Team:NYMU-Taipei/Project/Phosphate

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Contents

Background

Goal and Circuit Operation

  • Enhance and regulate the phosphate absorption of E.coli to balance phosphate concentration to a normal level in patients with kidney failure
  • This device operates when:
    • external pH is high and
    • external phosphate level is high
  • That is, this device will stop when:
    • external pH is low or
    • external phosphate level is low

Circuit Design

NYMU iGEM08 phosphate removal device v 1.0

NYMU IGEM08 phosphate device.png

  • over-expression of pst operon can enhance the total phosphate absorption by improving the cellular phosphate import
  • over-expression of ppK gene can enhance the total phosphate absorption by improving the cellular phosphate storage

Experimental Results

PhoB promoter - external phosphate sensory promoter

PhoB promoter under 40uM K2HPO4 (phosphate starvation)

NYMU 20081003 pPhoB 40uM pi growth.png

NYMU 20081003 pPhoB 40uM pi GFP.png

PhoB promoter under 40uM, 200uM and 1,000uM K2HPO4

PPhoB 20080927 + 20081003.png

PPK - synthase of Polyphosphate

PST - high affinity phosphate transporter

Simulation

References

Phosphate Metabolism and Regulation in E.coli

  • Rao NN, Torriani A (1990). "Molecular aspects of phosphate transport in Escherichia coli." Mol Microbiol 1990;4(7);1083-90.

PITA and PITB

  • Willsky GR, Malamy MH (1980). "Characterization of two genetically separable inorganic phosphate transport systems in Escherichia coli." J Bacteriol 1980;144(1);356-65.
  • Beard SJ, Hashim R, Wu G, Binet MR, Hughes MN, Poole RK (2000). "Evidence for the transport of zinc(II) ions via the pit inorganic phosphate transport system in Escherichia coli." FEMS Microbiol Lett 2000;184(2);231-5.
  • [http://jb.asm.org/cgi/content/abstract/183/17/5008 Characterization of PitA and PitB from Escherichia coli, JB 2001]

PST operon

  • Aguena M, Yagil E, Spira B (2002). "Transcriptional analysis of the pst operon of Escherichia coli." Mol Genet Genomics 268(4);518-24.
  • Amemura M, Makino K, Shinagawa H, Kobayashi A, Nakata A (1985). "Nucleotide sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli." J Mol Biol 184(2);241-50. PMID: 2993631
  • Kimura S, Makino K, Shinagawa H, Amemura M, Nakata A (1989). "Regulation of the phosphate regulon of Escherichia coli: characterization of the promoter of the pstS gene." Mol Gen Genet 1989;215(3);374-80. PMID: 2651888
  • Magota K, Otsuji N, Miki T, Horiuchi T, Tsunasawa S, Kondo J, Sakiyama F, Amemura M, Morita T, Shinagawa H (1984). "Nucleotide sequence of the phoS gene, the structural gene for the phosphate-binding protein of Escherichia coli." J Bacteriol 157(3);909-17. PMID: 6365894
  • Makino K, Shinagawa H, Amemura M, Kimura S, Nakata A, Ishihama A (1988). "Regulation of the phosphate regulon of Escherichia coli. Activation of pstS transcription by PhoB protein in vitro." J Mol Biol 1988;203(1);85-95. PMID: 3054125
  • Otsuka J, Watanabe H, Mori KT (1996). "Evolution of transcriptional regulation system through promiscuous coupling of regulatory proteins with operons; suggestion from protein sequence similarities in Escherichia coli." J Theor Biol 1996;178(2);183-204. PMID: 8729576
  • Salmon K, Hung SP, Mekjian K, Baldi P, Hatfield GW, Gunsalus RP (2003). "Global gene expression profiling in Escherichia coli K12. The effects of oxygen availability and FNR." J Biol Chem 278(32);29837-55. PMID: 12754220
  • Spira B, Yagil E (1999). "The integration host factor (IHF) affects the expression of the phosphate-binding protein and of alkaline phosphatase in Escherichia coli." Curr Microbiol 38(2);80-5. PMID: 9871104
  • Steed PM, Wanner BL (1993). "Use of the rep technique for allele replacement to construct mutants with deletions of the pstSCAB-phoU operon: evidence of a new role for the PhoU protein in the phosphate regulon." J Bacteriol 1993;175(21);6797-809. PMID: 8226621
  • Surin BP, Jans DA, Fimmel AL, Shaw DC, Cox GB, Rosenberg H (1984). "Structural gene for the phosphate-repressible phosphate-binding protein of Escherichia coli has its own promoter: complete nucleotide sequence of the phoS gene." J Bacteriol 1984;157(3);772-8. PMID: 6321434
  • Surin BP, Rosenberg H, Cox GB (1985). "Phosphate-specific transport system of Escherichia coli: nucleotide sequence and gene-polypeptide relationships." J Bacteriol 161(1);189-98. PMID: 3881386
  • Taschner NP, Yagil E, Spira B (2004). "A differential effect of sigmaS on the expression of the PHO regulon genes of Escherichia coli." Microbiology 150(Pt 9);2985-92. PMID: 15347756
  • Taschner NP, Yagil E, Spira B (2006). "The effect of IHF on sigmaS selectivity of the phoA and pst promoters of Escherichia coli." Arch Microbiol 185(3);234-7. PMID: 16404567

Phosphate-oriented regulation

  • [http://www.blackwell-synergy.com/doi/abs/10.1111/j.1574-6976.2008.00101.x The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis, FEMS Microbiology Reviews 2008]

Phosphate Removal by Engineered Microbials

  • [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=182526 Genetic improvement of Escherichia coli for enhanced biological removal of phosphate from wastewater, AEM 1993]
  • [http://www.ncbi.nlm.nih.gov/pubmed/10448683 Genetic improvement of bacteria for enhanced biological removal of phosphate from wastewater, Prog Mol Subcell Biol. 1999]
  • [http://aem.asm.org/cgi/content/full/68/8/4107?view=long&pmid=12147514 Accumulation of Inorganic Polyphosphate in phoU Mutants of Escherichia coli and Synechocystis sp. Strain PCC6803, AEM 2002]
  • [http://www.ncbi.nlm.nih.gov/pubmed/12187377 Enhanced phosphate uptake and polyphosphate accumulation in Burkholderia cepacia grown under low pH conditions, Microb Ecol. 2002] and abstract
  • [http://www.ncbi.nlm.nih.gov/pubmed/12964240 Microbial phosphate removal and polyphosphate production from wastewaters, Adv Appl Microbiol. 2003]
  • [http://www.springerlink.com/content/t6qkt5214pqncmjd/ Intelligent yeast strains with the ability to self-monitor the concentrations of intra- and extracellular phosphate or ammonium ion by emission of fluorescence from the cell surface, AMB 2004]
  • [http://www.jstage.jst.go.jp/article/jbb/95/6/95_637/_article/-char/en A method for screening polyphosphate-accumulating mutants which remove phosphate efficiently from synthetic wastewater, Journal of Bioscience and Bioengineering 2005]
  • [http://www.nature.com/nbt/journal/v24/n10/full/nbt1247.html Metagenomic analysis of two enhanced biological phosphorus removal (EBPR) sludge communities, nature 2006 (Héctor García Martín and et. al.)]

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