Difference between revisions of "Part:BBa K2549026"
(→Biology) |
|||
| Line 12: | Line 12: | ||
<!-- Add more about the biology of this part here --> | <!-- Add more about the biology of this part here --> | ||
===Biology=== | ===Biology=== | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
=====Synthetic promotor operators regulated by artificial zinc finger-based transcription factors===== | =====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>. | 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>. | ||
[[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.'']] | ||
| + | ===Characterization=== | ||
| + | =====It works as we designed.===== | ||
| + | [[File:aTF-test.png|none|480px|thumb|'''Fig.1 Interaction between transcriptional activators and their binding sites. A degradable EGFP (d2EGFP) is linked downstream the promoter to indicate the expression level of it. DBD, DNA binding domain which is zinc finger in our assay. AD or SD, activating- or silencing-form transcriptional domain. RE, responsive elements. RFI, relative fluorescence intensity.''']] | ||
| + | |||
| + | Flow cytometry results suggest that the transcriptional activators can effectively activate the responsive promoters with high specificity and high orthogonality. | ||
| + | |||
| + | [[File:ORgate.png|none|480px|thumb|'''Fig.2 OR gate based on 8*ZF21.16-minCMV. A degradable EGFP (d2EGFP) is linked downstream the promoter to indicate the expression level of it. DBD, DNA binding domain which is zinc finger in our assay. AD or SD, activating- or silencing-form transcriptional domain. RE, responsive elements. RFI, relative fluorescence intensity.''']] | ||
| + | |||
| + | Flow cytometry results indicate that when at least one of the transcriptional activators exist the expression of the promoter can be activated. | ||
| Line 27: | Line 31: | ||
<partinfo>BBa_K2549026 parameters</partinfo> | <partinfo>BBa_K2549026 parameters</partinfo> | ||
--> | --> | ||
| + | |||
===References=== | ===References=== | ||
Revision as of 13:27, 17 October 2018
8*ZF21.16-minCMV
This part is one of the response elements of our amplifier, also executing the combiner function. 8*ZF21.16 binding sites (Part:BBa_K2446015) is assembled using two 4*ZF21.16 binding sites (Part:BBa_K2446008) with a biobrick scar between them. Minimal CMV (Part:BBa_K2549049) is a promotor providing very low basal expression and high maximal expression after induction. This part can switch on the expression of gene downstream after induced by our zinc finger-based transcription activator.
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
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[1].
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.
Characterization
It works as we designed.
Fig.1 Interaction between transcriptional activators and their binding sites. A degradable EGFP (d2EGFP) is linked downstream the promoter to indicate the expression level of it. DBD, DNA binding domain which is zinc finger in our assay. AD or SD, activating- or silencing-form transcriptional domain. RE, responsive elements. RFI, relative fluorescence intensity.
Flow cytometry results suggest that the transcriptional activators can effectively activate the responsive promoters with high specificity and high orthogonality.
Fig.2 OR gate based on 8*ZF21.16-minCMV. A degradable EGFP (d2EGFP) is linked downstream the promoter to indicate the expression level of it. DBD, DNA binding domain which is zinc finger in our assay. AD or SD, activating- or silencing-form transcriptional domain. RE, responsive elements. RFI, relative fluorescence intensity.
Flow cytometry results indicate that when at least one of the transcriptional activators exist the expression of the promoter can be activated.
References
- ↑ 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