Difference between revisions of "Part:BBa K2549029"

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This part is one of the response elements of our amplifier, also executing the combiner function. 8*ZF21.16 binding sites is assembled using two 4*ZF21.16 binding sites ([[Part:BBa_K2446008]]) with a biobrick scar between them. CMV ([[Part:BBa_K2549050]]) is a promotor which has a high-level constitutive expression. This part can switch off the expression of gene downstream after induced by our zinc finger-based transcription repressor.
 
This part is one of the response elements of our amplifier, also executing the combiner function. 8*ZF21.16 binding sites is assembled using two 4*ZF21.16 binding sites ([[Part:BBa_K2446008]]) with a biobrick scar between them. CMV ([[Part:BBa_K2549050]]) is a promotor which has a high-level constitutive expression. This part can switch off the expression of gene downstream after induced by our zinc finger-based transcription repressor.
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This part is also served as an improvement version of [[Part:BBa_K2446030]] of <b>iGEM-team [http://2017.igem.org/Team:Fudan/Composite_Part Fudan 2017]</b>. We placed responsive elements of zinc finger-based transcription factors on the 5' end of the promotor to lower the impact they may have on the basal expression of the promotor. Besides, CMV promotor performs a higher-level expression than SV40 promotor. '''For more details, please refer to our <b>[http://2018.igem.org/Team:Fudan/Improve Parts improvement page]</b>.'''
  
 
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===Biology===
 
===Biology===
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=====It works as we designed =====
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=====Our characterization=====
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interaction between ZF21.16-KRAB and 8*ZF21.16-CMV promotor
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[[File:sTF-test.png|none|360px|thumb|'''Interaction between transcriptional repressors and their binding sites.''' A degradable EGFP (d2EGFP) is produced downstream the promoter to indicate the output strength. Experiments were conducted and analyzed as previous reported<ref>http://2017.igem.org/Team:Fudan/Demonstrate</ref>. DBD, DNA binding domain which is zinc finger in our assay. SD, silencing-form transcriptional domain; we used KRAB for the experiments in this figure. RE, responsive elements. MFI, median fluorescence intensity.]]
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Flow cytometry results suggest that the transcriptional repressors can effectively inhibit the promoters with responsive elements. Please visit http://2018.igem.org/Team:Fudan/Demonstration for a brief introduction of our project.
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[[File:NORgate.png|none|320px|thumb|'''Flow cytometry results of an intercellular NOR gate.''' RFI: output from the Combiner after activation was divided by the value before activation. The TRE3GV or URE2G promoters in the Amplifer receive signals from intercellular tTAA or GV2, respectively. Both Amplifiers would produce ZF21.16-KRAB, which could transduce signal to the Combiner which has ZF21.16 responsive elements. More details please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Measurement .]]
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Worked as designed, only when neither Amplifier produced the transcriptional repressor could the Combiner expresses d2EGFP with fluoresence.
  
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=====Synthetic promotor operators regulated by artificial zinc finger-based transcription factors=====
 
=====Synthetic promotor operators regulated by artificial zinc finger-based transcription factors=====
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[[File:ZF2.jpg|none|240px|thumb|Khalil AS et al stated:''sTFs constructed from OPEN-engineered ZFs are orthogonal to one another. sTF43-8 activated noncognate Promoter21-16 due to the fortuitous creation of a sequence that is significantly similar to the binding sequence of 43-8, when the downstream BamHI restriction site is considered.'']]
 
[[File:ZF2.jpg|none|240px|thumb|Khalil AS et al stated:''sTFs constructed from OPEN-engineered ZFs are orthogonal to one another. sTF43-8 activated noncognate Promoter21-16 due to the fortuitous creation of a sequence that is significantly similar to the binding sequence of 43-8, when the downstream BamHI restriction site is considered.'']]
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Latest revision as of 19:44, 17 October 2018

8*ZF21.16-CMV

This part is one of the response elements of our amplifier, also executing the combiner function. 8*ZF21.16 binding sites is assembled using two 4*ZF21.16 binding sites (Part:BBa_K2446008) with a biobrick scar between them. CMV (Part:BBa_K2549050) is a promotor which has a high-level constitutive expression. This part can switch off the expression of gene downstream after induced by our zinc finger-based transcription repressor.

This part is also served as an improvement version of Part:BBa_K2446030 of iGEM-team [http://2017.igem.org/Team:Fudan/Composite_Part Fudan 2017]. We placed responsive elements of zinc finger-based transcription factors on the 5' end of the promotor to lower the impact they may have on the basal expression of the promotor. Besides, CMV promotor performs a higher-level expression than SV40 promotor. For more details, please refer to our [http://2018.igem.org/Team:Fudan/Improve Parts improvement page].

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
    COMPATIBLE WITH RFC[1000]


Biology

Our characterization
Interaction between transcriptional repressors and their binding sites. A degradable EGFP (d2EGFP) is produced downstream the promoter to indicate the output strength. Experiments were conducted and analyzed as previous reported[1]. DBD, DNA binding domain which is zinc finger in our assay. SD, silencing-form transcriptional domain; we used KRAB for the experiments in this figure. RE, responsive elements. MFI, median fluorescence intensity.

Flow cytometry results suggest that the transcriptional repressors can effectively inhibit the promoters with responsive elements. Please visit http://2018.igem.org/Team:Fudan/Demonstration for a brief introduction of our project.

Flow cytometry results of an intercellular NOR gate. RFI: output from the Combiner after activation was divided by the value before activation. The TRE3GV or URE2G promoters in the Amplifer receive signals from intercellular tTAA or GV2, respectively. Both Amplifiers would produce ZF21.16-KRAB, which could transduce signal to the Combiner which has ZF21.16 responsive elements. More details please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Measurement .

Worked as designed, only when neither Amplifier produced the transcriptional repressor could the Combiner expresses d2EGFP with fluoresence.


Synthetic promotor operators regulated by artificial zinc finger-based transcription factors

Khalil AS et al have reported several synthetic promotor operators which can interact with artificial zinc finger-based transcription factors with high specificity and high orthogonality[2].

Khalil AS et al stated:sTFs constructed from OPEN-engineered ZFs are orthogonal to one another. sTF43-8 activated noncognate Promoter21-16 due to the fortuitous creation of a sequence that is significantly similar to the binding sequence of 43-8, when the downstream BamHI restriction site is considered.


References

  1. ↑ http://2017.igem.org/Team:Fudan/Demonstrate
  2. ↑ A synthetic biology framework for programming eukaryotic transcription functions. Khalil AS, Lu TK, Bashor CJ, ..., Joung JK, Collins JJ. Cell, 2012 Aug;150(3):647-58 PMID: 22863014; DOI: 10.1016/j.cell.2012.05.045