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

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. And at last we chose 8 copies of responsive elements to construct our NIMPLY gate to achieving both not-too-hard molecular cloning and pretty high signal-to-noise ratio.

Fig.2 NIMPLY gate constructed using 8*ZF21.16-minCMV-2*ZF43.8 or 8*ZF43.8-minCMV-2*ZF21.16. 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. RFI, relative fluorescence intensity.

It is shown that when transcriptional activator exists and repressor doesn't exist, d2EGFP performs a high-level expression. When the two co-exist, the expression level of d2EGFP is significantly turned down but still higher than circumstances the two don't exist or only the repressor exists. This result indicates that the repressor takes the predominant position when coexisting with the activator.