Composite

Part:BBa_K3111502:Design

Designed by: Matas Deveikis   Group: iGEM19_UCL   (2019-09-30)
Revision as of 11:24, 14 October 2019 by Matas deveikis (Talk | contribs) (→‎Source)


TmEncH_DARPin929_StrepII + miniSOG_TP


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 171
    Illegal BglII site found at 586
    Illegal BamHI site found at 950
    Illegal BamHI site found at 2101
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1677
    Illegal BsaI.rc site found at 2321
    Illegal SapI.rc site found at 520
    Illegal SapI.rc site found at 551


Design Notes

T. maritima encapsulin surface display and cargo loading were previously shown in BioBricks BBa_K3111501 and BBa_K3111402. In these experiments we looked at how we can use these properties for the construction of a drug delivery platform.

To induce cytotoxic action, we proceeded with loading of the encapsulin system with a fluorescent photosensitiser protein, miniSOG (BBa_K3111032). Under blue-light illumination, miniSOG is activated and generates reactive oxygen species which are damaging to cells (1). To achieve this we cloned BBa_K3111501 and BBa_K3111302 together to construct the plasmid observed in Figure 1.

Figure 1: pSB1C3 plasmid containing the T. maritima encapsulin monomer, DARPin929 and miniSOG coding sequences. t.p. - T. maritima encapsulin targeting peptide

Source

This composite part is made of several protein coding regions:

  • DARPin929 is a de novo protein created using mutagenesis and ribosome display by A. Plückthun, in 2008. Sequence was taken from 4HRL on RCSB Protein Data Bank.
  • Encapsulin was isolated from Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099). Sequence was taken from 3DKT on RCSB Protein Data Bank.
  • miniSOG is isolated from Arabidopsis thaliana. Sequence was taken from FPBase (https://www.fpbase.org/protein/minisog/)

All basic parts were sythesised by IDT.

References

[1] Xu S, Chisholm AD. Highly efficient optogenetic cell ablation in C. elegans using membrane-targeted miniSOG. Sci Rep [Internet]. 2016 Feb [cited 2019 Oct 14];6(1). Available from: http://www.nature.com/articles/srep21271

[2] Cassidy-Amstutz C, Oltrogge L, Going CC, Lee A, Teng P, Quintanilla D, et al. Identification of a Minimal Peptide Tag for in Vivo and in Vitro Loading of Encapsulin. Biochemistry. 2016 Jun 21;55(24):3461–

[3] Camacho-Leal MP, Sciortino M, Cabodi S. ErbB2 Receptor in Breast Cancer: Implications in Cancer Cell Migration, Invasion and Resistance to Targeted Therapy, Breast Cancer - From Biology to Medicine. Phuc Van Pham, IntechOpen.