Composite

Part:BBa_K1460001:Design

Designed by: Eric Holmes   Group: iGEM14_Cornell   (2014-08-14)


pT7 + RBS + GST (glutathione-S-transferase)-CRS5 (metallothionein) + Ter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 764
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 176


Design Notes

Flanking the T7 promoter are two KpnI cut sites with both the T7 promoter and a SacI cut site between them. These cut sites are present for easy removal of the T7 promoter for modulation of parts with other promoters. The SacI cut site can be used as a marker for successful removal of the T7 promoter from the part. When digested with SacI, part BBa_K1460001 in pSB1C3 will yield bands of size 2166bp and 931bp. If the T7 promoter is successfully removed, when digested with SacI the plasmid will yield the linearized length of the pSB1C3+BBa_K1460001


Source

T7 promoter is part BBa_I712074. RBS is from part BBa_J61101. Glutathione-S-transferase is from S. japonicum[1]. CRS5 is a metallothionein from S. cerevisiae[2]. Terminator is part BBa_B1006. The linker sequence is one that has been used in the past to make GST fusion proteins [3]. This sequence was synthesized by GenScript in the vector pUC57 and was then transferred using traditional cloning into pSB1C3.

References

[1] Smith, D., Davern, K., Board, P., Tiu, W., Garcia, E., & Mitchell, G. (1986). Mr 26,000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione S-transferase. Proc. Natl. Acad. Sci. U.S.A., 83(22), 8703-8707.
[2] Culotta, V., Howard, W., & Liu, X. (1994). CRS5 encodes a metallothionein-like protein in Saccharomyces cerevisiae. J. Biol. Chem., 269(41), 25295-25302.
[3] Johnsonb, K. S. (1988). Single-step purification S-transferase, 67, 31–40.