Composite
Lactobachi

Part:BBa_K2760010:Design

Designed by: iGEM18_TecMonterrey_GDL   Group: iGEM18_TecMonterrey_GDL   (2018-10-09)


Lactobachill Plan A (E. coli)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1924
    Illegal BamHI site found at 65
    Illegal BamHI site found at 1138
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1114
    Illegal AgeI site found at 799
    Illegal AgeI site found at 2647
    Illegal AgeI site found at 2759
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 731
    Illegal BsaI.rc site found at 1526
    Illegal SapI.rc site found at 1543
    Illegal SapI.rc site found at 1763


Design Notes

  • Optimized for E. coli


Source

In it’s full form the construct should contain contain the Pbad strong promoter (BBa_K206000), an E. coli strong ribosome binding site (registered before as Part:BBa_J34801), then the sequence for protein nsrR (BBa_K2760023) which is the PyeaR promoter repressor, two stop codons and then another ribosome binding site (BBa_J34801). After this ribosome binding site, follows a codon optimized for L. rhamnosus and E.coli iLOV sequence (BBa_K2760024), two stop codons and a terminator (BBa_B0015). A PyeaR promoter follows (BBa_K216005), the same ribosome binding site BBa_J34801, an attached signal peptide for excretion into the media (BBa_K2760000) in the gp130 (receptor IL-6 complex) human soluble protein (BBa_K2760002) and a His-Tag (BBa_K2760012), then two stop codons, the RBS (BBa_J34801) and finally the M-cherry (BBa_K2760029), stop codon, and a double terminator (BBa_B0015).

References

[1] Müller-Newen, G., Küster, A., Hemmann, U., Keul,R., Horsten, U., Martens, A., Graeve, L., Wijdenes, J., Heinrich, P. (1998) Soluble IL-6 Receptor Potentiates the Antagonistic Activity of Soluble gp130 on IL-6 Responses. The Journal American of Association Immunology. ISSN: 1550-6606.

[2] Han,S., Machhi, S., Berge,H., Xi, G., Linke, T., and Schoner, R. (2017). Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli. AMB Express. Doi: https://doi.org/10.1186/s13568-017-0394-1