Journal: JID Innovations

Article Title: En Route to Targeted Ribosome Editing to Replenish Skin Anchor Protein LAMB3 in Junctional Epidermolysis Bullosa

doi: 10.1016/j.xjidi.2023.100240

Figure Lengend Snippet: Treatment with RpL35 ligands ART and ATZ triggers increased production levels of LAMβ3FL in yeast cellular assays. Treatments of yeast vehicles transformed with dual luciferase reporter plasmids expressing C-terminally FF-tagged LAMB3 (Lamβ3 protein reporter) and REN (Renilla protein reporter) are shown in a , c , and e . Treatments of yeast vehicles transformed with dual luciferase reporter plasmids expressing C-terminally FF-tagged mutant LAMB3 protein (R635X), which reports the production of LAMB3FL reporter protein, and REN (Renilla protein reporter) are shown in panel b , d , and f . Vehicles were grown for 18 h under treatment with ART and ATZ or combined treatment of ATZ and ART. Reporter activity was measured in RLUs, and reporter protein expression levels were calculated as the FF signal to REN signal ratio and normalized to the mean FF signal to REN signal ratio set to 100% in untreated cells. Mean normalized reporter protein expression levels based on luciferase activity are shown with error bars, presenting the data as mean ± 1 SD. Varying concentrations as indicated on the x-axis were tested for ART, ATZ, and ATZ/ART. Three replicates per well were collected to assess RLUs for a given drug treatment condition, and at least 4 wells were measured per experiment (n = 12). The black arrow indicates treatment conditions delivering maximal response. ART, artesunate; ATZ, atazanavir; h, hour; LAMβ3FL, full-length LAMβ3; RLU, Relative Light Unit.

Article Snippet: Renilla (REN) luciferase control reporter sequence (Promega) was cloned into YCPlac111 vector, under the control of ADH1 promotor and ADH1 terminator, generating plasmid pLM164 ( ).

Techniques: Transformation Assay, Luciferase, Expressing, Mutagenesis, Activity Assay

Journal: JID Innovations

Article Title: En Route to Targeted Ribosome Editing to Replenish Skin Anchor Protein LAMB3 in Junctional Epidermolysis Bullosa

doi: 10.1016/j.xjidi.2023.100240

Figure Lengend Snippet: Treatment with RpL35 ligands ART and ATZ does not trigger increased production levels of FFFL in yeast cellular assays. Treatment of yeast vehicles transformed with luciferase reporter plasmids expressing Firefly (FF protein reporter) and Renilla (REN protein reporter) are shown in a , c , and e . Treatments of yeast vehicles transformed with dual luciferase reporter plasmids expressing mutant Firefly protein (Y53X), which reports production of full-length FF reporter protein (ie, FFFL) and REN (Renilla protein reporter) are shown in b , d , and f . Vehicles were grown for 18 h under treatment with ART and ATZ or combined treatment of ATZ and ART. Reporter activity was measured in RLUs, and reporter protein expression levels were calculated as the FF signal to REN signal ratio and normalized to the mean FF signal to REN signal ratio in untreated cells. Mean normalized reporter protein expression levels based on luciferase activity are shown with error bars, presenting the data as mean ± 1 SD. Varying concentrations as indicated on the x-axis were tested for ART, ATZ, and ATZ/ART. Three replicates per well were collected to assess RLUs for each drug treatment condition, and at least 4 wells were measured per experiment (n = 12). The black arrow indicates the treatment condition delivering maximal response. ART, artesunate; ATZ, atazanavir; h, hour; RLU, Relative Light Unit.

Article Snippet: Renilla (REN) luciferase control reporter sequence (Promega) was cloned into YCPlac111 vector, under the control of ADH1 promotor and ADH1 terminator, generating plasmid pLM164 ( ).

Techniques: Transformation Assay, Luciferase, Expressing, Mutagenesis, Activity Assay

Journal: JID Innovations

Article Title: En Route to Targeted Ribosome Editing to Replenish Skin Anchor Protein LAMB3 in Junctional Epidermolysis Bullosa

doi: 10.1016/j.xjidi.2023.100240

Figure Lengend Snippet: Treatment with macrolide antibiotic ERY nonselectively triggers increased production levels of LAMβ3FL and FFFL in cellular assays. ( a ) Treatment of yeast vehicles transformed with dual luciferase reporter plasmids expressing C-terminally FF-tagged LAMB3 (LAMB3 protein reporter) and REN (Renilla protein reporter). ( b ) Treatment of yeast vehicles transformed with dual luciferase reporter plasmids expressing C-terminally FF-tagged mutant LAMB3 protein (R635X), which reports the production of LAMB3FL reporter protein (LAMB3FL) and REN (Renilla protein reporter). ( c ) Treatment of yeast vehicles transformed with luciferase reporter plasmids expressing Firefly (FF protein reporter) and Renilla (REN protein reporter). ( d ) Treatment of yeast vehicles transformed with dual luciferase reporter plasmids expressing mutant Firefly protein (Y53X), which reports production of full-length FF reporter protein (FFFL) and REN (Renilla protein reporter). Vehicles were grown for 18 h under treatment with ERY. Reporter activity was measured in RLUs, and reporter protein expression levels were calculated as the FF signal to REN signal ratio and normalized to the mean FF signal to REN signal ratio in untreated cells. Mean normalized reporter protein expression levels based on luciferase activity are shown with error bars, presenting the data as mean ± 1 SD. Varying concentrations as indicated on the x-axis were tested for ERY. Three replicates per well were collected to assess RLUs for a given drug treatment condition, and at least 4 wells were measured per experiment (n = 12). The black arrow indicates the treatment condition delivering maximal response. ERY, erythromycin; h, hour; LAMβ3FL, full-length LAMβ3; RLU, Relative Light Unit.

Article Snippet: Renilla (REN) luciferase control reporter sequence (Promega) was cloned into YCPlac111 vector, under the control of ADH1 promotor and ADH1 terminator, generating plasmid pLM164 ( ).

Techniques: Transformation Assay, Luciferase, Expressing, Mutagenesis, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Importance of codon usage for the temporal regulation of viral gene expression

doi: 10.1073/pnas.1515387112

Figure Lengend Snippet: Influence of codon usage on SIV gp160 and RRV gH Induction. (A) Comparison of codon usage in SIV gp160 and RRV gH. The coding sequences of SIV239 gp160 and RRV gH were analyzed using the GCUA software (gcua.schoedl.de/) (40). The use of each indicated codon in RRV gH as percent of all codons for that particular amino acid is plotted vs. that for gp160 using a XY scatter plot with a linear regression line and a coefficient of determination (R2). Codons with the most skewed usage between the two sequences are those most extensively deviated from y = x line. To generate a codon-modified (gH-like) SIV gp160, some codons of SIV gp160 (empty colored circles) were changed into corresponding synonymous codons (filled colored circles) to reflect the codon usage of RRV gH. To generate a codon-modified (gp160-like) RRV gH, codon changes were performed in the exact opposite direction. (B) Schematic representation of expression cassettes with the codon-modified (gH-like) SIV gp160 and codon-modified (gp160-like) RRV gH sequence. Splicing donor (SD) sequence (indicated by gray arrow, aaacaagtaagt), and Rev-response element (RRE, red colored box) are shown. Each codon change is represented by a vertical line in the gp160 and gH box. Note that there were no codon changes in the RRE region. (C) Expression cassettes of SD-gp160 with the unmodified (U) (lanes 1, 2, and 3) or codon-modified (M, gH-like) SIV gp160 sequence (lanes 4, 5, and 6), and gp160 (no SD) with the unmodified (U) (lanes 7, 8, and 9) or codon-modified (M, gH-like) SIV gp160 sequence (lanes 10, 11, and 12) were transfected into HEK 293T cells together with either control, Rev, or ORF57 expression plasmid. The cells were harvested at 46 h after transfection and proteins were subjected to SDS/PAGE and immunoblotting. The expression of gp160 was detected using the 3.11H antibody. The expression of ORF57 and Rev was detected using a v5 antibody. (D) The total levels of gp160 RNA (copies per microgram) were measured by qPCR. (E) Expression cassettes of gH with the unmodified (U) or codon-modified (M, gp160-like) RRV gH sequence with or without flanking SD and RRE sequences was transfected into HEK 293T cells together with either control, Rev, or ORF57 expression plasmid. The cells were harvested at 46 h after transfection and proteins were subjected to SDS/PAGE and immunoblotting. The expressions of RRV gH, ORF57, and Rev were detected using a v5 antibody. The two bands for RRV gH appear to correspond to two bands previously noted for the gH of the closely related Kaposi sarcoma herpesvirus (46). (F) The total levels of gH RNA (copies per microgram) were measured by qPCR.

Article Snippet: Codon changes in firefly luciferase. ( A ) Sequence alignment of parental luciferase, gp160-like, and gH-like firefly luciferases was performed using MacVector.

Techniques: Comparison, Software, Modification, Expressing, Sequencing, Transfection, Control, Plasmid Preparation, SDS Page, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Importance of codon usage for the temporal regulation of viral gene expression

doi: 10.1073/pnas.1515387112

Figure Lengend Snippet: Codon Changes in SIV gp160 and RRV gH

Article Snippet: Codon changes in firefly luciferase. ( A ) Sequence alignment of parental luciferase, gp160-like, and gH-like firefly luciferases was performed using MacVector.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Importance of codon usage for the temporal regulation of viral gene expression

doi: 10.1073/pnas.1515387112

Figure Lengend Snippet: Sequence alignments of WT and codon-modified (c.m) SIV239 gp160 (A) and RRV gH (B). Sequence alignment was performed using MacVector program. Only the matched bases between the two sequences are shaded and boxed. The amino acids encoded by the mismatched bases are indicated on the top of each codon. The unmodified stretch between nucleotide numbers 1,520 and 1,876 in gp160 sequence corresponds to the RRE.

Article Snippet: Codon changes in firefly luciferase. ( A ) Sequence alignment of parental luciferase, gp160-like, and gH-like firefly luciferases was performed using MacVector.

Techniques: Sequencing, Modification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Importance of codon usage for the temporal regulation of viral gene expression

doi: 10.1073/pnas.1515387112

Figure Lengend Snippet: Nucleotide composition of gp160, gH, luciferase, and GFP

Article Snippet: Codon changes in firefly luciferase. ( A ) Sequence alignment of parental luciferase, gp160-like, and gH-like firefly luciferases was performed using MacVector.

Techniques: Luciferase

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Importance of codon usage for the temporal regulation of viral gene expression

doi: 10.1073/pnas.1515387112

Figure Lengend Snippet: Codon changes in firefly luciferase. (A) Sequence alignment of parental luciferase, gp160-like, and gH-like firefly luciferases was performed using MacVector. Nucleotide changes in gp160-like and gH-like firefly luciferase sequences are indicated, and unchanged ones are indicated with dots. (B) Gribskov codon preference plots of the luciferase sequences. The human codon usage was set as a bias with a 40-codon window. The coding frame is set as frame 1. The nucleotide residue number of each sequence was plotted on the x axis vs. the likelihood of a particular window of codons to a predetermined preferred codon usage in human exome on the y axis. Thus, a number lower than 1.0 indicates that more rare codons are being used in that specific region of sequence (i.e., the smaller the number is, the more skewed codon usage it has). The codon adaptation index (42) of each sequence is indicated under each sequence name.

Article Snippet: Codon changes in firefly luciferase. ( A ) Sequence alignment of parental luciferase, gp160-like, and gH-like firefly luciferases was performed using MacVector.

Techniques: Luciferase, Sequencing, Residue

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Importance of codon usage for the temporal regulation of viral gene expression

doi: 10.1073/pnas.1515387112

Figure Lengend Snippet: Induction of luciferase from the expression cassettes with unmodified and codon-modified luciferase sequences. The coding sequence of parental firefly luciferase was modified to make it gp160-like or gH-like. In addition to the six codon changes shown in Table S1, the following additional synonymous codon changes were made. For gp160-like, GCC→GCA, CGC→AGG, CGG→AGG, AAC→AAT, TGC→TGT, ATC→ATA, CTG→TTG, CCG→CCA, TAC→TAT, and GTT→GTA. For gH-like, GCC→GCA, GAC→GAT, TGC→TGT, ATC→ATA, CTC→TTG, CTG→TTA, AAG→AAA, TTC→TTT, TAC→TAT, and GTC→GTA. Each luciferase sequence was flanked by splicing donor (SD) sequence and the Rev-responsive element (RRE) in A. No SD and RRE were added in B. The levels of firefly luciferase expression from each expression cassette in the presence of empty vector, ORF57, or Rev were measured after transfection of corresponding plasmid DNAs into HEK 293T cells. The cells were harvested at 46 h after transfection. The levels of renilla luciferase expression from a pRL-SV plasmid (Promega) were used to calibrate the transfection efficiency among the samples. Shown are representative results for three independent experiments done in triplicate. Error bars indicate SD. RLU, relative light units. The calibrated level of firefly luciferase activity from the transfection of gp160-like luciferase sequence (with SD and RRE) in the presence of empty vector was set as 1 RLU. The RLU value of each transfection was indicated above the corresponding bars. Shown at the bottom are the corresponding luciferase RNA levels measured by qPCR.

Article Snippet: Codon changes in firefly luciferase. ( A ) Sequence alignment of parental luciferase, gp160-like, and gH-like firefly luciferases was performed using MacVector.

Techniques: Luciferase, Expressing, Modification, Sequencing, Plasmid Preparation, Transfection, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Importance of codon usage for the temporal regulation of viral gene expression

doi: 10.1073/pnas.1515387112

Figure Lengend Snippet: Summary of induction of gp160 by Rev and ORF57 using altered coding sequences. HEK 293T cells in each well of a six-well plate were transfected with 1.5 μg plasmid DNA of the gp160 constructs and 0.5 μg each inducer (Rev or ORF57) and cultured for 42–48 h before harvest. Protein input was normalized using a Bradford assay. The level of SIV239 gp160 in the harvested cell pellets was detected by immunoblotting. Very little or no expression is denoted with a minus (−) sign, and the relative degree of induction of each construct is represented with varying numbers of a plus (+) sign.

Article Snippet: Codon changes in firefly luciferase. ( A ) Sequence alignment of parental luciferase, gp160-like, and gH-like firefly luciferases was performed using MacVector.

Techniques: Transfection, Plasmid Preparation, Construct, Cell Culture, Bradford Assay, Western Blot, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Importance of codon usage for the temporal regulation of viral gene expression

doi: 10.1073/pnas.1515387112

Figure Lengend Snippet: Regions of gp160 sequence that contribute to Rev- or ORF57-mediated induction. Expression cassettes of gp160 with unmodified (U) or codon-modified (M, gH-like) SIV gp160 sequence and their derivatives, ORF57, or Rev plasmid were transfected into HEK 293T cells as indicated. The cells were harvested at 46 h after transfection, and the total cell lysate (TCL) from each cell pellet was subjected to SDS/PAGE and immunoblotting after protein normalization using a Bradford assay. The expression of each protein was detected using the corresponding antibodies. (A) Induction of gp160 from the expression cassette with the unmodified SIV gp160 sequence (U), constructs with the Arg + Leu (R/L) changes alone, the Glu + Gly (E/G) changes alone, the Pro + Thr (P/T) changes alone, and codon-modified gp160 (M) by Rev and ORF57. (B) Induction of gp160 from the expression cassette with the unmodified SIV gp160 sequence (U), constructs with 1/3 codon changes at the N terminus (nucleotides 1:879), 1/3 codon changes in the middle of the gp160 sequence (nucleotides 880:1758), 1/3 codon changes at the C terminus (nucleotides 1759:2650), and globally codon-modified gp160 (M) by Rev and ORF57. (C) Induction of gp160 from the expression cassette with the unmodified SIV gp160 sequence (U), 1/3 codon changes at the C terminus (nucleotides 1759:2640), 2/3 codon changes at the C terminus (nucleotides 880:2640), and globally codon-modified gp160 (M) by Rev and ORF57. (D and E) Induction of gp160 from the expression cassette with the unmodified SIV gp160 sequence (U), constructs with 1/12 codon changes at the N terminus (nucleotides 1:222), 1/6 codon changes at the N terminus (nucleotides 1:441), 1/4 codon changes at the N terminus (nucleotides 1:660), 1/3 codon changes at the N terminus (nucleotides 1:879), 2/3 codon changes at the N terminus (nucleotides 1:1,761), and globally codon-modified gp160 (M) by Rev (D) and ORF57 (E).

Article Snippet: Codon changes in firefly luciferase. ( A ) Sequence alignment of parental luciferase, gp160-like, and gH-like firefly luciferases was performed using MacVector.

Techniques: Sequencing, Expressing, Modification, Plasmid Preparation, Transfection, SDS Page, Western Blot, Bradford Assay, Construct

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Importance of codon usage for the temporal regulation of viral gene expression

doi: 10.1073/pnas.1515387112

Figure Lengend Snippet: Growth kinetics and Env expression of rRRVs. Early passage rhesus fibroblast cells (6.7 × 105) were seeded in T-25 tissue culture flasks, cultured for 1 d and then infected with the same amount of each rRRV (2 × 1010 genome copies) encoding either expression-optimized or codon-modified (gH-like) SIV gp160. Culture supernatants in all of the flasks were removed and fresh media were added at day 2 after infection. (A) RRV titers in the culture supernatants of days 3–7 after infection were measured by qPCR. (B) RF cells in the flasks of days 2–7 after infection were harvested, and the levels of Env expression were measured by immunoblotting.

Article Snippet: Codon changes in firefly luciferase. ( A ) Sequence alignment of parental luciferase, gp160-like, and gH-like firefly luciferases was performed using MacVector.

Techniques: Expressing, Cell Culture, Infection, Modification, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Importance of codon usage for the temporal regulation of viral gene expression

doi: 10.1073/pnas.1515387112

Figure Lengend Snippet: Elicitation of anti-env antibodies by recombinant RRV with codon-modified gp160 sequence. (A) Recombinant RRV (rRRV) harboring a codon-modified (gH-like) gp160 sequence was inoculated into two rhesus macaques (1 × 109 genome copies per animal), and levels of anti-SIV envelope antibodies were measured by an ELISA. (B) Neutralization of SIV316 by sera taken at weeks 17 and 33 after inoculation with rRRV with codon-modified gp160 sequence. SPF, negative control sera from specific pathogen-free animals; RLU, relative light units.

Article Snippet: Codon changes in firefly luciferase. ( A ) Sequence alignment of parental luciferase, gp160-like, and gH-like firefly luciferases was performed using MacVector.

Techniques: Recombinant, Modification, Sequencing, Enzyme-linked Immunosorbent Assay, Neutralization, Negative Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Importance of codon usage for the temporal regulation of viral gene expression

doi: 10.1073/pnas.1515387112

Figure Lengend Snippet: Codon usage of persistent and nonpersistent viruses. (A) The coding sequences of representative late gene products from six persistent viruses were analyzed using the GCUA software (gcua.schoedl.de/) (40). The usage (%) of each codon in RRV gH, SIV239 gp160, human papilloma virus 16 (HPV16) L1, simian virus 40 (SV40) VP1, human adenovirus 2 (hAV2) fiber, and parvovirus B19 VP1 was plotted on the x axis vs. the codon usage of human exome sequences on the y axis. Also shown is the coefficient of determination (R2). (B) The coding sequences of representative structural gene products from five acute, nonpersistent viruses were analyzed as in A. Listed are poliovirus P1, measles virus HA, Ebola virus gp, rabies virus gp, and yellow fever virus polyprotein sequences. The GeneBank accession number for each sequence is as follows: SIV gp160 (HM800148), RRV gH (AF210726), SV40 VP1 (EF579804), HPV16 L1 (K02718), hAV2 fiber (AY224420), parvovirus B19 vp1 (AY768535), poliovirus P1 (V01149), measles virus HA (AY445032), Ebola virus gp (L11365), rabies virus gp (S72465), and yellow fever virus polyprotein (KM388818).

Article Snippet: Codon changes in firefly luciferase. ( A ) Sequence alignment of parental luciferase, gp160-like, and gH-like firefly luciferases was performed using MacVector.

Techniques: Software, Virus, Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Importance of codon usage for the temporal regulation of viral gene expression

doi: 10.1073/pnas.1515387112

Figure Lengend Snippet: Expression of SIV239 gp160, RRV gH, SV40 VP1, and rhPV-1 L1 unmodified WT and codon-optimized (c.o) SIV239 gp160, RRV gH, SV40 VP1, and rhPV-1 L1 sequences were cloned into a pcDNA6-v5-hisA plasmid. HEK 293T cells in each well of a six-well plate were transfected with 1.5 μg each plasmid DNA and cultured for 42–48 h before harvest. The levels of SIV239 gp160, RRV gH, SV40 VP1, and rhPV-1 L1 protein in the harvested cell pellets were detected by immunoblotting.

Article Snippet: Codon changes in firefly luciferase. ( A ) Sequence alignment of parental luciferase, gp160-like, and gH-like firefly luciferases was performed using MacVector.

Techniques: Expressing, Clone Assay, Plasmid Preparation, Transfection, Cell Culture, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Importance of codon usage for the temporal regulation of viral gene expression

doi: 10.1073/pnas.1515387112

Figure Lengend Snippet: Prediction of secondary structure of RNA. Shown are predicted RNA secondary structures corresponding to WT gp160 (A), codon-modified (gH-like) gp160 (B), WT gH-RRE (C), and codon-modified (gp160-like) gH-RRE (D). The RRE region in each sequence is indicated with red color. Secondary structure prediction based on a minimum free energy model (MFE) was performed using the RNAfold algorithm (43). Presented is the secondary structure of each sequence that is computed to have its minimum free energy at 37 °C using a loop-based energy model and dynamic programming algorithm (47).

Article Snippet: Codon changes in firefly luciferase. ( A ) Sequence alignment of parental luciferase, gp160-like, and gH-like firefly luciferases was performed using MacVector.

Techniques: Modification, Sequencing

Journal: bioRxiv

Article Title: A general strategy to engineer high-performance mammalian Whole-Cell Biosensors

doi: 10.1101/2024.02.28.582526

Figure Lengend Snippet: a,d , Schematic representation of the constructs impementing the basic and TF-amplified biosensors. b,e , Dose-response curve of the Basic biosensor (red) and the TF-amplified biosensor (green) for increasing copper dichloride concentrations. n=9 biological replicates from three independent experiments. Firefly luciferase (FLuc) expression was evaluated by luminescence measurements normalised to Renilla firefly (RLuc) and reported either as Arbitrary Units [A.U.] (left panel), or as Fold Activation obtained by dividing each data point by the average luminescence in the absence of copper (right panel). c,f , Radar plot reporting the values of the five features of the Basic biosensor (red) and of the TF-amplified biosensor (green). Leak: leakiness, FC: fold change, S: sensitivity to input, OR: operation range, Lin: linearity. The score ranges from 0, which is assigned to the biosensor with the worst performance, to 1 for the one with the best performance. VPR-dCas9 : nuclease-deficient Cas9 fused to the transactivation domain VPR; FLuc : firefly Luciferase; gRNA _ B: gude RNA with sequence B; pMRE : synthetic promoter harbouring four metal responsive elements; p7B_min : a synthetic promoter consisting of 7 sequences complementary to gRNA_B upstream of a minimal CMV promoter .

Article Snippet: For the characterization of the NFL Motif, a Firefly Luciferase with two destabilization sequences has been used (Luc2CP, Promega), while for the biosensing experiments, a stable Firefly Luciferase (Luc2, Promega) has been used in order to compare it with the original biosensor.

Techniques: Construct, Amplification, Luciferase, Expressing, Activation Assay, Sequencing

Journal: bioRxiv

Article Title: A general strategy to engineer high-performance mammalian Whole-Cell Biosensors

doi: 10.1101/2024.02.28.582526

Figure Lengend Snippet: a , Schematic representation of the constructs transfected in HEK293T cells to assess the transcriptional activity of VPR-dCas9 and AcrIIA4, with or without the cognate coiled-coiled (CC) domains N7 and N8. The promoter p7B Min in the presence of the guide RNA gRNA_B drives expression of the firefly luciferase (FLuc). b , Fold-repression in luminescence when co-transfecting either the VPR-dCas9 (grey bars) or the coiled-coiled fusion variant VPR-dCas9-N8 (pink bars), in presence of AcrIIA4, or of the coiled-coiled fusion variant AcrIIA4-N7. Fold repression is computed by dividing the luminescence of FLuc in the absence of the AcrIIA4 (or AcrIIA4-N7) by the value in its presence. Statistical significance is computed via a two-population t-test. c , Implementation of the NFL motif. The VPR-dCas9-N8 is constitutively expressed from the pCMV promoter and, in the presence of the gRNA_B, binds to the 7B pMin promoter driving the rtTA transcription factor. In the presence of doxycycline, rtTA binds to the pTRE3G promoter upstream of the AcrIIA4-N7. The VPR-dCas9-N8 is inactivated by the AcrIIA4-N7, thus closing the loop. The firefly luciferase (FLuc) under the control of the p7B_Min promoter is used as a reporter. In the open loop configuration, the rtTA is under the control of the constitutive promoter pEF1 α . d , Dose-response curve to doxycycline of the NFL (blue circles) and of the open loop control (green squares). Relative Luciferase is the FLuc luminescence divided by its value in the absence of Doxycycline. The solid lines are the result of the numerical simulation of a mathematical model of the NFL. e , Experimental implementation of the NFL without the N7 and N8 coiled-coils. f , Dose-response curve to doxycycline of the NFL without CCs (blue circles) and of the open loop control (green squares). n=4 biological replicates. A minimum of n=3 when one of the measurements was identified as an outlier (Grubbs’ test, alpha=0 . 2). Statistics analysis has been conducted through a two-way ANOVA test. **** P ≤ 0 . 0001 VPR-dCas9-N8 : nuclease-deficient Cas9 fused to the transactivation domain VPR and to synthetic coiled-coil N8; AcrIIA4-N7 : Anti-CRISPR protein fused to the synthetic coiled-coil N7; rtTA : reverse tetracycline TransActivator 3G; pTRE3G : Tetracycline Responsive Element promoter 3G .

Article Snippet: For the characterization of the NFL Motif, a Firefly Luciferase with two destabilization sequences has been used (Luc2CP, Promega), while for the biosensing experiments, a stable Firefly Luciferase (Luc2, Promega) has been used in order to compare it with the original biosensor.

Techniques: Construct, Transfection, Activity Assay, Expressing, Luciferase, Variant Assay, CRISPR

Journal: bioRxiv

Article Title: A general strategy to engineer high-performance mammalian Whole-Cell Biosensors

doi: 10.1101/2024.02.28.582526

Figure Lengend Snippet: a , The Negative Feedback Loop (NFL) copper biosensor. b , The Mutual Inhibition (MI) copper biosensor. c , The Coherent Inhibitory Loop (CIL) copper biosensor. d-f , Dose-response curve of the indicated biosensor against the basic biosensor B (red line) for increasing copper dichloride concentrations. n=9 biological replicates from three independent experiments. Firefly luciferase (FLuc) expression was evaluated by luminescence measurements normalised to Renilla firefly (RLuc) luminescence and reported either as Arbitrary Units [A.U.] (upper panel), or as Fold Activation obtained by dividing each data point by the average luminescence in the absence of copper (lower panel). g-i , Radar plot reporting the values of the five features of each biosensor against the basal biosensor (red line). Leak: leakiness, FC: fold change, S: sensitivity to input, OR: operation range, Lin: linearity. The score ranges from 0, which is assigned to the biosensor with the worst performance, to 1 for the system with the best performance. CasRx : Cas13 RNA endonuclease RfxCas13d; DR : direct repeat sequence cleaved by CasRx; pCMV/2B : modified CMV promoter with two sequences complementary to gRNA_B flanking tha TATA box .

Article Snippet: For the characterization of the NFL Motif, a Firefly Luciferase with two destabilization sequences has been used (Luc2CP, Promega), while for the biosensing experiments, a stable Firefly Luciferase (Luc2, Promega) has been used in order to compare it with the original biosensor.

Techniques: Inhibition, Luciferase, Expressing, Activation Assay, Sequencing, Modification

Journal: bioRxiv

Article Title: A general strategy to engineer high-performance mammalian Whole-Cell Biosensors

doi: 10.1101/2024.02.28.582526

Figure Lengend Snippet: a , The NFL-MI copper biosensor. b , The NFL-CIL version 1 copper biosensor. c , The NFL-CIL version 2 copper biosensor. d-f , Dose-response curve of the indicated biosensor against the basic biosensor B (red line) for increasing copper dichloride concentrations. n=9 biological replicates from three independent experiments. Firefly luciferase (FLuc) expression was evaluated by luminescence measurements normalised to Renilla firefly (RLuc) luminescence and reported either as Arbitrary Units [A.U.] (upper panel), or as Fold Activation obtained by dividing each data point by the average luminescence in the absence of copper (lower panel). g-i , Radar plot reporting the values of the five features of each biosensor against the basal biosensor (red line). Leak: leakiness, FC: fold change, S: sensitivity to input, OR: operation range, Lin: linearity. The score ranges from 0, which is assigned to the biosensor with the worst performance, to 1 for the system with the best performance.

Article Snippet: For the characterization of the NFL Motif, a Firefly Luciferase with two destabilization sequences has been used (Luc2CP, Promega), while for the biosensing experiments, a stable Firefly Luciferase (Luc2, Promega) has been used in order to compare it with the original biosensor.

Techniques: Luciferase, Expressing, Activation Assay