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:
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
Flow cytometry results of an intercellular OR gate. RFI: output from the Combiner after activation was divided by the value before activation. Similar to tTAA, transcriptional activator GV2 works well. The TRE3GV or URE2G promoters in the Amplifer receive signals from intercellular tTAA or GV2, respectively. Both Amplifiers would produce ZF21.16-VP64, 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 .
Interaction between transcriptional activators 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. AD, activating-form transcriptional domain; we used VP64 for the experiments in this figure. RE, responsive elements. MFI, median fluorescence intensity.
Flow cytometry results suggest that the transcriptional activators can specifically activate the promoters with responsive elements, orthogonally. Please visit http://2018.igem.org/Team:Fudan/Demonstration for a brief introduction of our project.
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.
