Part:BBa_K2549034

8*ZF21.16-minCMV-2*ZF43.8

This part is one of the response elements of our amplifier, also executing the combiner function. 8*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 was designed to construct our NIMPLY logic gate and test our multiple binding sites amplifier model.

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

NIMPLY gate constructed with the Combiner using either 8*ZF21.16-minCMV-2*ZF43.8 or 8*ZF43.8-minCMV-2*ZF21.16. A degradable EGFP (d2EGFP) is produced downstream the promoter of the Combiner to indicate the output strength. 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. RFI, relative fluorescence intensity (comparing before and after activation).

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.

On the right, we show that when transcriptional activator (ZF-VP64) and repressor (ZF-KRAB) both were present, the expression level of d2EGFP was significantly turned down but still higher than neither one was present. This result indicates that the repressor with its responsive elements placed downstream of the promoter plays a predominant role over the transcriptional activator, which binds to the promoter upstream, in regulating transcription. We switched ZF in the experiment and observed a similar result, suggesting it was not due to specific DNA sequences within zinc finger binding domains. Next, we transfected different amount of DNA expressing either activating or repressing transcriptional factors, and examine their effects on the same Combiner 8*ZF21.16-minCMV-2*ZF43.8 (Part:BBa_K2549034).

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.

Khalil AS et al stated:Tuning up output strength by increasing ZF operator number in synthetic promoter (sTF43-8).