Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries -…

Step 1Obtaining the mutant insert by error-prone PCR and the control insert

The DsRed2 gene was isolated using the plasmid pDsRed2 (Clontech, Cat. No. 632404, UniProt Q9U6Y8) as a template (Fig.1A). Error-prone PCR of the DsRed2 gene was performed using the GeneMorph II Random Mutagenesis kit, following the manufacturers protocol The primers DsRed2-EcoRI-F and DsRed2-BamHI-R (Table 1) were used and the PCR conditions included one cycle at 94C for 2min, followed by 30 cycles at 94C for 15s, 68C for 30s, and 72C for 60s, with a final elongation step at 72C for 5min. The products of error-prone PCR are referred to as the mutant insert in this text. The DsRed2 gene without mutations (Fig.1A) was amplified using the same primers and high-fidelity polymerase (TAKARA LA Taq DNA, Clontech Cat. No. RR002A) as a control for the procedure (referred to as the control insert). The PCR conditions for this were 94C for 2min, followed by 30 cycles of 94C for 15s, 60C for 30s, 72C for 2min, and a final cycle at 72C for 5min. After PCR, the amplicons were verified on 1% agarose gel electrophoresis and purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare).

Graphic representation of the main methodological steps for comparing the Ligation-Dependent Process Cloning method (LDPC) and Circular Polymerase Extension Cloning (CPEC). (A) Step 1 Obtaining the Mutant Insert by Error-Prone PCR and the Control Insert. The DsRed2 gene was isolated from the plasmid pDsRed2 through error-prone PCR using specific primers and conditions, resulting in the mutant insert. The control insert was also isolated from the plasmid pDsRed2, but a high-fidelity polymerase was used. (B) Step 2Ligation-dependent process cloning. A vector was prepared by cleaving the pDsRed2 plasmid with BamHI-HF and EcoRI-HF enzymes, followed by digestion of all fragments (control insert and mutant insert from Step 1) using the same restriction enzymes, and ligation reactions were performed using T7 ligase. (C) Step 3Circular Polymerase Extension Cloning CPEC. The mutant insert, along with the control, was amplified via PCR, quantified, and cloned into the pCDF1b (GenBank Accession Number OR900361.1) expression vector using CPEC with overlapping primers.

Initially, the pDsRed2 plasmid was cleaved (Fig.1B) using the enzymes BamHI-HF (New England Biolabs, Cat. No. R3136) and EcoRI-HF (New England Biolabs, Cat. No. R3101) to get the vector. Afterwards, BamHI-HF and EcoRI-HF restriction enzymes were used to digest all fragments (control insert and mutant insert Step 1). This digestion took place over an incubation time of 2h at a temperature of 37C. The enzymes were inactivated for 20min at 65C. Digested fragments were quantified on the Qubit fluorimeter (Life Technologies, Brazil) using the Quant-iT dsDNA BR Assay kit (Invitrogen, Brazil). A 1:1 ratio was used for the ligation reactions. The vector (pDsRed2) was at a concentration of 81.7ng/L and the inserts were at a concentration of 84.1ng/L. The ligation using the T7 ligase (New Englands, Biolabs Cat. No M0318) was carried out according to the manufacturers protocol and was conducted in triplicate.

A total of the 1 L of product from each ligation was transformed into 40 L of electrocompetent Escherichia coli TOP 10 bacteria (0.2cm cuvette, 2.5kV/cm, 25 F, 200 , 1 pulse) using the Gene Pulser Xcell electroporation system (BioRad). The cells were grown in 480 L of SOC medium (2% tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5mM KCl, 20mM glucose) for 1:30h at 37C with constant shaking at 243g in a Stuart Shaking incubator SI500 orbital shaker (Stuart, Brazil). After incubation, the inoculants were seeded in plates containing Luria Bertani (LB) agar medium and antibiotic spectinomycin (100g/mL) and incubated for 16h at 37C. The bacteria transformed with the product of each ligation were screened for strong fluorescence using the Safe Imager 2.0 Blue Light Transilluminator (Invitrogen) with excitation at 470nm. The plates obtained were photographed and the total number of colonies on each plate was determined. The plates for the controls and mutants were quantified using microscopy and counted with ImageJ software.

We utilized the construct that was obtained and chosen from Step 2 (pDsRedmut) as a template for the construct that included the mutant insert. PCR reaction was performed using the primers Mut/Dsred2-F and Mut/Dsred2-R (Table 1) and the TAKARA LA taq high fidelity DNA polymerase (5U/L TAKARA LA Taq, 10X LA PCR buffer II (Mg2+ free, 25mM MgCl2, 0.25mM dNTP). The PCR conditions were one cycle of 94C for 2min (initial denaturation) followed by 30 cycles of 94C for 15s, 66C for 30s, and 68C for 3min, and a final elongation of 72C for 10min. After PCR, the fragments (hereafter mutant) were quantified using the Quant-iT dsDNA HS Assay kit (Invitrogen, Brasil). The same procedure was done in the DsRed2 gene as a control. The mutant gene and the control were cloned into the pCDF1b expression vector (Novagen, Cat. No. 71330-3) (Fig.1C). The ligation of fragments (DsRed2 and DsRed mut) with vector (pCDF1b) was done via CPEC with the primers PCDF-F and PCDF-R (Table 1). These oligonucleotides have an overlapping sequence (bases under-arrayed in the sequence) with the product mutant for CPEC to occur.

The PCR for CPEC was carried out using the TAKARA LA Taq enzyme (Clontech Cat. No. RR002A), following the conditions: 94C/2min, 30 cycles of 94C/15s, 63C/30s, 68C/4min and 1 final cycle 72C/5min. The template DNA for the CPEC reaction was the double-stranded fragments of the mutant and the vector pCDF1b was added in a 1:1 ratio. In the first PCR cycle, the fragments are denatured. In the following cycles, the single strands are ringed in the sequence in which they overlap, and it is from this overlap that the fragments extend to form the double strand of the circular plasmid pCDF1b-DsRed2mut and pCDF1b-DsRed2, respectively. The fragments were analyzed using 1% agarose gel electrophoresis.

The expression vectors produced (pCDF1b-Mutant and pCDF1b-DsRed2) were transformed into electrocompetent Escherichia coli BL21-DE3 by electroporation (0.2cm cuvette, 2.5kV/cm, 25 F, 200 , 1 pulse) using the Gene Pulser Xcell electroporation system (BioRad). The transformed bacteria were seeded in plates containing Luria Bertani (LB) agar medium and antibiotic spectinomycin (100g/mL) and incubated for 16h at 37C. After transformation, bacterial colonies were inoculated into liquid Luria Bertani (LB) medium, using antibiotic spectinomycin (100g/mL) as a selective agent, incubated for 16h at 37C with constant shaking at 243g in a Stuart Shaking incubator SI500 (Stuart, Brazil). Subsequently, the plasmids were purified using the Ilustra- Plasmid Prep Mini Spin Kit (GE Healthcare). The plates obtained were photographed and the total number of colonies on each plate was determined. The plates for the controls and mutants were quantified using microscopy and counted with ImageJ software.

The selected bacterial colonies were inoculated into liquid Luria Bertani (LB) medium, using the antibiotic spectinomycin (100g/mL) as a selective agent, incubated for 16h at 37C with constant shaking at 243g in a Stuart Shaking incubator SI500 orbital shaker (Stuart, Brazil). Subsequently, the plasmids with mutant and control inserts were purified using the Ilustra- Plasmid Prep Mini Spin Kit (GE Healthcare). After purification, the plasmids were sequenced using the oligonucleotides PCDFBGL-Seq-F and PCDFBGL-Seq-R (Table 1) to confirm binding using the CPEC and LDCP methodologies.

One-way ANOVA was used for the statistical analysis of the data, with a significance threshold of p<0.05. To make sure the test assumptions were met, tests for homogeneity of variances and residuals normality were performed before to the ANOVA. Specifically, Levenes test was used to assess the homogeneity of variances, and the ShapiroWilk test was employed to evaluate the normality of residuals.

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Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries -...