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: General CPR concept, (a) Schematic of CPR principle. A gene circuit is generated in which a partner-gene activity allows the expression of a DNA polymerase in bacterial cells. Inactive gene variants lead to no expression of the DNA polymerase. The genetic circuit containing the diversified partner-gene pool is expressed in vivo, allowing DNA polymerase production only in cells with active partner-gene variants. The live cells are subsequently emulsified to produce no more than a single cell per emulsion droplet. The initial boiling step of the ePCR lyses the cells, releasing the produced DNA polymerase protein as well as the partner-gene-encoding plasmid into the aqueous solution of the emulsion droplet. Ensuing thermal cycling amplifies only the active partner-gene variant, which is recovered and used in the next round of CPR selection, (b) Examples of genetic circuits for CPR. Partner-gene function can be linked to expression of a DNA polymerase in a number of ways, as is demonstrated by the example of T7 RNAP, pol, polymerase; tRNA synthetase, and tRNA engineering.

Article Snippet: 5Prime Phase Lock Gel Heavy tubes (QuantaBio, cat. no. 2302830) Restriction enzyme Dpnl (NEB, cat. no. R0176) CutSmart Buffer, 10× (NEB, cat. no. B7204) QIAquick PCR Purification Kit (Qiagen, cat. no. 28104) Sodium acetate (Sigma-Aldrich, cat. no. S2889) Glacial acetic acid (Fisher, cat. no. A38) Recovery PCR list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Phusion high-fidelity DNA polymerase (supplied with 5× HF buffer; NEB, cat. no. M0530) PCR primers (Integrated DNA Technologies; sequences listed in ) table ft1 table-wrap mode="anchored" t5 TABLE 2 | caption a7 Primer Sequence 5’−3’ Purpose Rec-For B (AJM.15) GATACAATACAACCAG CGATAGACTCAGG Recovery T7 RNAP Rec-Rev B (AJM.16) CAATACCATACTTTTCA TGTGCCCAGACCACTG Recovery T7 RNAP Rec-For A (JWE.46) CCAAATCAGAAAGAA ACTCAACATCGTCAC Recovery tRNA Rec-Rev A (JWE.47) CGTCTACCCGAAGCCTC TACTATGATCAGT Recovery tRNA Open in a separate window Primers for Recovery PCR. dNTP mix, 10 mM each (Thermo Fisher, cat. no. R0192) TAE, 50× liquid concentrate (Apex Bioresearch Products, cat. no. 20–194) Seakem LE Agarose (Lonza, cat. no. 50004) Ethidium bromide (Thermo Fisher, cat. no. 15585011) !

Techniques: Generated, Activity Assay, Expressing, In Vivo, Produced, Plasmid Preparation, Variant Assay, Selection

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: Overview and time line of experiments. A CPR procedure encompasses bacterial expression of the DNA polymerase and partner genes for several hours, emulsification of the bacterial cells in water-oil droplets, in vitro amplification of active partner genes via ePCR, breaking of the emulsions, purification of amplified DNA from cell debris and PCR primers, purification of amplified DNA from background plasmid DNA by Dpnl digestion, and final recovery and bulk amplification of the partner gene using an additional recovery PCR. A single round of CPR typically takes 3–5 d, and the procedure can be repeated 3–10 times until no more enrichment is observed.

Article Snippet: 5Prime Phase Lock Gel Heavy tubes (QuantaBio, cat. no. 2302830) Restriction enzyme Dpnl (NEB, cat. no. R0176) CutSmart Buffer, 10× (NEB, cat. no. B7204) QIAquick PCR Purification Kit (Qiagen, cat. no. 28104) Sodium acetate (Sigma-Aldrich, cat. no. S2889) Glacial acetic acid (Fisher, cat. no. A38) Recovery PCR list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Phusion high-fidelity DNA polymerase (supplied with 5× HF buffer; NEB, cat. no. M0530) PCR primers (Integrated DNA Technologies; sequences listed in ) table ft1 table-wrap mode="anchored" t5 TABLE 2 | caption a7 Primer Sequence 5’−3’ Purpose Rec-For B (AJM.15) GATACAATACAACCAG CGATAGACTCAGG Recovery T7 RNAP Rec-Rev B (AJM.16) CAATACCATACTTTTCA TGTGCCCAGACCACTG Recovery T7 RNAP Rec-For A (JWE.46) CCAAATCAGAAAGAA ACTCAACATCGTCAC Recovery tRNA Rec-Rev A (JWE.47) CGTCTACCCGAAGCCTC TACTATGATCAGT Recovery tRNA Open in a separate window Primers for Recovery PCR. dNTP mix, 10 mM each (Thermo Fisher, cat. no. R0192) TAE, 50× liquid concentrate (Apex Bioresearch Products, cat. no. 20–194) Seakem LE Agarose (Lonza, cat. no. 50004) Ethidium bromide (Thermo Fisher, cat. no. 15585011) !

Techniques: Expressing, In Vitro, Amplification, Purification, Plasmid Preparation

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: 5Prime Phase Lock Gel Heavy tubes (QuantaBio, cat. no. 2302830) Restriction enzyme Dpnl (NEB, cat. no. R0176) CutSmart Buffer, 10× (NEB, cat. no. B7204) QIAquick PCR Purification Kit (Qiagen, cat. no. 28104) Sodium acetate (Sigma-Aldrich, cat. no. S2889) Glacial acetic acid (Fisher, cat. no. A38) Recovery PCR list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Phusion high-fidelity DNA polymerase (supplied with 5× HF buffer; NEB, cat. no. M0530) PCR primers (Integrated DNA Technologies; sequences listed in ) table ft1 table-wrap mode="anchored" t5 TABLE 2 | caption a7 Primer Sequence 5’−3’ Purpose Rec-For B (AJM.15) GATACAATACAACCAG CGATAGACTCAGG Recovery T7 RNAP Rec-Rev B (AJM.16) CAATACCATACTTTTCA TGTGCCCAGACCACTG Recovery T7 RNAP Rec-For A (JWE.46) CCAAATCAGAAAGAA ACTCAACATCGTCAC Recovery tRNA Rec-Rev A (JWE.47) CGTCTACCCGAAGCCTC TACTATGATCAGT Recovery tRNA Open in a separate window Primers for Recovery PCR. dNTP mix, 10 mM each (Thermo Fisher, cat. no. R0192) TAE, 50× liquid concentrate (Apex Bioresearch Products, cat. no. 20–194) Seakem LE Agarose (Lonza, cat. no. 50004) Ethidium bromide (Thermo Fisher, cat. no. 15585011) !

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

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:

Article Snippet: 5Prime Phase Lock Gel Heavy tubes (QuantaBio, cat. no. 2302830) Restriction enzyme Dpnl (NEB, cat. no. R0176) CutSmart Buffer, 10× (NEB, cat. no. B7204) QIAquick PCR Purification Kit (Qiagen, cat. no. 28104) Sodium acetate (Sigma-Aldrich, cat. no. S2889) Glacial acetic acid (Fisher, cat. no. A38) Recovery PCR list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Phusion high-fidelity DNA polymerase (supplied with 5× HF buffer; NEB, cat. no. M0530) PCR primers (Integrated DNA Technologies; sequences listed in ) table ft1 table-wrap mode="anchored" t5 TABLE 2 | caption a7 Primer Sequence 5’−3’ Purpose Rec-For B (AJM.15) GATACAATACAACCAG CGATAGACTCAGG Recovery T7 RNAP Rec-Rev B (AJM.16) CAATACCATACTTTTCA TGTGCCCAGACCACTG Recovery T7 RNAP Rec-For A (JWE.46) CCAAATCAGAAAGAA ACTCAACATCGTCAC Recovery tRNA Rec-Rev A (JWE.47) CGTCTACCCGAAGCCTC TACTATGATCAGT Recovery tRNA Open in a separate window Primers for Recovery PCR. dNTP mix, 10 mM each (Thermo Fisher, cat. no. R0192) TAE, 50× liquid concentrate (Apex Bioresearch Products, cat. no. 20–194) Seakem LE Agarose (Lonza, cat. no. 50004) Ethidium bromide (Thermo Fisher, cat. no. 15585011) !

Techniques: Concentration Assay

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:

Article Snippet: 5Prime Phase Lock Gel Heavy tubes (QuantaBio, cat. no. 2302830) Restriction enzyme Dpnl (NEB, cat. no. R0176) CutSmart Buffer, 10× (NEB, cat. no. B7204) QIAquick PCR Purification Kit (Qiagen, cat. no. 28104) Sodium acetate (Sigma-Aldrich, cat. no. S2889) Glacial acetic acid (Fisher, cat. no. A38) Recovery PCR list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Phusion high-fidelity DNA polymerase (supplied with 5× HF buffer; NEB, cat. no. M0530) PCR primers (Integrated DNA Technologies; sequences listed in ) table ft1 table-wrap mode="anchored" t5 TABLE 2 | caption a7 Primer Sequence 5’−3’ Purpose Rec-For B (AJM.15) GATACAATACAACCAG CGATAGACTCAGG Recovery T7 RNAP Rec-Rev B (AJM.16) CAATACCATACTTTTCA TGTGCCCAGACCACTG Recovery T7 RNAP Rec-For A (JWE.46) CCAAATCAGAAAGAA ACTCAACATCGTCAC Recovery tRNA Rec-Rev A (JWE.47) CGTCTACCCGAAGCCTC TACTATGATCAGT Recovery tRNA Open in a separate window Primers for Recovery PCR. dNTP mix, 10 mM each (Thermo Fisher, cat. no. R0192) TAE, 50× liquid concentrate (Apex Bioresearch Products, cat. no. 20–194) Seakem LE Agarose (Lonza, cat. no. 50004) Ethidium bromide (Thermo Fisher, cat. no. 15585011) !

Techniques: Concentration Assay