Difference between revisions of "Part:BBa K782085"

Revision as of 11:46, 26 September 2012

10x[TALA] operator_minimal promoter_TALA:NLS:VP16_t2a_BFP

TALA labels represents TAL effector 1257 from zebrafish experiments (Sander et al., 2011)

Introduction

Our construct contain 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. TAL repressor and fluorescent reporter were connected by an intermediate t2A sequence, causing the ribosome to skip the formation of a peptide bond during protein translation, producing the activator and reporter as separate proteins in equimolar amounts (recently also used by Garg et al., 2012) (Kim et al., 2011, de Felipe et al., 2006).

BFP is a monomeric fluorescent protein with excitation maximum at 402 nm and emision maximum at 457 nm.

Figure 1: Schematic representation of the construct.

Characterization

Results: Specific TAL binding sites were further characterized with other reporter constructs.

BFP was obtained from Evrogen.

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