Difference between revisions of "Part:BBa K2549033"
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This part is one of the response elements of our amplifier, also executing the combiner function. 6*ZF21.16 binding sites and 2*ZF43.8 binding sites ([[Part:BBa_K2549051]]) can bind to different zinc finger-based transcription activator ZF21.16-VP64 ([[Part:BBa_K2549023]]) and zinc finger-based repressor ZF43.8-KRAB ([[Part:BBa_K2446041]]), respectively, with high orthogonality. Minimal CMV ([[Part:BBa_K2549049]]) is a promotor providing very low basal expression and high maximal expression after induction. This part is designed to construct our NIMPLY logic gate and test our multiple binding sites amplifier model<ref>http://2018.igem.org/Team:Fudan/Model#Transcriptional_Amplifer</ref>. | This part is one of the response elements of our amplifier, also executing the combiner function. 6*ZF21.16 binding sites and 2*ZF43.8 binding sites ([[Part:BBa_K2549051]]) can bind to different zinc finger-based transcription activator ZF21.16-VP64 ([[Part:BBa_K2549023]]) and zinc finger-based repressor ZF43.8-KRAB ([[Part:BBa_K2446041]]), respectively, with high orthogonality. Minimal CMV ([[Part:BBa_K2549049]]) is a promotor providing very low basal expression and high maximal expression after induction. This part is designed to construct our NIMPLY logic gate and test our multiple binding sites amplifier model<ref>http://2018.igem.org/Team:Fudan/Model#Transcriptional_Amplifer</ref>. | ||
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| + | <span class='h3bb'>Sequence and Features</span> | ||
| + | <partinfo>BBa_K2549033 SequenceAndFeatures</partinfo> | ||
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<!-- Add more about the biology of this part here --> | <!-- Add more about the biology of this part here --> | ||
===Biology=== | ===Biology=== | ||
| − | ==== | + | =====Our characterization===== |
| − | + | [[File:model.png|none|440px|thumb|'''Flow cytometry results of transcriptional activator interacts with multiple binding sites.''' RFI: output from the Combiner after activation was divided by the value before activation. A degradable EGFP (d2EGFP) is produced downstream the promoter of the Combiner to indicate the output strength. ZF21.16 fused with VP64 was the transcriptional activator. There was no transcriptional repressor in the experiment. More details please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Measurement .]] | |
| − | + | Worked as designed, copy numbers of responsive elements have an impact on tuning output. By increasing the copy number of transcriptional activator responsive elements upstream of the promoter, its ability to activate the transcription increases, despite there were extra responsive elements were placed downstream of the promoter, which provides a platform for transcriptional competition. Please note that [[Part:BBa_K2549035]] has 8 copies of responsive elements downstream of the promoter, but [[Part:BBa_K2549034]] only has 2 copies. | |
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| − | + | =====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<ref>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</ref>. | |
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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.'']] | ||
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| + | [[File:ZF3.jpg|none|240px|thumb|Khalil AS et al stated:''Tuning up output strength by increasing ZF operator number in synthetic promoter (sTF43-8).'']] | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K2549033 parameters</partinfo> | <partinfo>BBa_K2549033 parameters</partinfo> | ||
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===References=== | ===References=== | ||
Latest revision as of 20:38, 17 October 2018
6*ZF21.16-minCMV-2*ZF43.8
This part is one of the response elements of our amplifier, also executing the combiner function. 6*ZF21.16 binding sites and 2*ZF43.8 binding sites (Part:BBa_K2549051) can bind to different zinc finger-based transcription activator ZF21.16-VP64 (Part:BBa_K2549023) and zinc finger-based repressor ZF43.8-KRAB (Part:BBa_K2446041), respectively, with high orthogonality. Minimal CMV (Part:BBa_K2549049) is a promotor providing very low basal expression and high maximal expression after induction. This part is designed to construct our NIMPLY logic gate and test our multiple binding sites amplifier model[1].
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Biology
Our characterization
Flow cytometry results of transcriptional activator interacts with multiple binding sites. RFI: output from the Combiner after activation was divided by the value before activation. A degradable EGFP (d2EGFP) is produced downstream the promoter of the Combiner to indicate the output strength. ZF21.16 fused with VP64 was the transcriptional activator. There was no transcriptional repressor in the experiment. More details please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Measurement .
Worked as designed, copy numbers of responsive elements have an impact on tuning output. By increasing the copy number of transcriptional activator responsive elements upstream of the promoter, its ability to activate the transcription increases, despite there were extra responsive elements were placed downstream of the promoter, which provides a platform for transcriptional competition. Please note that Part:BBa_K2549035 has 8 copies of responsive elements downstream of the promoter, but Part:BBa_K2549034 only has 2 copies.
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.
