Composite

Part:BBa_K782085

Designed by: Fedja Pavlovec   Group: iGEM12_Slovenia   (2012-09-24)
Revision as of 23:51, 26 September 2012 by Rjerala (Talk | contribs) (Introduction)

10x[TALA] operator_minimal promoter_TALA:NLS:VP16_t2a_BFP

  • TALA labels represents TAL effector 1257 from zebrafish experiments (Sander et al., 2011).
  • DNA binding sites for individual TAL effectors are indicated with square brackets [ ].


Introduction

Our construct contains TALA binding sites that are cloned upstream of minimal promoter. Downstream of promoter are TALA:VP16 and blue fluorescent protein.

To enable monitoring level of expression of TAL activator, we linked it with a fluorescent reporter. BFP is a monomeric fluorescent protein with excitation maximum at 402 nm and emision maximum at 457 nm. TAL activator and fluorescent reporter were connected by an intermediate t2A sequence. This sequence causes the ribosome to skip formation of a peptide bond during protein translation, producing the activator and reporter as separate proteins in equimolar amounts (Garg et al., 2012, Kim et al., 2011, de Felipe et al., 2006).


BFP.png

Figure 1: Schematic representation of the construct.

Characterization

We extensively characterized this construct, since it represents essential part of innovative [http://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch bistable switch based on a positive feedback loop]. Part contains self-activator, necessary for creating positive feedback loop.


Uf.png

Figure 2:

Left: Addition of Pristinamycine triggers dissociation of PIP:KRAB from its DNA-binding site. Consequently TALB repressor and TALA activator are transcribed. Activator causes transcription of another pair of TALB:KRAB and TALA:VP16, responsible for the positive feedback loop. Meanwhile TALB:KRAB represses the other state of the switch. Right: When the inducer is removed, the inducible repressor binds back to its DNA-binding site, but since TALA:VP16 is still present, auto-activation is achieved, resulting in a stable state.

References

Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nature Biotechnology 29, 697–698.

Kim, J. H., Lee, S.-R., Li, L.-H., Park, H.-J., Park, J.-H., Lee, K. Y., Kim, M.-K., et al. 2011. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PloS one 6,e18556.

de Felipe, P., Luke, G. a, Hughes, L. E., Gani, D., Halpin, C., Ryan, M. D. 2006. E unum pluribus: multiple proteins from a self-processing polyprotein. Trends in biotechnology 24,68–75.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 750
    Illegal BamHI site found at 720
    Illegal BamHI site found at 3262
    Illegal XhoI site found at 788
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 147
    Illegal NgoMIV site found at 507
    Illegal AgeI site found at 12
    Illegal AgeI site found at 347
    Illegal AgeI site found at 372
    Illegal AgeI site found at 707
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 4207


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