Difference between revisions of "Part:BBa K2549038"

 
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<partinfo>BBa_K2549038 short</partinfo>
 
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This part is one of the downstream elements of our amplifier. It is constructed by fusing CfaC, ZF21.16C and NLS, from N terminal to C terminal. CfaC is the C-terminal fragment of Cfa which is a consensus sequence from an alignment of 73 naturally occurring DnaE inteins that are predicted to have fast splicing rates. ZF21.16C is the C-terminal fragment of the zinc finger whose recognition helices for three-finger arrays are substituted by the reported synthetic zinc finger 21.16 residues on the basis of the BCR_ABL-1 artificial zinc finger. NLS is a short nuclear location sequence from SV40 large T antigen. When coexpressed with VP64-ZF21.16N-CfaN in the same cell, both fusions are formed and a transcription activating function is executed.
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This part is one of the downstream elements of our amplifier. It was constructed by fusing CfaC ([[Part:BBa_K2549010]]), ZF21.16C ([[Part:BBa_K2549012]]) and NLS ([[Part:BBa_K2549054]]), from N terminal to C terminal. CfaC is the C-terminal fragment of Cfa which is a consensus sequence from an alignment of 73 naturally occurring DnaE inteins that are predicted to have fast splicing rates. ZF21.16C is the C-terminal fragment of the zinc finger whose recognition helices for three-finger arrays are substituted by the reported synthetic zinc finger 21.16 residues on the basis of the BCR_ABL-1 artificial zinc finger<ref>A tunable zinc finger-based framework for Boolean logic computation in mammalian cells. Lohmueller JJ, Armel TZ, Silver PA. Nucleic Acids Res, 2012 Jun;40(11):5180-7  PMID: 22323524; DOI: 10.1093/nar/gks142</ref>. NLS is a short nuclear location sequence from SV40 large T antigen. When coexpressed with VP64-ZF21.16N-CfaN ([[Part:BBa_K2549036]]) in the same cell, both fusions will be produced and a transcription activating function will be executed. Also, when coexpressed with KRAB-ZF21.16N-CfaN ([[Part:BBa_K2549037]]) in the same cell, both fusions will be produced formed and a transcription repressing function will be executed.
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===Usage and Biology===
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<partinfo>BBa_K2549038 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2549038 SequenceAndFeatures</partinfo>
  
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===Biology===
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=====Our characterization=====
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[[File:AND-test.png|none|480px|thumb|'''CfaN intein-based AND gate.'''  A degradable EGFP (d2EGFP) is produced downstream the promoter of the Combiner to indicate the output (CusP) strength. DBD, DNA binding domain which is zinc finger in our assay. AD, activating-form transcriptional domain, was VP64. RE, responsive elements. RFI, relative fluorescence intensity (comparing before and after activation). More details please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Measurement .]]
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We show that when VP64-ZF21.16N-CfaN co-expressed with CfaC-ZF21.16C-NLS, the expression level of d2EGFP is relatively turned up due to the formation of VP64-ZF21.16 after auto-plicing and ligation. The signal-noise-ratio is still not optimal, and we are improving the design by switching the split position as well as adding linkers.
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[[File:NAND-test.png|none|480px|thumb|'''CfaN intein-based NAND gate.'''  A degradable EGFP (d2EGFP) is produced downstream the promoter of the Combiner to indicate the output (CusP) strength. DBD, DNA binding domain which is zinc finger in our assay. SD, silencing-form transcriptional domain, was KRAB. RE, responsive elements. RFI, relative fluorescence intensity (comparing before and after activation).]]
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We show that when KRAB-ZF21.16N-CfaN co-expressed with CfaC-ZF21.16C-NLS, the expression level of d2EGFP is relatively turned down due to the formation of KRAB-ZF21.16 after auto-plicing and ligation.
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The signal-noise-ratio is ok when both present. However, we are not sure why CfaC-ZF21.16C-NLS alone has effect. We have formed a couple of hypothesis and in the process of optimizing this gate. Please note [[Part:BBa_K2549036]] contains a transcriptional activation domain VP64, while [[Part:BBa_K2549037]] has a transcriptional repression domain KRAB, both need to ligate with CfaC-ZF21.16C-NLS ([[Part:BBa_K2549038]]) before functional.
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=====Boolean logic gates via split zinc finger-based transcription factors=====
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Lohmueller JJ et al have demonstrated the split ZF-TF reconstitution process. Please note that we used Cfa split intein ([[Part:BBa_K2549009]] and [[Part:BBa_K2549010]]) but not dnaB reported below.
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[[File:ZF-TF.jpg|none|400px|thumb|Lohmueller JJ et al demonstrated: ''After expression, the two split ZF-intein fragments bind together and undergo protein splicing to cleave away intein fragments and reconstitute the full ZF activator leading to activation of the BCR_ABL reporter.'']]
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[[File:zfAND.jpg|none|400px|thumb|Lohmueller JJ et al demonstrated: ''For AND gates, a ZF activator is spliced and the logical operation is computed as TRUE only when both input signals are present. For the response data shown BCR_ABL-1:GCN4 activator split fragments were used and the response promoter contains 6 copies of the BCR_ABL target site. CFP expression was measured by flow cytometry and expressed as fold change over an off-target expression control.'']]
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[[File:zfNAND.jpeg|none|400px|thumb|Lohmueller JJ et al demonstrated: ''For NAND gates, the computational module splices a ZF repressor, and the logical operation is computed as TRUE as long as both inputs are not present together. For the response data shown BCR_ABL-1:GCN4 repressor split fragments were used and the response promoter contains 6 copies of the BCR_ABL target site. CFP expression was measured by flow cytometry and expressed as fold change over an off-target expression control.'']]
  
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2549038 parameters</partinfo>
 
<partinfo>BBa_K2549038 parameters</partinfo>
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===References===

Latest revision as of 21:36, 17 October 2018


CfaC-ZF21.16C-NLS

This part is one of the downstream elements of our amplifier. It was constructed by fusing CfaC (Part:BBa_K2549010), ZF21.16C (Part:BBa_K2549012) and NLS (Part:BBa_K2549054), from N terminal to C terminal. CfaC is the C-terminal fragment of Cfa which is a consensus sequence from an alignment of 73 naturally occurring DnaE inteins that are predicted to have fast splicing rates. ZF21.16C is the C-terminal fragment of the zinc finger whose recognition helices for three-finger arrays are substituted by the reported synthetic zinc finger 21.16 residues on the basis of the BCR_ABL-1 artificial zinc finger[1]. NLS is a short nuclear location sequence from SV40 large T antigen. When coexpressed with VP64-ZF21.16N-CfaN (Part:BBa_K2549036) in the same cell, both fusions will be produced and a transcription activating function will be executed. Also, when coexpressed with KRAB-ZF21.16N-CfaN (Part:BBa_K2549037) in the same cell, both fusions will be produced formed and a transcription repressing function will be executed.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 91
    Illegal SapI.rc site found at 21


Biology

Our characterization
CfaN intein-based AND gate. A degradable EGFP (d2EGFP) is produced downstream the promoter of the Combiner to indicate the output (CusP) strength. DBD, DNA binding domain which is zinc finger in our assay. AD, activating-form transcriptional domain, was VP64. RE, responsive elements. RFI, relative fluorescence intensity (comparing before and after activation). More details please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Measurement .

We show that when VP64-ZF21.16N-CfaN co-expressed with CfaC-ZF21.16C-NLS, the expression level of d2EGFP is relatively turned up due to the formation of VP64-ZF21.16 after auto-plicing and ligation. The signal-noise-ratio is still not optimal, and we are improving the design by switching the split position as well as adding linkers.

CfaN intein-based NAND gate. A degradable EGFP (d2EGFP) is produced downstream the promoter of the Combiner to indicate the output (CusP) strength. DBD, DNA binding domain which is zinc finger in our assay. SD, silencing-form transcriptional domain, was KRAB. RE, responsive elements. RFI, relative fluorescence intensity (comparing before and after activation).

We show that when KRAB-ZF21.16N-CfaN co-expressed with CfaC-ZF21.16C-NLS, the expression level of d2EGFP is relatively turned down due to the formation of KRAB-ZF21.16 after auto-plicing and ligation.

The signal-noise-ratio is ok when both present. However, we are not sure why CfaC-ZF21.16C-NLS alone has effect. We have formed a couple of hypothesis and in the process of optimizing this gate. Please note Part:BBa_K2549036 contains a transcriptional activation domain VP64, while Part:BBa_K2549037 has a transcriptional repression domain KRAB, both need to ligate with CfaC-ZF21.16C-NLS (Part:BBa_K2549038) before functional.

Boolean logic gates via split zinc finger-based transcription factors

Lohmueller JJ et al have demonstrated the split ZF-TF reconstitution process. Please note that we used Cfa split intein (Part:BBa_K2549009 and Part:BBa_K2549010) but not dnaB reported below.

Lohmueller JJ et al demonstrated: After expression, the two split ZF-intein fragments bind together and undergo protein splicing to cleave away intein fragments and reconstitute the full ZF activator leading to activation of the BCR_ABL reporter.
Lohmueller JJ et al demonstrated: For AND gates, a ZF activator is spliced and the logical operation is computed as TRUE only when both input signals are present. For the response data shown BCR_ABL-1:GCN4 activator split fragments were used and the response promoter contains 6 copies of the BCR_ABL target site. CFP expression was measured by flow cytometry and expressed as fold change over an off-target expression control.
Lohmueller JJ et al demonstrated: For NAND gates, the computational module splices a ZF repressor, and the logical operation is computed as TRUE as long as both inputs are not present together. For the response data shown BCR_ABL-1:GCN4 repressor split fragments were used and the response promoter contains 6 copies of the BCR_ABL target site. CFP expression was measured by flow cytometry and expressed as fold change over an off-target expression control.


References

  1. ↑ A tunable zinc finger-based framework for Boolean logic computation in mammalian cells. Lohmueller JJ, Armel TZ, Silver PA. Nucleic Acids Res, 2012 Jun;40(11):5180-7 PMID: 22323524; DOI: 10.1093/nar/gks142