fli1  (Santa Cruz Biotechnology)

Journal: Communications Biology

Article Title: TNAP dephosphorylates phosphocholine and phosphoethanolamine and participates in triglyceride transport from the liver to the bloodstream

doi: 10.1038/s42003-025-08901-3

Figure Lengend Snippet: PC is sequentially hydrolyzed in the intestine by sPLA 2 -IB, PLB, and GDE5, or in the bloodstream by Lp-PLA 2 , lyso-PLA 1 , ENPP2, and/or ENPP6. TNAP could be the phosphatase dephosphorylating extracellular phosphocholine and phosphoethanolamine, allowing cellular choline and ethanolamine uptake. In hepatocytes, choline and ethanolamine generate PC and PEA via parallel metabolic pathways, and PEA can be methylated into PC by PEMT. PC is hydrolyzed into Lyso-PC and GPC in the endoplasmic reticulum by the sequential activity of PNPLA8 and PNPLA7, or by PLA 2 G15 in lysosomes. Choline serves in hepatocytes as a methyl donor via the production of SAM. BHMT betaine homocysteine methyltransferase, CDP-choline cytidine diphosphate choline, CTP cytidine triphosphate, DH dehydrogenase, ENPP ectonucleotide pyrophosphatase phosphodiesterase, GDE5 glycerophosphodiesterase 5, GPC glycerophosphocholine, Lp-PLA 2 lipoprotein-associated phospholipase A, PC phosphatidylcholine, PLB phospholipase B, PEA phosphatidylethanolamine, PEMT phosphatidylethanolamine N-methyltransferase, PLA 2 G15 phospholipase A, group XV, PNPLA patatin-like phospholipase domain containing, SAH S-Adenosyl-homocysteine, SAM S-Adenosyl-methionine, sPLA 2 -IB secreted phospholipase A IB.

Article Snippet: The sequence of the plasmid used to produce recombinant TNAP has been deposited at Addgene (ID 246682).

Techniques: Methylation, Activity Assay

Journal: Communications Biology

Article Title: TNAP dephosphorylates phosphocholine and phosphoethanolamine and participates in triglyceride transport from the liver to the bloodstream

doi: 10.1038/s42003-025-08901-3

Figure Lengend Snippet: A Michaelis-Menten representation, i.e., reaction rate (mM.h −1 ) of phosphocholine, phosphoethanolamine, and PP i hydrolysis by human TNAP, depending on the substrate concentration. Experimental data are presented, as well as the fit by a model with substrate inhibition, i.e., following the equation V 0 = V max [ S ]/([ S ]+ K m + [ S ] 2 / K i ), where V 0 is the initial reaction rate, V max the maximal reaction rate, and [ S ] the substrate concentration. All experiments were repeated at least three times. For the sake of clarity, individual data points are not presented in ( A ). However, source data are available in the . B Kinetic parameters ( K m , k cat , k cat / K m , and K i ) are given for phosphocholine, phosphoethanolamine, and PP i hydrolysis.

Article Snippet: The sequence of the plasmid used to produce recombinant TNAP has been deposited at Addgene (ID 246682).

Techniques: Concentration Assay, Inhibition

Journal: Communications Biology

Article Title: TNAP dephosphorylates phosphocholine and phosphoethanolamine and participates in triglyceride transport from the liver to the bloodstream

doi: 10.1038/s42003-025-08901-3

Figure Lengend Snippet: Substrate accommodation in TNAP’s active site. The best docking poses for phosphocholine and PP i obtained with the Apo enzyme are superimposed onto the TNAP structure containing P i : A Secondary structures and key residues that define the negatively charged zones (D109 and E452) and positively charged zones (R168 and R184). B The electrostatic surface is color-coded to depict the charge distribution, with red representing negatively charged regions (the groove) and blue representing positively charged regions (the pockets). C Two examples demonstrate the orientation of phosphoethanolamine within the active site: one positioned in the positively charged pocket and the other within the negatively charged groove. D Structure of the TNAPi MLS-0038949 (MLS) and global representation of MLS interaction with TNAP’s surface. E MLS forms P i –cation interactions between the para-di-methoxy-benzene group and Zn²⁺ (Zn2). This interaction is further stabilized by a hydrogen bond between His341 and the sulfonamide group of MLS. Additionally, cation– P i –stacking interactions are observed between Glu342, His338, and the quinolin-3-yl group. Finally, His338 contributes to the binding through a P i –stacking interaction with the quinolin-3-yl group of the MLS. F MLS positioning relative to the different substrates of TNAP. The structure in the presence of MLS was superimposed onto the structure containing P i , as well as the structure used for docking with phosphocholine and PP i . This comparison highlights the steric hindrance caused by the para-di-methoxy-benzene part of the inhibitor in relation to the phosphate group of the different substrates. G Superposition of the TNAP structure (in green) containing MLS with the AlphaFold model of IAP (in gray), illustrating the interaction specificity between TNAP and the MLS. The three residues interacting with MLS, which differ between TNAP and IAP, are indicated in the respective colors of the two structures.

Article Snippet: The sequence of the plasmid used to produce recombinant TNAP has been deposited at Addgene (ID 246682).

Techniques: Binding Assay, Comparison

Journal: Nature Communications

Article Title: EWS::FLI1 expression in human embryonic mesenchymal stem cells leads to transcriptional reprograming, defective DNA damage repair and Ewing sarcoma

doi: 10.1038/s41467-025-64475-y

Figure Lengend Snippet: A EWS::FLI1 and CD99 expression in heMSCs at 48 h after infection and in A673 cells. Actin, loading control. At the bottom, densitometric quantification of western blot signals normalized to Actin intensity. RT-qPCR data obtained from two independent experiments performed in triplicate are expressed as mean ± s.d. Statistics performed by two-tailed unpaired t -test. B Western blot detection of proteins of the p53-p21-RB1 axis in EF-heMSCs ( n = 2 independent experiments). Actin, loading control. C RT-qPCR to detect the expression of some of the induced and repressed oncogene targets in heMSC-1 cells at 48 h after EWS::FLI1 infection ( n = 2 independent experiments performed in triplicate). Data express mean ± s.d. Statistics performed by two-tailed unpaired t -test. D Gene-concept networks of the top 5 significantly enriched terms in EF-heMSCs vs . control heMSCs. Data was analyzed as indicated in Fig. . E RT-qPCR to determine the expression of 15 of the 30 top genes most potently induced by EWS::FLI1 in heMSC-1 cells at 48 h. Data from three independent experiments performed in triplicate are expressed as mean ± s.d. A two-tailed unpaired t -test was performed. F Expression values obtained from DepMap of the genes shown in ( E ) in Ewing sarcoma and other cancer cell lines (log2(TPM + 1)). A two-tailed Mann–Whitney U test was performed. G GSEA of EF-heMSCs transcriptomes in EWS::FLI1 signatures (EWS::FLI1 expression in UET-13 mesenchymal progenitors , rhabdomyosarcoma RD cells , and hMSCs ). H GSEA of EF-heMSCs transcriptomes in Ewing sarcoma signatures , . I , J Heatmap and PCA representation of the unsupervised clustering analysis of gene expression signatures in heSCs, hMSCs, and Ewing sarcoma samples. K GSEA of EF-heMSCs transcriptomes in the 400 most expressed and under-represented genes identified by the unsupervised clustering analysis of Ewing sarcomas. In ( G , H , K ), a two-sided test was performed with GSEA based on limma-derived statistics (−log( p value) × signFC). Running score plots show the cumulative enrichment of the gene set across the ranked gene list. The peak of the curve indicates the maximum enrichment score (ES). P values are adjusted for multiple testing using the Benjamini–Hochberg (FDR < 0.05).

Article Snippet: Antibodies used for these techniques were: FLI1, p53, p21, ATM, hHLA, PRKCB (Santa Cruz); CD99, hKu80(Cell Signaling); β-actin (Abcam); RB1 (BD Biosciences); human nucleus, BRCA1, pS1981ATM(Millipore); pS1423-BRCA1, ATR, pT1989-ATR, DNA-PKc, pS2056-DNA-PKcs (Abclonal); Flag (Merck Life Science); BCL11B (Biolegend) and ITM2A (Fisher Scientific).

Techniques: Expressing, Infection, Control, Western Blot, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Gene Expression, Derivative Assay

Journal: Nature Communications

Article Title: EWS::FLI1 expression in human embryonic mesenchymal stem cells leads to transcriptional reprograming, defective DNA damage repair and Ewing sarcoma

doi: 10.1038/s41467-025-64475-y

Figure Lengend Snippet: A Left, genomic annotation of oncogene-bound regions in heMSC-1 cells 48 h after infection with a Flag-tagged EWS::FLI1, identified by ChIP-seq performed with a Flag antibody. Right, overlapping of peaks. Peak calling using the input as control was performed with MACS2 . B Chromatin states associated with EWS::FLI1-bound peaks in heMSC-1 cells, performed with MACS2 tools using five core histone modification marks : H3K27me3 (Polycomb repression, ReprPC); H3K9me3 (heterochromatin regions, Het); H3K4me1 (enhancer regions, Enh); H3K4me3 (promoter regions, TssA); and H3K36me3 (transcribed regions, Tx). Statistical significance of the relative frequency of EWS::FLI1 peaks in each chromatin state was assessed using a two-sided Fisher’s exact test. Numbers in the bars indicate P values, odds ratios, and confidence intervals. Quiet (Quiescent/Low) chromatin state was excluded from this graph to better visualize the data. C Percentage of EWS::FLI1-binding sites upstream and downstream from the transcriptional start sites (TSS) of the nearest genes. D Identification of EWS::FLI1-binding motifs by MEME tools . E Overlap of genes associated with EWS::FLI1-binding peaks in heMSC and A673 cells. Bottom panel, annotations of EWS::FLI1 peaks in heMSCs corresponding to oncogene-bound genes. F Genome browser tracks depicting EWS::FLI1 binding to the PRKCB locus in heMSCs (top) and A673 cells (bottom) . In blue, heMSC-1 cells infected with EWS::FLI1; in gray, heMSC-1 cells infected with control supernatants. Scale, 0-23. Bottom left panel, validation of EWS::FLI1 binding to intron 7 of PRKCB in heMSCs, detected by ChIP-qPCR in EF-heMSC-1 cells. Values referred to the percentage of input and were normalized with respect to the control condition. ACAT1 , negative control. Bottom right panel, PRKCB induction is abolished after EWS::FLI1 knockdown. Data from two independent experiments performed in triplicate are expressed as mean ± s.d. Statistics performed by two-tailed unpaired t -test. G Sankey plots showing annotations of peaks corresponding to genes bound by EWS::FLI1 in both heMSC and A673 cells. In each of the plots, the transitions from distal intergenic (on the left), first intron (in the middle), and other introns (on the right) of the oncogene peaks in heMSCs to the peaks in A673 cells have been highlighted.

Article Snippet: Antibodies used for these techniques were: FLI1, p53, p21, ATM, hHLA, PRKCB (Santa Cruz); CD99, hKu80(Cell Signaling); β-actin (Abcam); RB1 (BD Biosciences); human nucleus, BRCA1, pS1981ATM(Millipore); pS1423-BRCA1, ATR, pT1989-ATR, DNA-PKc, pS2056-DNA-PKcs (Abclonal); Flag (Merck Life Science); BCL11B (Biolegend) and ITM2A (Fisher Scientific).

Techniques: Infection, ChIP-sequencing, Control, Modification, Binding Assay, Biomarker Discovery, ChIP-qPCR, Negative Control, Knockdown, Two Tailed Test

Journal: Nature Communications

Article Title: EWS::FLI1 expression in human embryonic mesenchymal stem cells leads to transcriptional reprograming, defective DNA damage repair and Ewing sarcoma

doi: 10.1038/s41467-025-64475-y

Figure Lengend Snippet: A Top panel, genome browser screenshot illustrating EWS::FLI1 binding to the BRCA1 locus in control and EF-heMSC cells. The scale of the tracks is the same size for the control and the EF. Lower panel, chromatin immunoprecipitation of BRCA1 exons 11 and 15 by EWS::FLI1 in heMSC-1 cells infected with EWS::FLI1. Values were referred to the percentage of input and normalized with respect to the control condition. Data correspond to two independent experiments performed in duplicate and are expressed as mean ± s.d. ACAT1, negative control. A two-tailed unpaired t -test was performed. B BRCA1 expression in heMSC-1 cells infected with EWS::FLI1, detected by RT-qPCR and Western blot. Data were obtained from three independent experiments performed in triplicate and expressed as mean ± s.d. A two-tailed unpaired t -test was performed. C BRCA1 induction is abolished after EWS::FLI1 knockdown. Data obtained from two independent experiments performed in triplicate are expressed as mean ± s.d. A two-tailed unpaired t -test was performed. D Representative images of the alkaline comet assay performed with control and EF-heMSC-1 cells. Magnification bar: 50 μm. Below, Box-Whisker plot representation of the quantification of the product of the tail length and the fraction of total DNA in the tail (Olive tail moment) in control and EF-heMSC cells. Box-Whisker plot represents: center line = median; box = 25th–75th percentiles; the lower whisker corresponds to the minimum and the upper whisker to 1,5(75th percentile). Outliers are plotted as individual points. The difference between groups was analyzed by using a multiple regression model and a log( x + 0.1) transformation. E Western blot analysis to detect the expression and phosphorylation status of BRCA1 and kinases involved in DNA damage repair in control and EF-heMSC cells under basal conditions and after treatment with 5 µM etoposide. At the bottom, densitometric quantification of Western blot signals, normalized to Actin intensity ( n = 2 independent experiments). F Dose-response curves and IC50 values for etoposide in control heMSC-1 and EF-heMSC-1 cells. Representative values of three independent experiments are expressed as mean ± s.d. A two-tailed unpaired t -test was performed.

Article Snippet: Antibodies used for these techniques were: FLI1, p53, p21, ATM, hHLA, PRKCB (Santa Cruz); CD99, hKu80(Cell Signaling); β-actin (Abcam); RB1 (BD Biosciences); human nucleus, BRCA1, pS1981ATM(Millipore); pS1423-BRCA1, ATR, pT1989-ATR, DNA-PKc, pS2056-DNA-PKcs (Abclonal); Flag (Merck Life Science); BCL11B (Biolegend) and ITM2A (Fisher Scientific).

Techniques: Binding Assay, Control, Chromatin Immunoprecipitation, Infection, Negative Control, Two Tailed Test, Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Alkaline Single Cell Gel Electrophoresis, Whisker Assay, Transformation Assay, Phospho-proteomics

Journal: Nature Communications

Article Title: EWS::FLI1 expression in human embryonic mesenchymal stem cells leads to transcriptional reprograming, defective DNA damage repair and Ewing sarcoma

doi: 10.1038/s41467-025-64475-y

Figure Lengend Snippet: A H&E staining of an abdominal mass and a lung metastasis, and identification of cell clusters based on their corresponding UMAPs. Magnification bars: 400 μm. B Expression maps of some of the most relevant genes that determine clusterization in the above UMAPs. C Expression levels in Ewing sarcoma cell lines and in other tumor cell lines, extracted from the DepMap portal ( https://depmap.org/ ), of the most relevant genes identified by computation of cell clusters from UMAP dimensional reduction. Legends in bold highlight genes directly bound by EWS::FLI1 in A673 cells in promoters or enhancers . A two-tailed Mann–Whitney U test was performed.

Article Snippet: Antibodies used for these techniques were: FLI1, p53, p21, ATM, hHLA, PRKCB (Santa Cruz); CD99, hKu80(Cell Signaling); β-actin (Abcam); RB1 (BD Biosciences); human nucleus, BRCA1, pS1981ATM(Millipore); pS1423-BRCA1, ATR, pT1989-ATR, DNA-PKc, pS2056-DNA-PKcs (Abclonal); Flag (Merck Life Science); BCL11B (Biolegend) and ITM2A (Fisher Scientific).

Techniques: Staining, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: EWS::FLI1 expression in human embryonic mesenchymal stem cells leads to transcriptional reprograming, defective DNA damage repair and Ewing sarcoma

doi: 10.1038/s41467-025-64475-y

Figure Lengend Snippet: A Expression maps of some of the genes differentially expressed in Ewing sarcomas (Supplementary Data ) (adjusted p value < 0.05, log2(FC), top 100). B IHC of the ES distinctive markers BCL11B and ITM2A in EF-heMSC-derived tumors. Thymus, spleen, and liver sections correspond to normal tissues. Magnification bar, 50 μm. C Expression levels of differentially expressed genes in Ewing sarcoma cell lines and in other tumor cell lines, extracted from the DepMap portal ( https://depmap.org/ ). Legends in bold highlight genes directly bound by EWS::FLI1 in A673 cells in promoters or enhancers . A two-tailed Mann–Whitney U test was performed. D Spatial images of the gene set activity scores calculated using the singscore R package , , which implements a rank-based single-sample scoring method. Scores were computed using unidirectional gene signatures with known direction (knownDirection = TRUE). The resulting scores are directly interpretable as a normalized mean percentile rank. As a reference gene set, the top 400 (for abdominal mass) or 200 (for pulmonary metastasis) differentially expressed genes in Ewing sarcoma (Supplementary Data ) were ordered by the log2(FC) (adjusted p value < 0.5).

Article Snippet: Antibodies used for these techniques were: FLI1, p53, p21, ATM, hHLA, PRKCB (Santa Cruz); CD99, hKu80(Cell Signaling); β-actin (Abcam); RB1 (BD Biosciences); human nucleus, BRCA1, pS1981ATM(Millipore); pS1423-BRCA1, ATR, pT1989-ATR, DNA-PKc, pS2056-DNA-PKcs (Abclonal); Flag (Merck Life Science); BCL11B (Biolegend) and ITM2A (Fisher Scientific).

Techniques: Expressing, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Activity Assay

Journal: Current Protocols

Article Title: Directed Evolution in Escherichia coli for Novel Ligand‐Binding Regulators: Evolving a Progesterone‐Responsive Transcription Factor to Bind Cortisol

doi: 10.1002/cpz1.70218

Figure Lengend Snippet: Overview of the protocols described in this workflow for enhancing the ligand response of an aTF. ( A ) In Basic Protocol , the aTF library is assembled. This is done by selecting the residues that will be randomized, designing primers for mutagenesis, and then amplifying the aTF gene to introduce mutations before reassembling the DNA into a plasmid. ( B ) Basic Protocol outlines the steps to transform DH10B cells that are expressing the reporter plasmid (pSELIS) with the mutagenized regulator plasmid from Basic Protocol (pREG library). Library diversity is determined, and glycerol stocks of the libraries are saved. ( C ) Basic Protocol describes the functional assay of the libraries. This is achieved by selectively enriching aTF variants that bind to the operator by the addition of an antibiotic (zeocin) to the growth medium. Post selection, libraries are plated on LB plates with the target ligand, and the most highly fluorescent colonies are picked for the microplate assay. The fold change in induction is determined, and the best‐performing variants are cultured for further analysis. ( D ) Basic Protocol outlines the procedure for genotyping the variants, subcloning them into new expression vectors, and assaying with the target ligand and cross‐reactive ligands in a dose‐response assay.

Article Snippet: Glycerol stock of E. coli DH10B cells containing pSELIS (transcription factor reporter plasmid containing SRTF1‐responsive promoter, Addgene, cat. no. ID 246681) LB agar plates with (see recipe): 20 μg/ml Cam 20 μg/ml Cam and 100 μg/ml Carb LB broth with (see recipe): 20 μg/ml Cam 20 μg/ml Cam and 100 μg/ml Carb Superior broth with 20 μg/ml Cam, sterile (see recipe) 10% glycerol, sterile, ice‐cold (see recipe) Liquid nitrogen DNA from Basic Protocol SOC outgrowth medium (New England Biolabs, cat. no. B9020S) 50% glycerol, sterile (see recipe) Qiagen Miniprep kit (Qiagen, cat. no. 27104) Inoculation loop, 10‐μl, 200‐mm, PS (Grenier Bio‐One, cat. no. 731170) 37°C incubator shaker (VWR, New Brunswick Innova 44/44R, cat. no. 75874‐524, or equivalent) 14‐ml culture tubes (Falcon, cat. no. 352057) 500‐ml Erlenmeyer baffled cell culture flask, sterile Cell density meter (WPA, CO8000) Cuvette (FisherBrand, cat. no. 14955127) Benchtop centrifuge, 4°C (Beckman Coulter, cat. no. B06314 , or equivalent) Ice bucket and ice 50‐ml serological pipettes, sterile (Avantar, cat. no. GILSF110131) 20‐, 200‐, and 1000‐μl single‐channel pipettes (Rainin, or equivalent) 20‐, 200‐, and 1000‐μl pipette tips, filtered, sterile, (Rainin, or equivalent) 1.5‐ml microtubes (Axygen, cat. no. MCT‐150‐C‐S) Gene Pulser electroporation cuvettes (Bio‐Rad, cat. no. 165‐2086) Electroporator (Bio‐Rad, cat. no. 1652101) Plating beads, 4.5‐mm (Zymo Research, cat. no. S1001), sterile AeraSeal film (Excel Scientific, cat. no. BS‐25) Cryogenic tubes (National Scientific Supply, cat. no. BC20NA‐PS), sterile Tabletop centrifuge (Eppendorf, cat. no. 5415R, or equivalent)

Techniques: Mutagenesis, Introduce, Plasmid Preparation, Expressing, Functional Assay, Selection, Cell Culture, Subcloning

Journal: Current Protocols

Article Title: Directed Evolution in Escherichia coli for Novel Ligand‐Binding Regulators: Evolving a Progesterone‐Responsive Transcription Factor to Bind Cortisol

doi: 10.1002/cpz1.70218

Figure Lengend Snippet: Graphical representation of Basic Protocol . This protocol is separated into two parts: (1) competent cell preparation and (2) transformations. In part one, a colony of DH10B containing pSELIS is cultured overnight and then subcultured into fresh superior broth and grown to mid‐log phase before being chilled, washed with ice‐cold glycerol, and concentrated to generate electrocompetent cells. In part two, the electrocompetent cells are transformed with the pREG library from Basic Protocol . Library size is determined, and sequencing QC is performed before saving glycerol stocks of the library.

Article Snippet: Glycerol stock of E. coli DH10B cells containing pSELIS (transcription factor reporter plasmid containing SRTF1‐responsive promoter, Addgene, cat. no. ID 246681) LB agar plates with (see recipe): 20 μg/ml Cam 20 μg/ml Cam and 100 μg/ml Carb LB broth with (see recipe): 20 μg/ml Cam 20 μg/ml Cam and 100 μg/ml Carb Superior broth with 20 μg/ml Cam, sterile (see recipe) 10% glycerol, sterile, ice‐cold (see recipe) Liquid nitrogen DNA from Basic Protocol SOC outgrowth medium (New England Biolabs, cat. no. B9020S) 50% glycerol, sterile (see recipe) Qiagen Miniprep kit (Qiagen, cat. no. 27104) Inoculation loop, 10‐μl, 200‐mm, PS (Grenier Bio‐One, cat. no. 731170) 37°C incubator shaker (VWR, New Brunswick Innova 44/44R, cat. no. 75874‐524, or equivalent) 14‐ml culture tubes (Falcon, cat. no. 352057) 500‐ml Erlenmeyer baffled cell culture flask, sterile Cell density meter (WPA, CO8000) Cuvette (FisherBrand, cat. no. 14955127) Benchtop centrifuge, 4°C (Beckman Coulter, cat. no. B06314 , or equivalent) Ice bucket and ice 50‐ml serological pipettes, sterile (Avantar, cat. no. GILSF110131) 20‐, 200‐, and 1000‐μl single‐channel pipettes (Rainin, or equivalent) 20‐, 200‐, and 1000‐μl pipette tips, filtered, sterile, (Rainin, or equivalent) 1.5‐ml microtubes (Axygen, cat. no. MCT‐150‐C‐S) Gene Pulser electroporation cuvettes (Bio‐Rad, cat. no. 165‐2086) Electroporator (Bio‐Rad, cat. no. 1652101) Plating beads, 4.5‐mm (Zymo Research, cat. no. S1001), sterile AeraSeal film (Excel Scientific, cat. no. BS‐25) Cryogenic tubes (National Scientific Supply, cat. no. BC20NA‐PS), sterile Tabletop centrifuge (Eppendorf, cat. no. 5415R, or equivalent)

Techniques: Cell Culture, Transformation Assay, Sequencing

Journal: Current Protocols

Article Title: Directed Evolution in Escherichia coli for Novel Ligand‐Binding Regulators: Evolving a Progesterone‐Responsive Transcription Factor to Bind Cortisol

doi: 10.1002/cpz1.70218

Figure Lengend Snippet: Genetic constructs used in this study. ( A ) pREG expresses the aTF from a medium‐strength promoter. ( B ) pSELIS contains an aTF‐responsive promoter upstream of both the λ cI repressor (for negative selection) and sfGFP (for screening). ( C ) P aTF construct used in this study. The SRTF1 operator sequence is placed downstream of a synthetic promoter.

Article Snippet: Glycerol stock of E. coli DH10B cells containing pSELIS (transcription factor reporter plasmid containing SRTF1‐responsive promoter, Addgene, cat. no. ID 246681) LB agar plates with (see recipe): 20 μg/ml Cam 20 μg/ml Cam and 100 μg/ml Carb LB broth with (see recipe): 20 μg/ml Cam 20 μg/ml Cam and 100 μg/ml Carb Superior broth with 20 μg/ml Cam, sterile (see recipe) 10% glycerol, sterile, ice‐cold (see recipe) Liquid nitrogen DNA from Basic Protocol SOC outgrowth medium (New England Biolabs, cat. no. B9020S) 50% glycerol, sterile (see recipe) Qiagen Miniprep kit (Qiagen, cat. no. 27104) Inoculation loop, 10‐μl, 200‐mm, PS (Grenier Bio‐One, cat. no. 731170) 37°C incubator shaker (VWR, New Brunswick Innova 44/44R, cat. no. 75874‐524, or equivalent) 14‐ml culture tubes (Falcon, cat. no. 352057) 500‐ml Erlenmeyer baffled cell culture flask, sterile Cell density meter (WPA, CO8000) Cuvette (FisherBrand, cat. no. 14955127) Benchtop centrifuge, 4°C (Beckman Coulter, cat. no. B06314 , or equivalent) Ice bucket and ice 50‐ml serological pipettes, sterile (Avantar, cat. no. GILSF110131) 20‐, 200‐, and 1000‐μl single‐channel pipettes (Rainin, or equivalent) 20‐, 200‐, and 1000‐μl pipette tips, filtered, sterile, (Rainin, or equivalent) 1.5‐ml microtubes (Axygen, cat. no. MCT‐150‐C‐S) Gene Pulser electroporation cuvettes (Bio‐Rad, cat. no. 165‐2086) Electroporator (Bio‐Rad, cat. no. 1652101) Plating beads, 4.5‐mm (Zymo Research, cat. no. S1001), sterile AeraSeal film (Excel Scientific, cat. no. BS‐25) Cryogenic tubes (National Scientific Supply, cat. no. BC20NA‐PS), sterile Tabletop centrifuge (Eppendorf, cat. no. 5415R, or equivalent)

Techniques: Construct, Selection, Sequencing