Difference between revisions of "Part:BBa K2549040"
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<partinfo>BBa_K2549040 short</partinfo>
 
<partinfo>BBa_K2549040 short</partinfo>
  
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This part is one of the downstream elements of our amplifier. It is constructed by fusing VP64, dNLS and ZF21.16, from N terminal to C terminal. KRAB is a strong transcription repressor. dNLS, which is short for destroyable nuclear location sequence, is able to guide the fusion protein to be located to the nucleus without TEV protease. It is cleaved and unable to guide the fusion protein when TEV protease exist. Thus when coexpressed with NLS-TEVp, it is cleaved and the transcription activation function cannot be conducted.
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This part is one of the downstream elements of our amplifier. It is constructed by fusing KRAB ([[Part:BBa_BBa_K2549055]]), dNLS ([[Part:BBa_K2549046]]) and ZF21.16 ([[Part:BBa_K2549054]]), from N terminal to C terminal. KRAB is a strong transcription repressor. dNLS, which is short for destroyable nuclear localization sequence, is able to guide the fusion protein to be located to the nucleus without TEV protease. It is cleaved and unable to guide the fusion protein when TEV protease exist. Thus when coexpressed with NLS-TEVp ([[Part:BBa_K2549041]]), it is cleaved and the transcription repression function cannot be conducted.
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===Usage and Biology===
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===Biology===
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====TEV protease-based transcription regulation or proteolysis-only activity====
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TEV protease is widely used in synthetic biology for its high cleavage specificity (targetting amino acids sequence ENLYFQG/S between QG or QS)<ref>Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase. Parks TD, Leuther KK, Howard ED, Johnston SA, Dougherty WG. Anal Biochem, 1994 Feb;216(2):413-7  PMID: 8179197; DOI: 10.1006/abio.1994.1060</ref> and cleaves and high efficiency (optimized by RB Kapust et al to remove autolysis)<ref>Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Kapust RB, Tözsér J, Fox JD, ..., Copeland TD, Waugh DS. Protein Eng, 2001 Dec;14(12):993-1000  PMID: 11809930</ref>. Rossner MJ et al have demonstrated the TEV activity-dependent activation of several reporters<ref>Monitoring regulated protein-protein interactions using split TEV. Wehr MC, Laage R, Bolz U, ..., Nave KA, Rossner MJ. Nat Methods, 2006 Dec;3(12):985-93  PMID: 17072307; DOI: 10.1038/nmeth967</ref>. '''In our system, we set the TEV protease specific cleavage site into the spacer region of the nuclear localization sequence<ref>Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Robbins J, Dilworth SM, Laskey RA, Dingwall C. Cell, 1991 Feb;64(3):615-23  PMID: 1991323</ref>, which is the core design of the TEV protease-based complex logic gates.'''
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[[File:TEVp.jpg|none|400px|thumb|Rossner MJ et al stated:''TEV protease activity can be monitored with either inactivated
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transcription factors (‘transcription-coupled’ reporters) or inactivated reporter proteins (‘proteolysis-only’ reporters). We designed a ‘transcription-coupled’ TEV reporter at the membrane (TM-GV) by fusing a transcription factor composed of a yeast Gal4 DNA-binding domain and the herpes simplex VP16 transactivation domain (GV) to an hemagglutinin (HA)-tagged transmembrane region from the human PDGF-a receptor via a TEV-protease cleavage site ENLYFQ'G . After TEV protease cleavage, GV can translocate into the nucleus and induce reporter gene expression via five clustered Gal4-responsive cis elements. We constructed membrane localized ‘proteolysis-only’ reporters containing a luciferase moiety. We flanked GV N- and C-terminally by tevS sites and ERT2 domains (GV-2ER) thereby trapping the GV transcription factor reporter unit in the cytoplasm.'']]
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For more details about TEV protease, please refer to our TEVp ([[Part:BBa_K2549013]]).
  
 
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<partinfo>BBa_K2549040 parameters</partinfo>
 
<partinfo>BBa_K2549040 parameters</partinfo>
 
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===References===

Revision as of 02:57, 12 October 2018


KRAB-dNLS-ZF21.16

This part is one of the downstream elements of our amplifier. It is constructed by fusing KRAB (Part:BBa_BBa_K2549055), dNLS (Part:BBa_K2549046) and ZF21.16 (Part:BBa_K2549054), from N terminal to C terminal. KRAB is a strong transcription repressor. dNLS, which is short for destroyable nuclear localization sequence, is able to guide the fusion protein to be located to the nucleus without TEV protease. It is cleaved and unable to guide the fusion protein when TEV protease exist. Thus when coexpressed with NLS-TEVp (Part:BBa_K2549041), it is cleaved and the transcription repression function cannot be conducted.

Biology

TEV protease-based transcription regulation or proteolysis-only activity

TEV protease is widely used in synthetic biology for its high cleavage specificity (targetting amino acids sequence ENLYFQG/S between QG or QS)[1] and cleaves and high efficiency (optimized by RB Kapust et al to remove autolysis)[2]. Rossner MJ et al have demonstrated the TEV activity-dependent activation of several reporters[3]. In our system, we set the TEV protease specific cleavage site into the spacer region of the nuclear localization sequence[4], which is the core design of the TEV protease-based complex logic gates.

Rossner MJ et al stated:TEV protease activity can be monitored with either inactivated transcription factors (‘transcription-coupled’ reporters) or inactivated reporter proteins (‘proteolysis-only’ reporters). We designed a ‘transcription-coupled’ TEV reporter at the membrane (TM-GV) by fusing a transcription factor composed of a yeast Gal4 DNA-binding domain and the herpes simplex VP16 transactivation domain (GV) to an hemagglutinin (HA)-tagged transmembrane region from the human PDGF-a receptor via a TEV-protease cleavage site ENLYFQ'G . After TEV protease cleavage, GV can translocate into the nucleus and induce reporter gene expression via five clustered Gal4-responsive cis elements. We constructed membrane localized ‘proteolysis-only’ reporters containing a luciferase moiety. We flanked GV N- and C-terminally by tevS sites and ERT2 domains (GV-2ER) thereby trapping the GV transcription factor reporter unit in the cytoplasm.

For more details about TEV protease, please refer to our TEVp (Part:BBa_K2549013).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 577
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

  1. ↑ Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase. Parks TD, Leuther KK, Howard ED, Johnston SA, Dougherty WG. Anal Biochem, 1994 Feb;216(2):413-7 PMID: 8179197; DOI: 10.1006/abio.1994.1060
  2. ↑ Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Kapust RB, Tözsér J, Fox JD, ..., Copeland TD, Waugh DS. Protein Eng, 2001 Dec;14(12):993-1000 PMID: 11809930
  3. ↑ Monitoring regulated protein-protein interactions using split TEV. Wehr MC, Laage R, Bolz U, ..., Nave KA, Rossner MJ. Nat Methods, 2006 Dec;3(12):985-93 PMID: 17072307; DOI: 10.1038/nmeth967
  4. ↑ Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Robbins J, Dilworth SM, Laskey RA, Dingwall C. Cell, 1991 Feb;64(3):615-23 PMID: 1991323