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A split intein T7 RNA polymerase for transcriptional AND-logic.

Schaerli Y, Gili M, Isalan M - Nucleic Acids Res. (2014)

Bottom Line: Only when both domains are expressed does the split intein mediate protein trans-splicing, yielding a full-length T7 RNA polymerase that can transcribe genes via a T7 promoter.We demonstrate an AND gate with the new part: the signal-to-background ratio is very high, resulting in an almost digital signal.The split intein approach should be widely applicable for engineering artificial gene circuit parts.

View Article: PubMed Central - PubMed

Affiliation: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003 Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain yolanda.schaerli@ieu.uzh.ch.

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Application of the split intein T7 RNAP in a band-pass filter circuit. (A) Schematic view of the network and its implementation. PJ23106: constitutive promoter; TetO: tet operator; aTc: anhydrotetracycline. (B) Bacteria carrying the network show a band-pass behaviour in an arabinose gradient (green). In the presence of aTc (orange), to prevent repression by TetR, the same circuit shows increasing fluorescence with increasing arabinose. Mean and SD from three biological replicates.
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Figure 2: Application of the split intein T7 RNAP in a band-pass filter circuit. (A) Schematic view of the network and its implementation. PJ23106: constitutive promoter; TetO: tet operator; aTc: anhydrotetracycline. (B) Bacteria carrying the network show a band-pass behaviour in an arabinose gradient (green). In the presence of aTc (orange), to prevent repression by TetR, the same circuit shows increasing fluorescence with increasing arabinose. Mean and SD from three biological replicates.

Mentions: A split intein T7 RNAP for transcriptional AND-signal integration. (A) Schematic depiction of the designed construct and splicing. PBAD: arabinose inducible promoter; boxes: genes; grey ellipses: ribosomal binding sites; PT7(−3G): T7 promoter with a G at position −3. (B) The construct depicted in (A) and control constructs (c1, c2, c3; (C)) were transformed into E. coli, together with the PT7-GFP reporter construct. The cells were grown for 8 h in the presence of the indicated amount of arabinose, and the fluorescence was measured. Mean and SD from three biological replicates. (C) Schematic depiction of control constructs. (D) Schematic depiction of an AND logic gate using the split intein T7 RNAP. (E) The construct depicted in (D) was transformed into E. coli, the cells were grown for 8 h in the presence of the indicated amounts of arabinose and IPTG, and their fluorescence was measured. Mean and SD from three biological replicates. (F) Flow cytometry data for all input states. The dark line is for the [11] sample.


A split intein T7 RNA polymerase for transcriptional AND-logic.

Schaerli Y, Gili M, Isalan M - Nucleic Acids Res. (2014)

Application of the split intein T7 RNAP in a band-pass filter circuit. (A) Schematic view of the network and its implementation. PJ23106: constitutive promoter; TetO: tet operator; aTc: anhydrotetracycline. (B) Bacteria carrying the network show a band-pass behaviour in an arabinose gradient (green). In the presence of aTc (orange), to prevent repression by TetR, the same circuit shows increasing fluorescence with increasing arabinose. Mean and SD from three biological replicates.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231753&req=5

Figure 2: Application of the split intein T7 RNAP in a band-pass filter circuit. (A) Schematic view of the network and its implementation. PJ23106: constitutive promoter; TetO: tet operator; aTc: anhydrotetracycline. (B) Bacteria carrying the network show a band-pass behaviour in an arabinose gradient (green). In the presence of aTc (orange), to prevent repression by TetR, the same circuit shows increasing fluorescence with increasing arabinose. Mean and SD from three biological replicates.
Mentions: A split intein T7 RNAP for transcriptional AND-signal integration. (A) Schematic depiction of the designed construct and splicing. PBAD: arabinose inducible promoter; boxes: genes; grey ellipses: ribosomal binding sites; PT7(−3G): T7 promoter with a G at position −3. (B) The construct depicted in (A) and control constructs (c1, c2, c3; (C)) were transformed into E. coli, together with the PT7-GFP reporter construct. The cells were grown for 8 h in the presence of the indicated amount of arabinose, and the fluorescence was measured. Mean and SD from three biological replicates. (C) Schematic depiction of control constructs. (D) Schematic depiction of an AND logic gate using the split intein T7 RNAP. (E) The construct depicted in (D) was transformed into E. coli, the cells were grown for 8 h in the presence of the indicated amounts of arabinose and IPTG, and their fluorescence was measured. Mean and SD from three biological replicates. (F) Flow cytometry data for all input states. The dark line is for the [11] sample.

Bottom Line: Only when both domains are expressed does the split intein mediate protein trans-splicing, yielding a full-length T7 RNA polymerase that can transcribe genes via a T7 promoter.We demonstrate an AND gate with the new part: the signal-to-background ratio is very high, resulting in an almost digital signal.The split intein approach should be widely applicable for engineering artificial gene circuit parts.

View Article: PubMed Central - PubMed

Affiliation: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003 Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain yolanda.schaerli@ieu.uzh.ch.

Show MeSH
Related in: MedlinePlus