pselis  (Addgene inc)

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