Team:UPO-Sevilla/Demonstrate

DEMONSTRATE

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 population3,4. 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.