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We took ppk1 in Citrobacter freundii ATCC 8090 as a comparison and validated its activity through the experiment. We found that like in  Citrobacter freundii ATCC 8090, ppk1 can also produce polyP in the E.coli, which proved our hypothesis.After incubation in the  polyphosphorous culture medium for 20 min, we found that polyP was produced by PPK1, which was proved by ashing and spectrum.
 
We took ppk1 in Citrobacter freundii ATCC 8090 as a comparison and validated its activity through the experiment. We found that like in  Citrobacter freundii ATCC 8090, ppk1 can also produce polyP in the E.coli, which proved our hypothesis.After incubation in the  polyphosphorous culture medium for 20 min, we found that polyP was produced by PPK1, which was proved by ashing and spectrum.
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We also measured the curve of OD value CPVM in PA medium. Here is our result:
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Revision as of 07:40, 10 October 2021


ppk1 in Citrobacter freundii ATCC 8090

Natural function of part: The polyphosphate kinase in Citrobacter freundii ATCC 8090 is responsible for its intracellular inorganic polyphosphate (polyP) production via reversibly catalyzing the transfer of terminal phosphate from ATP to a growing polyP chain.

Wild-type Citrobacter freundii ATCC 8090 was purchased from China Center of Industrial Culture Collection (CICC, China). The ppk1 gene was acquired by PCR. This organism is our chasiss, in which its native PPK1 will be overexpressed with a plasmid of medium-copy numbers.

Although PPKs from E. coli and C. freundii shares 96% amino acids’ identity, the C. freundii PPK has a glutamate and a lysine residue in positions 327 and 328, where in E. coli they are substituted by much less strongly charged alanine and glutamine residues, respectively. Although these natural occurred mutation sites found in C. freundii PPK are distant from the enzymes’ active site, they lie in the interfaces among monomers of the PPK tetramer. Benefit from this difference, a dramatic increase of intracellular polyP accumulation can be achieved with C. freundii.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


iGEM2021_Nanjing-China Experiment

Group: Nanjing-China 2021
Author: Hao Yin

We took ppk1 in Citrobacter freundii ATCC 8090 as a comparison and validated its activity through the experiment. We found that like in Citrobacter freundii ATCC 8090, ppk1 can also produce polyP in the E.coli, which proved our hypothesis.After incubation in the polyphosphorous culture medium for 20 min, we found that polyP was produced by PPK1, which was proved by ashing and spectrum. We also measured the curve of OD value CPVM in PA medium. Here is our result:


iGEM2020_Nanjing-China Experiment

Group: Nanjing-China 2020
Author: Guangyu Hu

We found that polyP productivity would reach the highest after inoculation for 14 hours and 100ml of synthetic wastewater in a 200ml Erlenmeyer flask has the highest output and the highest conversion efficiency.

PolyP formation is closely associated with ppk1 gene in CPP. This means the cultivation time and volume will affect P consuming a lot. After comparing different cultivation conditions, we found that polyP productivity will reach the highest after inoculation for 14 hours, that is approximately 20mg/L. Besides, 100ml of synthetic wastewater in a 200ml Erlenmeyer flask has the highest output and the highest conversion efficiency. Without bacterial contamination and normal bacterial viability，it can almost convert all inorganic phosphates into polyphosphates.


iGEM2019_Nanjing China Experiment

This year our team develops a simple solo medium-copy plasmid-based polyphosphate kinase (PPK1) overexpression strategy for achieving maximum intracellular polyphosphate accumulation.

We test plasmid-borne ppk1 expression in CPP via qRT-PCR analysis, supernatant Pi concentration and optical density of CPP and CWT in Synthetic municipal wastewater(SMW).

Ps: SMW means Synthetic municipal wastewater

CPP means solo medium-copy C. f reundii ATCC8090 ppk in C. f reundii ATCC8090

CWT means wild type C. f reundii ATCC8090

Figure 1）qRT-PCR analysis of plasmid-borne ppk1 expression in CPP

Figure 2）Pi absorption&release

Figure 3）Optical density of CPP grown in SMW compared with CWT

Reference: Wang X , Wang X , Hui K , et al. Highly Effective Polyphosphate Synthesis, Phosphate Removal and Concentration Using Engineered Environmental Bacteria Based on a Simple Solo Medium-copy Plasmid Strategy[J]. Environmental Science & Technology, 2017:acs.est.7b04532.