Composite

Part:BBa_K2633000:Design

Designed by: Julian Liber   Group: iGEM18_MichiganState   (2018-10-10)
Revision as of 16:57, 10 October 2018 by Liberjul (Talk | contribs) (→‎Source)


proB + RBS + Pseudomonas acdS+ linker + GFPmut3b + term


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 824
    Illegal XhoI site found at 1882
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 601
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 39
    Illegal BsaI.rc site found at 1811


Design Notes

The part is designed to produce a fusion protein of the plant-growth promoting enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) and enhanced green fluorescent protein (eGFP or GFPmut3b). Organisms which express ACCD have been implicating in promoting resilience to osmotic stresses such as drought and soil salinization. The attachment of the eGFP domain by a flexible linker allows for visualization of bacteria carrying the plasmid in plant tissues. It also improves ability to select transformed colonies in bacterial strains with high number of non-transformed colonies. There is a His tag for optional purification of the protein.


Source

The amino acid sequence of acdS comes from isolated endophytic bacteria Pseudomonas sp. 1C2P-04, and primers for its amplification were based on /_Pseudomonas_/ sp. UW4. The promoter, RBS, eGFP with His-tag, and terminator are derived from pJK_proB_eGFP (Bienick et al. 2014).

References

Bienick MS, Young KW, Klesmith JR, Detwiler EE, Tomek KJ, Whitehead TA. The interrelationship between promoter strength, gene expression, and growth rate. PLoS One. 2014 Oct 6;9(10):e109105.