Team:UMaryland/Contribution

Home
Team
Team Attributions Collaborations
Project
Communication Contribution Description Engineering Experiments Implementation Notebook Proof of Concept Results
Parts
Parts
Safety
Human Practices
Awards
Education Model




Contribution

Having benefited from the contributions of the 2016 Purdue iGEM team, we set out to make helpful contributions for future iGEM teams specifically interested in tackling problems related to mitigating excess phosphorus pollution.

Here are some contributions we made for future iGEM teams:

  1. New information learned from literature
  2. Design Improvements: Modularity in genetic construct design

New information learned from literature

According to the 2016 Purdue iGEM team, the protein, polyphosphate-dependent glucokinase (PPGK) was deemed necessary for a phosphorus reclamation module with the function of removing excess phosphorus from bodies of water1. The PPGK enzyme phosphorylates glucose with a phosphate from polyphosphate or a nucleotide triphosphate (catalyzes step one in glycolysis); however, based on our own literature, we learned new information that suggested it was not necessary to include PPGK from M. phosphovorus in our phosphorus sequestration units2. Rudat et. al indicates that E. coli has its own glucose phosphorylation systems that catalyze the first step of glycolysis2. Additionally, in their experimentation of using the PPGK system instead of endogenous glucose phosphorylation systems in E. coli, they found that “expression of PPGK alone did not improve growth,” whereas expression of the endogenous system in E. coli did permit “robust growth”2. This suggests that the inclusion of the PPGK gene in a genetic construct for phosphorus sequestration may not be as imperative.

Design Improvements: Modularity in genetic construct design

Another contribution we made for future iGEM teams interested in phosphorus pollution mitigation was through our improved designs for our genetic construct. In the 2016 Purdue iGEM’s team project, their focus was to primarily characterize key phosphorus-metabolic genes from M. phosphovorus1. As a result, their part designs for each gene were similar (all were His-tagged gene sequences regulated by the constitutive weak Anderson promoter). We wanted to improve this design by specifically designing constructs with built-in modularity for characterizing experiments as well as design aspects that would facilitate ease of assembly.

To do this, we put each gene (PPK1, PPK2 homolog A, PPX2 homolog, Pit homolog A, Pit homolog B, Pit homolog C) into functional groups instead of testing each gene the same way as Purdue first did. Our functional gene groups were:

  1. Uptake of Inorganic Phosphorus (Pi): Pit homolog A, Pit homolog B, Pit homolog C
  2. Storage of Pi as PolyP: PPK1, PPK2 homolog A
  3. Release of Pi: PPX2 homolog

Since the 2016 Purdue team’s characterization did not have statistical significance for all their results, we designed our constructs such that they could be tested in a modular, combinatorial approach1. As mentioned previously, the ideal end goal of our project (though we were ultimately unable to achieve it during this cycle) was to use the best gene combinations and transform the best plasmid from each group (three total) into a single cell. We designed it the “three-plasmid transformation” way to prioritize the modular testing component. As a result, another design improvement we initiated (that we hope will benefit future iGEM teams) is making each functional gene group combination a different antibiotic resistance to facilitate easy later assembly. In our design, we made the Pit gene functional group ampicillin-resistant, the PPK gene functional group as chloramphenicol-resistant, and the PPX2 gene functional group as kanamycin-resistant.

Figure 1. The Pit gene functional group regulated by the pLac promoter is responsible for the uptake of inorganic phosphorus. The purpose of these constructs designs is to determine which gene combination facilitates the best inorganic phosphate transport into the cell. Since these genes are regulated by the pLac promoter, these plasmids will be transformed into XL1-Blue cells that overexpress the lac repressor.

Figure 2. The PPK gene functional group is regulated by the constitutive weak Anderson promoter and is responsible for storing phosphorus in the cells as polyphosphate chains. The purpose of these constructs designs is to determine which gene combination facilitates the best intracellular polyphosphate storage in the cell.

Figure 3. The PPX2 homolog gene functional group is regulated by the inducible pBAD promoter and is responsible for phosphorus release.The purpose of this construct design is to determine how well the PPX2 homolog hydrolyzes the terminal phosphate of the intracellular polyphosphate chains.

References

  1. 2016 Purdue iGEM: Protein Profiles. Retrieved from https://2016.igem.org/Team:Purdue/ProteinProfiles
  2. Rudat AK, Pokhrel A, Green TJ, Gray MJ. Mutations in Escherichia coli Polyphosphate Kinase That Lead to Dramatically Increased In Vivo Polyphosphate Levels. J Bacteriol. 2018;200(6):e00697-17. Published 2018 Feb 23. doi:10.1128/JB.00697-17