Coding

Part:BBa_K1152009

Designed by: Ralf Beer, Konrad Herbst, Nikolaos Ignatiadis   Group: iGEM13_Heidelberg   (2013-09-19)

Sfp 4'-Phosphopanthetheinyl-transferase

Figure 1: Phenotypes of TOP10 producig indigoidine. As negative control untransformed TOP10 were used.

Sfp (Figure 1) encodes for a 4'-Phosphopanthetheinyl-transferase (PPTase) which transfers a 4'-PPT residue from CoA to a conserved serine residue in the T-Domain of NRPS modules, thus activating these enzymes (Figure 2).

Figure 2: General mechanism of activation the activation of NRP Synthetases from apo- to holo-form.

We hereby improve parts BBa_K802006 and BBa_K302010 which also encode for sfp from Bacillus subtilis. Both parts are equal in sequence despite the fact that obviously BBa_K802006 includes additional bases upstream from the B. subtilis genome. we improved these parts in availability to the community as well as in characterization.

By MSA we can conclude that BBa_K1152009 is different from BBa_K802006 and BBa_K302010 at 3 different amino acid sites: MSA sfp BioBricks.

We can also conclude that BBa_K802006 is actually sfp even when not annotated like this.

Figure 3 shows the analytical restiction digest we performed for part validation:

Figure 3: Restriction digest with EcoRI, XbaI, SpeI, PstI.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 651
    Illegal SapI.rc site found at 661

References

  • Lambalot RH, Gehring AM, Flugel RS, Zuber P, LaCelle M, Marahiel MA, Reid R, Khosla C, Walsh CT (1996) A new enzyme superfamily - the phosphopantetheinyl transferases. Chem Biol 3: 923-936.
  • Nakano, M. M., Corbell, N., Besson, J., & Zuber, P. (1992). Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis. Molecular & general genetics : MGG, 232(2), 313–21. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1557038
  • Pfeifer, B. a, Admiraal, S. J., Gramajo, H., Cane, D. E., & Khosla, C. (2001). Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science (New York, N.Y.), 291(5509), 1790–2. doi:10.1126/science.1058092


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