid. Sadly, it didn’t go well. However, Metabolic pathway modeling software analysis told us the bottleneck in the glycerol assimilation is glpF, and its expression is enough to improve it in high rates. As it was no necessary to express oprB, we focused on cloning, integrating and expressing glpF and we didn’t keep on trying to clone oprB.

Also, we did amplified glpK gene by PCR (which codes for a enzyme involved in glycerol catabolism) and tried to clone it into pSB1K3. This first attempt didn’t go well, but, as in the first case, we didn’t tried it anymore as we saw it is not necessary.

Biofilm Module (LapG and expression system xylS2/Pm)

DISCUSSION

The protease LapG cleaves the adhesin LapA (main protein of the biofilm adhesion) what reduce the numbers of the adhesion interactions and then the amount of biofilm is reduced, too. The expression of LapG under the expression system nahR-Psal (BBa_K1973006) in the presence of salicylate influence lightly in the biofilm development because it is only reduced the maximum amount of biofilm. The activity of the added LapG in KT2442 is not counteracted by the inner activity of LapD, that degrades LapG, that causes a lightly reduction of the biofilm. However, the increase of the LapG concentration in KT2442 lapG^- produces a modification in the biofilm formation and dispersal what cause a delay of the biofilm formation due to the degradation of LapA by LapG, and a reduction of the maximum and minimum amount of biofilm. The biofilm dispersal of lapG mutant does not produce, but when we complemented the mutation with the expression of LapG, the strain disperse like the wild type. Even so, we could not complement the LapG deficiency in the lapG mutant. This might be because the expression under nahR-Psal is not regulated at the same way as the wild type, the expression level of our system is constant during all the biofilm development, but the level of LapG in the wild type vary during this process. In both strains the basal expression of the expression system is not enough to cause a change in the biofilm development. To sum up this section we have achieved a strain that is able to disperse the biofilm only when we induce our expression system.

The expression system nahR-Psal-nasF-lapG (BBa_K1973007) has demonstrated that it works correctly. The planktonic and biofilm curves of KT2442 lapG^--TpMRB133 have the same values when we test them in both conditions: adding or not salicylate. That means that the lapG gen does not express, so the nasF attenuator achieve its function, blocking the expression of any gen downstream the Psal promoter. Expressing the transcription antiterminator protein NasR under the Psal promoter would be the next experiment to finish the study of this system expression behavior . After doing this experiment, we will get a expression system of lapG with minimum basal expression levels and when we induce the promoter the expression will raise hugely.

The beta-galactosidase assay permits us to probe the functionality of the mutant Pm promoters and XylS2 protein. The xylS2 gene is under the Psal or Psal-nasF promoter, so when we induce the expression system, it express this gene and XylS2 protein actives the Pm promoter. The functionality of XylS2 is checked when the assay works because we express a GFP under the Pm promoter. Pm1 promoter (BBa_K1973013) is the one whose expression is higher in the strain with nahR-Psal-xylS2 (BBa_K1973024) in the genome. Its expression have a huge difference at induce and no induce conditions, getting an activity of 6000 Miller units at no induce conditions and 47000 Miller units. The expression system with the attenuator nasF (BBa_K1973021) also works correctly because the expression of the fusion protein LacZ-GFPmut3 decreases with this system. The mutations in the promoter Pm have not eliminated its expression, but the expression of the original promoter Pm is similar to the control in the assay. This should be a fail in the introduction of the original promoter Pm in the plasmid because its natural activity is very high. To sum up, we have achieved creating the expression system xylS2/Pm in a Biobrick format and it works successfully.

THEY DIDN’T GO WELL…

We did not get testing the activity of the original promoter Pm by the beta-galactosidase assay, so we do not know the specific effect of the mutation in this promoter. It is not a real problem to finish the project, but that would give an idea to us about how good or bad it is our mutant promoters.