Part:BBa_K2549033

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.

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 different transcription factors interaction 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, and ZF43.8 fused with KRAB was the transcriptionl repressor. 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 overcome the transcriptional suppression increases, whose responsive elements were placed downstream of the promoter and only two copies were used.

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).

Characterization

It works as we designed

Fig.1 Flow cytometry results of different transcription factors interaction with multiple binding sites. A degradable EGFP (d2EGFP) is linked downstream the promoter to indicate the expression level of it. RFI, relative fluorescence intensity.

Thus it is verified by experiment that promoter operator numbers can have an impact on tuning output. By changing the copy number of transcriptional activator binding sites, others can tune the maximum activation level as they desire.