Difference between revisions of "Part:BBa K4257000:Design"
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===Design Notes===
 
===Design Notes===
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PPK-M is a mutant of E.coli polyphosphate kinase 1 (PPK1), which catalyzes the synthesis of polyphosphate (polyP) using cellular ATP as the substrate. Compared to native E.coli PPK1 that has an alanine and a glutamine residue in position 327 and 328, PPK-M has much more strongly charged glutamate and lysine residues. It has been documented that expression of PPK-M leads to substantially higher levels of polyP accumulation in vivo by disrupting intracellular PPK-repressing interactions, as compared to the situation found with expression of PPK1. PolyP consists of inorganic phosphate (Pi), which is essentially derived from the uptake of exogenous phosphorous by the host cell. For this reason, if enhanced uptake of exogenous phosphorous by the host cell is desired, PPK-M will be a better option. This year, our team wants to develop an engineered E.coli K12 strain that can use phosphite as a raw material to manufacture Pi, therefore, highly active PPK-M was picked.
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Highly active version of E. coli native PPK.
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===Source===
 
===Source===
  
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The EcPPK coding sequence was obtained by PCR amplification using the genomic DNA of E.coli K12 as the template.
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The PPK-M coding sequence was obtained by PCR-mediated site-directed mutagenesis using the genomic DNA of E.coli K12 as the template.
  
 
===References===
 
===References===
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Rudat, A. K., et al. (2018). "Mutations in Escherichia coli polyphosphate kinase that lead to dramatically increased in vivo polyphosphate levels." 200(6): e00697-00617.

Latest revision as of 16:18, 26 September 2022


PPK-M


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]


Design Notes

Highly active version of E. coli native PPK.

Source

The PPK-M coding sequence was obtained by PCR-mediated site-directed mutagenesis using the genomic DNA of E.coli K12 as the template.

References

Rudat, A. K., et al. (2018). "Mutations in Escherichia coli polyphosphate kinase that lead to dramatically increased in vivo polyphosphate levels." 200(6): e00697-00617.