Step 1—Obtaining 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 manufacturer’s protocol The primers DsRed2-EcoRI-F and DsRed2-BamHI-R (Table 1) were used and the PCR conditions included one cycle at 94 °C for 2 min, followed by 30 cycles at 94 °C for 15 s, 68 °C for 30 s, and 72 °C for 60 s, with a final elongation step at 72 °C for 5 min. 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 94 °C for 2 min, followed by 30 cycles of 94 °C for 15 s, 60 °C for 30 s, 72 °C for 2 min, and a final cycle at 72 °C for 5 min. 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 2—Ligation-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 3—Circular 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.
Step 2—Ligation-dependent process cloning
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 2 h at a temperature of 37 °C. The enzymes were inactivated for 20 min at 65 °C. 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 81.7 ng/µL and the inserts were at a concentration of 84.1 ng/µL. The ligation using the T7 ligase (New Englands, Biolabs Cat. No M0318) was carried out according to the manufacturer’s protocol and was conducted in triplicate.
Bacterial transformation for T7 ligation products
A total of the 1 µL of product from each ligation was transformed into 40 µL of electrocompetent Escherichia coli TOP 10 bacteria (0.2 cm cuvette, 2.5 kV/cm, 25 µF, 200 Ω, 1 pulse) using the Gene Pulser Xcell™ electroporation system (BioRad). The cells were grown in 480 µL of SOC medium (2% tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 20 mM glucose) for 1:30 h at 37 °C with constant shaking at 243 g 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 (100 µg/mL) and incubated for 16 h at 37 °C. 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 470 nm. 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.
Step 3—Circular polymerase extension cloning—CPEC
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/µL TAKARA LA Taq, 10X LA PCR buffer II (Mg2+ free, 25 mM MgCl2, 0.25 mM dNTP). The PCR conditions were one cycle of 94 °C for 2 min (initial denaturation) followed by 30 cycles of 94 °C for 15 s, 66 °C for 30 s, and 68 °C for 3 min, and a final elongation of 72 °C for 10 min. 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: 94 °C/2 min, 30 cycles of 94 °C/15 s, 63 °C/30 s, 68 °C/4 min and 1 final cycle 72 °C/5 min. 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.
Bacterial transformation for CPEC products
The expression vectors produced (pCDF1b-Mutant and pCDF1b-DsRed2) were transformed into electrocompetent Escherichia coli BL21-DE3 by electroporation (0.2 cm cuvette, 2.5 kV/cm, 25 μF, 200 Ω, 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 (100 μg/mL) and incubated for 16 h at 37 °C. After transformation, bacterial colonies were inoculated into liquid Luria Bertani (LB) medium, using antibiotic spectinomycin (100 μg/mL) as a selective agent, incubated for 16 h at 37 °C with constant shaking at 243 g 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 (100 μg/mL) as a selective agent, incubated for 16 h at 37 °C with constant shaking at 243 g 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.
Statistical analysis
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, Levene’s test was used to assess the homogeneity of variances, and the Shapiro–Wilk test was employed to evaluate the normality of residuals.
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- Source: https://www.nature.com/articles/s41598-024-66584-y