Difference between revisions of "Part:BBa K782085"

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==Introduction==
 
==Introduction==
  
Our construct contain [https://parts.igem.org/Part:BBa_K782070 TALA binding sites] that are cloned upstream of [https://parts.igem.org/Part:BBa_K782087 minimal promoter]. Downstream of promoter [https://parts.igem.org/Part:BBa_K782065 TALA:VP16] and blue fluorescent protein.  
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Our construct contain [https://parts.igem.org/Part:BBa_K782070 TALA binding sites] that are cloned upstream of [https://parts.igem.org/Part:BBa_K782087 minimal promoter]. Downstream of promoter are [https://parts.igem.org/Part:BBa_K782065 TALA:VP16] and blue fluorescent protein.
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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).
  
  
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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
 
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
  
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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.
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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.
  
  

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.

BFP.png

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


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