Part:BBa_K782085
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. 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, 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).
Figure 1: Schematic representation of the construct
Characterization
Construct was further characterized by [http://2012.igem.org/Team:Slovenia iGEM 2012 Team Slovenia], where 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.
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 activator B is still present, autoa-ctivation is achieved, resulting in a stable state.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE 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 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 4207
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