Part:BBa_K2549028

8*ZF43.8-minCMV

This part is one of the response elements of our amplifier, also executing the combiner function. 8*ZF43.8 binding (Part:BBa_K2446013) sites is assembled using two 4*ZF43.8 binding sites (Part:BBa_K2446006) 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

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. 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.

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.

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. MFI, median fluorescence intensity.

Flow cytometry results suggest that the transcriptional activators can effectively activate the responsive promoters with high specificity and high orthogonality.