Journal: Nature protocols

Article Title: Compartmentalized partnered replication for the directed evolution of genetic parts and circuits

doi: 10.1038/nprot.2017.119

Figure Lengend Snippet: Anticipated results, (a-f) Appearance of mixtures in the process of emulsification/de-emulsification, (a) A 2 ml-tube containing cells suspended in the ePCR buffer, oil mixture, and the rubber stopper from a 1-ml syringe before agitation on TissueLyser. The mixture appears clear and is separated into two phases, (b) Same mixture as in a after agitation on TissueLyser. The emulsion is viscous, and appears homogeneous and of milky white color, (c) The appearance of the mixture after being aliquoted in 12 × 100-μl PCR samples, subjected to thermal cycling, and pooled together in a 1.5 ml tube, (d) The appearance of the mixture in c after 10-min centrifugation. The mixture separated into two visible layers, with a top cloudy oil phase and a bottom remaining emulsion layer. The top oil phase is to be discarded. The remaining bottom emulsion layer appears as an amorphous white solid, (e) The appearance of a broken emulsion after phenol/chloroform/isoamyl alcohol addition and vortexing. The mixture is still cloudy but exhibits a greatly reduced viscosity. The bottom amorphous solid-like layer is no longer present, (f) The same mixture as in e after 2-min centrifugation. The mixture separated into two clear phases: the top aqueous phase (to be transferred to a new tube) and the bottom organic phase (to be discarded), (g) Example of a gradient of emulsion stability that can be generated under different emulsification conditions. After 30 rounds of PCR thermal cycles, the emulsions were visually analyzed for stability. A gradient of emulsion stabilities is observed, in which unstable emulsions separated into two phases (left), while stable emulsions remained opaque, with minimal phase separation (right). Green squares, intact emulsion; red squares, oil phase separated from disrupted emulsion droplets, (h) Phase-contrast microscopy image of 50x-diluted emulsion. Scale bar, 10 μlη. (i) Superimposed GFP fluorescence/phase-contrast microscopy image of emulsified GFP-expressing DH10B(DE3) E. coli cells under 40× magnification. Scale bar, 10 μlη. (j,k) Example of mock selection data. E. coli expressing either wild-type tyrosil-tRNA synthetase from Methanocatdcococcus jannaschii (MjYRS) or its nonfunctional variant (containing a stop codon and a Notl restriction site) were mixed at the indicated ratios and subjected to a single round of ePCR. (j) After the mock selection samples were amplified by re-amp PCR and equal amounts of DNA were restriction-digested by Notl, the DNA fragments were analyzed by gel electrophoresis to distinguish active (uncut) from inactive (cut) variants of MjYRS. Several thousandfold enrichment of active enzyme variant is observed. Star, active variant fragment size; arrows, inactive variant fragment sizes. Adapted with permission from ref. 29, American Chemical Society, (k) Gel-electrophoresis image of recovery PCR. 1: pure active MjYRS amplicon; 0: pure inactive MjYRS amplicon; 10_1-10−4: amplicons of activeiinactive MjYRS dilutions. (1) Monitoring enrichment progress by GFP assay. BL21 E. coli cells carrying pACYC-GFPmut2 plasmid (in which PT7 drives GFP expression) and plasmid ligations from the initial T7 RNAP selection rounds were assayed in a microplate reader for GFP fluorescence. XX, negative control T7 RNAP with two premature stop codons; WT, parental T7-RSS plasmid reported; R0, naive library; R1–R12, the output for each subsequent round during the selections for use of PT7; CGG-R7–8, a single clone from round 7 (this mutant was subject to error-prone PCR, yielding CGG-R7 epPCR); CGG-R12-KI, a single clone from R12; other CGG-R12 variants are selected combinations of mutations seen in the round 12 population. Data represent averages of three independently grown samples. Error bars represent 1 s.d. Adapted with permission from ref. 28, Nature Publishing Group.

Article Snippet: General equipment Water bath (Fisher, cat. no. FSGPD02) Incubator/shaker (New Brunswick, model no. Innova 44) Microcentrifuge (Eppendorf, model no. 5418) Vortex mixer (Scientific Industries, model no. SI-0236) Thermocycler (Bio-Rad, model no. T100) Microwave (LG, model no. LCS1112ST) 2-ml Microtubes (Eppendorf, cat. no. 022431048) 1.5-ml Microtubes (Eppendorf, cat. no. 022431021) PCR strip tubes (Genesee Scientific, cat. no. 27–125) GFP assay 96-Well black microplates (Corning, cat. no. CLS3915) 96-Well clear microplates (Corning, cat. no. 3370) Microplate reader (Tecan, M200 PRO) Library Transformation and Expression Electroporation cuvettes (0.2 cm gap; Bio-Rad, cat. no. 1652086) Electroporation apparatus (Bio-Rad, cat. no. 1652662) Petri dishes (Thermo Scientific, cat. no. 249964) Rattler plating beads (Zymo Research, cat. no. SI001) Spectrophotometer (Biochrom WPA, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"C08000","term_id":"1533071","term_text":"C08000"}} C08000 ) Falcon 50-rnl conical centrifuge tubes (Corning, cat. no. 352070) AirPore Tape Sheets (Qiagen, cat. no. 19571) Round-bottom polystyrene tubes (Corning, cat. no. 14–959–IB) Emulsion PCR Spectrophotometer (Biochrom WPA, model no. {"type":"entrez-nucleotide","attrs":{"text":"C08000","term_id":"1533071","term_text":"C08000"}} C08000 ) 1 ml Syringes (Covidien-Medtronic, cat. no. 1180100555) TissueLyser LT (Qiagen, model no. 69980) Microman pipette for viscous liquids (Gilson, cat. no. F148504) Capillary pistons (Gilson, cat. no. CP 100) Inverted Epi-Fluorescence Phase Contrast Microscope (Olympus, model no. 1X51) FITC/GFP filter cube (Chroma, cat. no. 41001) Cellometer cell counting chambers (Nexcelom, cat. no. SD100) High-performance near-infrared charge-coupled device (CCD) camera, IEEE 1394 FireWire (Qlmaging, model RoleraXR) Microscope camera calibration slide (0.01-mm stage micrometer; OMAX, cat. no. CS-A36CALM1) ImageJ ( https://imagej.nih.gov ) Recovery PCR Gel electrophoresis equipment (Bio-Rad, cat. nos.

Techniques: Emulsification, Emulsion, Centrifugation, Viscosity, Generated, Microscopy, Fluorescence, Expressing, Selection, Variant Assay, Amplification, Nucleic Acid Electrophoresis, Plasmid Preparation, Negative Control, Mutagenesis

Journal: Nature protocols

Article Title: Compartmentalized partnered replication for the directed evolution of genetic parts and circuits

doi: 10.1038/nprot.2017.119

Figure Lengend Snippet: Anticipated results, (a-f) Appearance of mixtures in the process of emulsification/de-emulsification, (a) A 2 ml-tube containing cells suspended in the ePCR buffer, oil mixture, and the rubber stopper from a 1-ml syringe before agitation on TissueLyser. The mixture appears clear and is separated into two phases, (b) Same mixture as in a after agitation on TissueLyser. The emulsion is viscous, and appears homogeneous and of milky white color, (c) The appearance of the mixture after being aliquoted in 12 × 100-μl PCR samples, subjected to thermal cycling, and pooled together in a 1.5 ml tube, (d) The appearance of the mixture in c after 10-min centrifugation. The mixture separated into two visible layers, with a top cloudy oil phase and a bottom remaining emulsion layer. The top oil phase is to be discarded. The remaining bottom emulsion layer appears as an amorphous white solid, (e) The appearance of a broken emulsion after phenol/chloroform/isoamyl alcohol addition and vortexing. The mixture is still cloudy but exhibits a greatly reduced viscosity. The bottom amorphous solid-like layer is no longer present, (f) The same mixture as in e after 2-min centrifugation. The mixture separated into two clear phases: the top aqueous phase (to be transferred to a new tube) and the bottom organic phase (to be discarded), (g) Example of a gradient of emulsion stability that can be generated under different emulsification conditions. After 30 rounds of PCR thermal cycles, the emulsions were visually analyzed for stability. A gradient of emulsion stabilities is observed, in which unstable emulsions separated into two phases (left), while stable emulsions remained opaque, with minimal phase separation (right). Green squares, intact emulsion; red squares, oil phase separated from disrupted emulsion droplets, (h) Phase-contrast microscopy image of 50x-diluted emulsion. Scale bar, 10 μlη. (i) Superimposed GFP fluorescence/phase-contrast microscopy image of emulsified GFP-expressing DH10B(DE3) E. coli cells under 40× magnification. Scale bar, 10 μlη. (j,k) Example of mock selection data. E. coli expressing either wild-type tyrosil-tRNA synthetase from Methanocatdcococcus jannaschii (MjYRS) or its nonfunctional variant (containing a stop codon and a Notl restriction site) were mixed at the indicated ratios and subjected to a single round of ePCR. (j) After the mock selection samples were amplified by re-amp PCR and equal amounts of DNA were restriction-digested by Notl, the DNA fragments were analyzed by gel electrophoresis to distinguish active (uncut) from inactive (cut) variants of MjYRS. Several thousandfold enrichment of active enzyme variant is observed. Star, active variant fragment size; arrows, inactive variant fragment sizes. Adapted with permission from ref. 29, American Chemical Society, (k) Gel-electrophoresis image of recovery PCR. 1: pure active MjYRS amplicon; 0: pure inactive MjYRS amplicon; 10_1-10−4: amplicons of activeiinactive MjYRS dilutions. (1) Monitoring enrichment progress by GFP assay. BL21 E. coli cells carrying pACYC-GFPmut2 plasmid (in which PT7 drives GFP expression) and plasmid ligations from the initial T7 RNAP selection rounds were assayed in a microplate reader for GFP fluorescence. XX, negative control T7 RNAP with two premature stop codons; WT, parental T7-RSS plasmid reported; R0, naive library; R1–R12, the output for each subsequent round during the selections for use of PT7; CGG-R7–8, a single clone from round 7 (this mutant was subject to error-prone PCR, yielding CGG-R7 epPCR); CGG-R12-KI, a single clone from R12; other CGG-R12 variants are selected combinations of mutations seen in the round 12 population. Data represent averages of three independently grown samples. Error bars represent 1 s.d. Adapted with permission from ref. 28, Nature Publishing Group.

Article Snippet: General equipment Water bath (Fisher, cat. no. FSGPD02) Incubator/shaker (New Brunswick, model no. Innova 44) Microcentrifuge (Eppendorf, model no. 5418) Vortex mixer (Scientific Industries, model no. SI-0236) Thermocycler (Bio-Rad, model no. T100) Microwave (LG, model no. LCS1112ST) 2-ml Microtubes (Eppendorf, cat. no. 022431048) 1.5-ml Microtubes (Eppendorf, cat. no. 022431021) PCR strip tubes (Genesee Scientific, cat. no. 27–125) GFP assay 96-Well black microplates (Corning, cat. no. CLS3915) 96-Well clear microplates (Corning, cat. no. 3370) Microplate reader (Tecan, M200 PRO) Library Transformation and Expression Electroporation cuvettes (0.2 cm gap; Bio-Rad, cat. no. 1652086) Electroporation apparatus (Bio-Rad, cat. no. 1652662) Petri dishes (Thermo Scientific, cat. no. 249964) Rattler plating beads (Zymo Research, cat. no. SI001) Spectrophotometer (Biochrom WPA, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"C08000","term_id":"1533071","term_text":"C08000"}} C08000 ) Falcon 50-rnl conical centrifuge tubes (Corning, cat. no. 352070) AirPore Tape Sheets (Qiagen, cat. no. 19571) Round-bottom polystyrene tubes (Corning, cat. no. 14–959–IB) Emulsion PCR Spectrophotometer (Biochrom WPA, model no. {"type":"entrez-nucleotide","attrs":{"text":"C08000","term_id":"1533071","term_text":"C08000"}} C08000 ) 1 ml Syringes (Covidien-Medtronic, cat. no. 1180100555) TissueLyser LT (Qiagen, model no. 69980) Microman pipette for viscous liquids (Gilson, cat. no. F148504) Capillary pistons (Gilson, cat. no. CP 100) Inverted Epi-Fluorescence Phase Contrast Microscope (Olympus, model no. 1X51) FITC/GFP filter cube (Chroma, cat. no. 41001) Cellometer cell counting chambers (Nexcelom, cat. no. SD100) High-performance near-infrared charge-coupled device (CCD) camera, IEEE 1394 FireWire (Qlmaging, model RoleraXR) Microscope camera calibration slide (0.01-mm stage micrometer; OMAX, cat. no. CS-A36CALM1) ImageJ ( https://imagej.nih.gov ) Recovery PCR Gel electrophoresis equipment (Bio-Rad, cat. nos.

Techniques: Emulsification, Emulsion, Centrifugation, Viscosity, Generated, Microscopy, Fluorescence, Expressing, Selection, Variant Assay, Amplification, Nucleic Acid Electrophoresis, Plasmid Preparation, Negative Control, Mutagenesis

Journal: Nature protocols

Article Title: Compartmentalized partnered replication for the directed evolution of genetic parts and circuits

doi: 10.1038/nprot.2017.119

Figure Lengend Snippet: Troubleshooting table.

Article Snippet: General equipment Water bath (Fisher, cat. no. FSGPD02) Incubator/shaker (New Brunswick, model no. Innova 44) Microcentrifuge (Eppendorf, model no. 5418) Vortex mixer (Scientific Industries, model no. SI-0236) Thermocycler (Bio-Rad, model no. T100) Microwave (LG, model no. LCS1112ST) 2-ml Microtubes (Eppendorf, cat. no. 022431048) 1.5-ml Microtubes (Eppendorf, cat. no. 022431021) PCR strip tubes (Genesee Scientific, cat. no. 27–125) GFP assay 96-Well black microplates (Corning, cat. no. CLS3915) 96-Well clear microplates (Corning, cat. no. 3370) Microplate reader (Tecan, M200 PRO) Library Transformation and Expression Electroporation cuvettes (0.2 cm gap; Bio-Rad, cat. no. 1652086) Electroporation apparatus (Bio-Rad, cat. no. 1652662) Petri dishes (Thermo Scientific, cat. no. 249964) Rattler plating beads (Zymo Research, cat. no. SI001) Spectrophotometer (Biochrom WPA, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"C08000","term_id":"1533071","term_text":"C08000"}} C08000 ) Falcon 50-rnl conical centrifuge tubes (Corning, cat. no. 352070) AirPore Tape Sheets (Qiagen, cat. no. 19571) Round-bottom polystyrene tubes (Corning, cat. no. 14–959–IB) Emulsion PCR Spectrophotometer (Biochrom WPA, model no. {"type":"entrez-nucleotide","attrs":{"text":"C08000","term_id":"1533071","term_text":"C08000"}} C08000 ) 1 ml Syringes (Covidien-Medtronic, cat. no. 1180100555) TissueLyser LT (Qiagen, model no. 69980) Microman pipette for viscous liquids (Gilson, cat. no. F148504) Capillary pistons (Gilson, cat. no. CP 100) Inverted Epi-Fluorescence Phase Contrast Microscope (Olympus, model no. 1X51) FITC/GFP filter cube (Chroma, cat. no. 41001) Cellometer cell counting chambers (Nexcelom, cat. no. SD100) High-performance near-infrared charge-coupled device (CCD) camera, IEEE 1394 FireWire (Qlmaging, model RoleraXR) Microscope camera calibration slide (0.01-mm stage micrometer; OMAX, cat. no. CS-A36CALM1) ImageJ ( https://imagej.nih.gov ) Recovery PCR Gel electrophoresis equipment (Bio-Rad, cat. nos.

Techniques: Growth Assay, Concentration Assay, Positive Control, Western Blot, Sequencing, Expressing, Amplification, Emulsion, Plasmid Preparation, Selection, Variant Assay, Emulsification, Functional Assay