plh sgrna1 ms2 pp7  (Addgene inc)

Journal: Journal of Biological Engineering

Article Title: Protein-inducible ribosomal frameshifting enables programmable translational control for genetic circuit design in Escherichia coli

doi: 10.1186/s13036-026-00629-w

Figure Lengend Snippet: Logic gate construction and multilayered regulation using PIRF. ( A ) Design of two-input OR gate for PP7cp and MS2cp. GFP reporter encoded in − 1 frame can be expressed when −1 PRF event occurs on either PP7 PIRF or MS2 PIRF. ( B ) GFP fluorescence output of the two-input OR gate for different combinations of inducers for PP7cp and MS2cp inputs. ‘Ng’ represents the negative control using the construct lacking the slippery sequence, in which no frameshifting occurs. ( C ) Design of two-input AND gate for PP7cp and MS2cp. GFP reporter encoded in − 2 frame can be expressed when −1 PRF event occurs at both PP7 PIRF and MS2 PIRF. ( D ) GFP fluorescence output of the two-input AND gate for different inducer conditions. ( E ) Design of the multilayer regulatory circuit using SWT and PIRF. The presence of the trigger RNA disrupts the transcriptional terminator structure within SWT, thereby enabling expression of the downstream PP7cp, which in turn can activate the PP7 PIRF-controlled GFP reporter output. ( F ) GFP fluorescence output of the multilayer circuit in the presence and absence of each component. IPTG induction was omitted as a negative control for trigger RNA expression. SWT–MS2cp construct was used in place of the SWT–PP7cp as a negative control for PP7cp expression. Trg: trigger RNA for SWT. ‘Auto’ indicates autofluorescence measured from the decoy trigger, SWT–MS2cp, and PP7 ng PIRF–GFP set, where PP7 ng serves as a negative control containing a scrambled slippery sequence to determine background fluorescence. The number of biological replicates is three for all experiments. Error bars are the standard deviation of three biological replicate measurements

Article Snippet: The PP7 coat protein (PP7cp) gene was obtained from pET283xFlagPP7CPHis (Addgene plasmid #28174) and inserted into a pCDFDuet backbone downstream of the pLlacO promoter.

Techniques: Fluorescence, Negative Control, Construct, Sequencing, Expressing, RNA Expression, Standard Deviation

Journal: Journal of Biological Engineering

Article Title: Protein-inducible ribosomal frameshifting enables programmable translational control for genetic circuit design in Escherichia coli

doi: 10.1186/s13036-026-00629-w

Figure Lengend Snippet: Design and characterization of PP7 and MS2 PIRF switches. ( A ) Schematic illustration of the PP7 PIRF mechanism and sequence layout. The red box indicates the slippery sequence (AAAAAG), and the orange codon (UAG) represents the in-frame stop codon. The blue hairpin marks the aptamer located between the slippery sequence and the in-frame stop codon. The dimeric structure represents the RNA binding protein (RBP) that binds to the aptamer. (Top) In the absence of RBP, translation terminates at the in-frame stop codon, resulting in a short polypeptide. (Bottom) Upon binding of RBP to the aptamer, the stabilized stem-loop structure induces ribosome stalling and −1 ribosomal frameshifting, allowing translation of the downstream GFP. ( B ) Schematic of the PP7 PIRF system, where frameshifting is induced by PP7cp binding to its cognate aptamer. ( C ) GFP fluorescence of the PP7 PIRF system at different IPTG concentrations to induce PP7cp expression. ( D ) Flow cytometry analysis of GFP fluorescence from the PP7 PIRF system. PIRF constructs were tested with (navy) and without (light blue) IPTG induction (1 mM and 0 mM, respectively). A negative control containing a scrambled slippery sequence was used to measure background fluorescence (gray). ( E ) Schematic of the MS2 PIRF system, where −1 ribosomal frameshifting is triggered by binding of MS2cp to its corresponding aptamer. ( F ) GFP fluorescence of the MS2 PIRF system in response to increasing concentrations of aTc to induce MS2cp expression. ( G ) Flow cytometry analysis of GFP fluorescence from cells expressing MS2 PIRF constructs with (burgundy) and without (light orange) aTc induction (100 ng/mL and 0 ng/mL, respectively). A negative control containing a scrambled slippery sequence was used to measure background fluorescence (gray). The GFP fluorescence datasets shown in panels (C) and (F), acquired under 0.2% L-arabinose induction, correspond to those presented in Supplementary Figure and , respectively. The number of biological replicates is three for all experiments. Error bars are the standard deviation of three biological replicate measurements

Article Snippet: The PP7 coat protein (PP7cp) gene was obtained from pET283xFlagPP7CPHis (Addgene plasmid #28174) and inserted into a pCDFDuet backbone downstream of the pLlacO promoter.

Techniques: Sequencing, RNA Binding Assay, Binding Assay, Fluorescence, Expressing, Flow Cytometry, Construct, Negative Control, Standard Deviation

Journal: Journal of Biological Engineering

Article Title: Protein-inducible ribosomal frameshifting enables programmable translational control for genetic circuit design in Escherichia coli

doi: 10.1186/s13036-026-00629-w

Figure Lengend Snippet: PIRF-mediated control of fusion protein expression. ( A ) Schematic of the fusion construct, consisting of an upstream mCherry, a PIRF module, and a downstream GFP coding sequence. In the absence of RBP, only mCherry is translated and GFP remains untranslated due to the premature stop codon in frame 0. Upon RBP binding, a −1 frameshifting event repositions GFP in-frame, resulting in the expression of an mCherry-GFP fusion protein. (B, C) Fluorescence analysis of the PP7 PIRF ( B ) and MS2 PIRF ( C ) inserted between mCherry and GFP coding regions. PP7cp or MS2cp expression was induced with 100 ng/mL aTc via an aTc-inducible pLtetO promoter. Analysis of mCherry-only and PIRFng constructs as controls further indicated that there are leaky expression of GFP that corresponds to 10 ~ 15% of the maximum induction level beyond the intrinsic PIRF leakage (Supplementary Fig. ). The fluorescence outputs of mCherry and GFP were measured by flow cytometry to assess the expression levels of fusion proteins. The number of biological replicates is three for all experiments. Error bars are the standard deviation of three biological replicate measurements

Article Snippet: The PP7 coat protein (PP7cp) gene was obtained from pET283xFlagPP7CPHis (Addgene plasmid #28174) and inserted into a pCDFDuet backbone downstream of the pLlacO promoter.

Techniques: Control, Expressing, Construct, Sequencing, Binding Assay, Fluorescence, Flow Cytometry, Standard Deviation

Journal: Journal of Biological Engineering

Article Title: Protein-inducible ribosomal frameshifting enables programmable translational control for genetic circuit design in Escherichia coli

doi: 10.1186/s13036-026-00629-w

Figure Lengend Snippet: PIRF-mediated translational control of intracellular protein aggregation. ( A ) Schematic illustration of PIRF-regulated ELK16 fusion construct. (Left) In the absence of − 1 frameshifting, only mCherry is translated without an ELK16 tag, resulting in distributed fluorescence throughout the cell. (Right) Upon frameshifting triggered by RBP binding, ELK16 is expressed as a C-terminal fusion, leading to protein aggregation. ( B , C ) Fluorescence microscopy image and demographic analysis of mCherry fluorescence distribution in E. coli harboring PIRF constructs designed to express mCherry-ELK16 fusions upon frameshifting, regulated by PP7cp ( B ) and MS2cp ( C ). All constructs were induced with 100 ng/mL aTc for coat protein expression. Demographs represent the spatial distribution of mCherry fluorescence along the cell axis, where 0 denotes the center of the cell, and cells are sorted by length. Scale bar: 5 μm

Article Snippet: The PP7 coat protein (PP7cp) gene was obtained from pET283xFlagPP7CPHis (Addgene plasmid #28174) and inserted into a pCDFDuet backbone downstream of the pLlacO promoter.

Techniques: Control, Construct, Fluorescence, Binding Assay, Microscopy, Expressing

Journal: Journal of Biological Engineering

Article Title: Protein-inducible ribosomal frameshifting enables programmable translational control for genetic circuit design in Escherichia coli

doi: 10.1186/s13036-026-00629-w

Figure Lengend Snippet: PIRF enables translational control of periplasmically localized protein fusions. ( A ) Schematic illustration of periplasmic localization control system using PIRF. An N-terminal DsbA signal peptide is fused to sfGFP via a PIRF module. (Left) In the absence of coat protein input, sfGFP remains untranslated due to the premature stop codon encountered. (Right) Upon RBP binding, −1 frameshifting restores the reading frame of sfGFP downstream of DsbA peptide, enabling DsbA-mediated secretion of sfGFP to the periplasm. ( B , C ) Fluorescence microscopy images of E. coli expressing DsbA-PIRF-sfGFP constructs regulated by PP7cp ( B ) and MS2cp ( C ). Under non-induced conditions, minimal sfGFP signal is observed. Upon induction of coat protein expression, peripheral sfGFP localization appears, indicative of periplasmic targeting. Scale bar: 5 μm. ( D , E ) Flow cytometry analysis of total sfGFP fluorescence of cell populations as in (B,C). Both PP7cp and MS2cp were expressed under the control of a pLtetO and induced with 100 ng/mL aTc. A 4.0-fold increase for PP7 PIRF ( D ) and a 2.1-fold increase for MS2 PIRF ( E ) were observed upon induction. The number of biological replicates is three for all experiments. Error bars are the standard deviation of three biological replicate measurements

Article Snippet: The PP7 coat protein (PP7cp) gene was obtained from pET283xFlagPP7CPHis (Addgene plasmid #28174) and inserted into a pCDFDuet backbone downstream of the pLlacO promoter.

Techniques: Control, Binding Assay, Fluorescence, Microscopy, Expressing, Construct, Flow Cytometry, Standard Deviation