Difference between revisions of "Part:BBa K5034223:Design"
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===Design Notes===
 
===Design Notes===
  
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PPK2 and NADK genes were PCR-amplified and inserted into the plasmid pYYDT(after NdeI and XhoI digestion) to get the expression vector. It then got transformed into <i>E. coli</i> DH5&#945; to amplify, and was purified by picking monoclonal culture and sequencing.
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PPK2 and NADK genes were PCR-amplified and inserted into the plasmid pBBR1MCS-terminator(after EcoRI and XhoI digestion) to get the expression vector. It then got transformed into <i>E. coli</i> DH5&#945; to amplify, and was purified by picking monoclonal culture and sequencing.
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The terminator(BBa_B0015) is on the plasmid backbone(BBa_K5034201).
  
 
===Source===
 
===Source===

Revision as of 11:24, 30 September 2024


Pi <-> Poly P Poly P -> NADP


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 4981
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4981
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 2834
    Illegal NotI site found at 4987
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4981
    Illegal BglII site found at 3580
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 4981
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 4981
    Illegal XbaI site found at 4996
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 562
    Illegal NgoMIV site found at 4244
    Illegal NgoMIV site found at 4527
    Illegal AgeI site found at 402
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

PPK2 and NADK genes were PCR-amplified and inserted into the plasmid pBBR1MCS-terminator(after EcoRI and XhoI digestion) to get the expression vector. It then got transformed into E. coli DH5α to amplify, and was purified by picking monoclonal culture and sequencing.

The terminator(BBa_B0015) is on the plasmid backbone(BBa_K5034201).

Source

BBa_K5034205: Polyphosphate kinase 2(PPK2) from Pseudomonas paraeruginosa. NCBI reference sequence: NZ_CP020560.1:c164262-163366

BBa_K5034206: Inorganic polyphosphate/ATP-NAD kinase(PPNK) from Mycobacterium tuberculosis H37Rv. NCBI reference sequence: NC_000962.3:1918746-1919669

References

Zhang, H., Ishige, K., & Kornberg, A. (2002). A polyphosphate kinase (PPK2) widely conserved in bacteria. Proceedings of the National Academy of Sciences, 99(26), 16678-16683. doi:10.1073/pnas.262655199

Neville, N., Roberge, N., & Jia, Z. (2022). Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence. International Journal of Molecular Sciences, 23(2), 670. doi:10.3390/ijms23020670

Itoh, H., & Shiba, T. (2004). Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. Journal of Bacteriology, 186(15), 5178-5181. doi:10.1128/jb.186.15.5178-5181.2004