Difference between revisions of "Part:BBa K2549038"

Revision as of 21:15, 17 October 2018

CfaC-ZF21.16C-NLS

This part is one of the downstream elements of our amplifier. It was constructed by fusing CfaC (Part:BBa_K2549010), ZF21.16C (Part:BBa_K2549012) and NLS (Part:BBa_K2549054), from N terminal to C terminal. CfaC is the C-terminal fragment of Cfa which is a consensus sequence from an alignment of 73 naturally occurring DnaE inteins that are predicted to have fast splicing rates. ZF21.16C is the C-terminal fragment of the zinc finger whose recognition helices for three-finger arrays are substituted by the reported synthetic zinc finger 21.16 residues on the basis of the BCR_ABL-1 artificial zinc finger^[1]. NLS is a short nuclear location sequence from SV40 large T antigen. When coexpressed with VP64-ZF21.16N-CfaN (Part:BBa_K2549036) in the same cell, both fusions will be produced and a transcription activating function will be executed. Also, when coexpressed with KRAB-ZF21.16N-CfaN (Part:BBa_K2549037) in the same cell, both fusions will be produced formed and a transcription repressing function will be executed.

Sequence and Features

Biology

Our characterization

CfaN intein-based AND gate. A degradable EGFP (d2EGFP) is produced downstream the promoter of the Combiner to indicate the output (CusP) strength. DBD, DNA binding domain which is zinc finger in our assay. AD, activating-form transcriptional domain, was VP64. RE, responsive elements. RFI, relative fluorescence intensity (comparing before and after activation).

We show that when VP64-ZF21.16N-CfaN co-expressed with CfaC-ZF21.16C-NLS, the expression level of d2EGFP is relatively turned up due to the formation of VP64-ZF21.16 after auto-plicing and ligation. The signal-noise-ratio is still not optimal, and we are improving the design by switching the split position as well as adding linkers.

CfaN intein-based NAND gate. A degradable EGFP (d2EGFP) is produced downstream the promoter of the Combiner to indicate the output (CusP) strength. DBD, DNA binding domain which is zinc finger in our assay. SD, silencing-form transcriptional domain, was KRAB. RE, responsive elements. RFI, relative fluorescence intensity (comparing before and after activation).

We show that when KRAB-ZF21.16N-CfaN co-expressed with CfaC-ZF21.16C-NLS, the expression level of d2EGFP is relatively turned down due to the formation of KRAB-ZF21.16 after auto-plicing and ligation.

The signal-noise-ratio is ok when both present. However, we are not sure why CfaC-ZF21.16C-NLS alone has effect. We have formed a couple of hypothesis and in the process of optimizing this gate. Please note Part:BBa_K2549036 contains a transcriptional activation domain VP64, while Part:BBa_K2549037 has a transcriptional repression domain KRAB, both need to ligate with CfaC-ZF21.16C-NLS (Part:BBa_K2549038) before functional.

Boolean logic gates via split zinc finger-based transcription factors

Lohmueller JJ et al have demonstrated the split ZF-TF reconstitution process. Please note that we used Cfa split intein (Part:BBa_K2549009 and Part:BBa_K2549010) but not dnaB reported below.

Lohmueller JJ et al demonstrated: After expression, the two split ZF-intein fragments bind together and undergo protein splicing to cleave away intein fragments and reconstitute the full ZF activator leading to activation of the BCR_ABL reporter.

Lohmueller JJ et al demonstrated: For AND gates, a ZF activator is spliced and the logical operation is computed as TRUE only when both input signals are present. For the response data shown BCR_ABL-1:GCN4 activator split fragments were used and the response promoter contains 6 copies of the BCR_ABL target site. CFP expression was measured by flow cytometry and expressed as fold change over an off-target expression control.

Lohmueller JJ et al demonstrated: For NAND gates, the computational module splices a ZF repressor, and the logical operation is computed as TRUE as long as both inputs are not present together. For the response data shown BCR_ABL-1:GCN4 repressor split fragments were used and the response promoter contains 6 copies of the BCR_ABL target site. CFP expression was measured by flow cytometry and expressed as fold change over an off-target expression control.

References

1. ↑ A tunable zinc finger-based framework for Boolean logic computation in mammalian cells. Lohmueller JJ, Armel TZ, Silver PA. Nucleic Acids Res, 2012 Jun;40(11):5180-7 PMID: 22323524; DOI: 10.1093/nar/gks142