In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1

Marc Blanch-Asensio; Sourik Dey; Shrikrishnan Sankaran

doi:10.1371/journal.pone.0281625

. 2023 Feb 16;18(2):e0281625. doi: 10.1371/journal.pone.0281625

In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1

Marc Blanch-Asensio ^1,^#, Sourik Dey ^1,^#, Shrikrishnan Sankaran ^1,^*

Editor: Hari S Misra²

PMCID: PMC9934402 PMID: 36795741

Abstract

Lactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due to their complex and thick cell walls limiting our capability to transform them with exogenous DNA. To overcome this, large amounts of DNA (>1 μg) are normally required to successfully transform these bacteria. An intermediate host, like E. coli, is often used to amplify recombinant DNA to such amounts although this approach poses unwanted drawbacks such as an increase in plasmid size, different methylation patterns and the limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on in-vitro assembly and PCR amplification to yield recombinant DNA in significant quantities for successful transformation in L. plantarum WCFS1. The advantage of this method is demonstrated in terms of shorter experimental duration and the possibility to introduce a gene incompatible with E. coli into L. plantarum WCFS1.

Introduction

Lactobacilli are a group of Gram-positive bacteria of great importance to the food and healthcare industries with numerous strains identified as being beneficial for humans, and used as probiotics [1–3]. Furthermore, since they naturally colonize almost every site of the human body that hosts a healthy microbiome, e.g. the gastrointestinal tract [4,5], urogenital tracts [6], oral cavity [7] and nasal cavity [8], lactobacilli are an excellent foundational candidate for the development of live biotherapeutic products (LBPs) [9]. Beyond their natural health benefits, there is considerable interest in engineering them with heterologous genes for therapeutic applications like drug delivery [10,11] and mucosal vaccinations [12,13]. However, one of the crucial factors slowing down progress in lactobacilli engineering is difficulties in transforming them with exogenous DNA [14]. This is largely due to their thick and complex cell wall structures, which prevent successful bacterial transformation if the concentration of plasmid DNA is less than >1 μg [15]. To obtain such high plasmid DNA quantities, shuttle vectors are often used that can be amplified in intermediate hosts, predominantly E. coli [16]. To facilitate the construction of recombinant plasmids, several shuttle vectors have been identified, which can undergo stable replication in both the cloning host, E. coli and the relevant Lactobacilli strains [17–19]. Nevertheless, since E. coli is a Gram-negative bacterium that is phylogenetically distant from Lactobacillus genera, this strategy can lead to genetic sequence incompatibilities due to GC-content differences [20], DNA methylation [21], repetitive sequence insertions [22] and toxic protein buildup in the E. coli cloning host [23]. Alternatively, the Gram-positive lactic acid bacterium, Lactococcus lactis, can also be used as an intermediate host for recombinant plasmid construction [24]. However, the availability of functional replication origins in L. lactis is limited [25] and inclusion of additional broad-range replicons significantly increases the size of the plasmid. The excessive increase in the size of the plasmid might lead to segregational instability [26] and thereby limit the size of the heterologous genes that can be included in it. Hence, it is desirable to be able to directly transform circular plasmid dsDNA into the lactobacilli strains without relying on intermediate bacterial hosts like E. coli and L. lactis. To avoid the need for an intermediate host, Spath et al. developed a direct cloning approach based on the assembly of PCR-amplified DNA fragments by restriction digestion and ligation to obtain optimal quantities of circular dsDNA for transformation in Lactiplantibacillus plantarum CD033 [27]. They further show that the unmethylated plasmid DNA allowed for transformation in a strain (L. plantarum DSM20174) that could not be transformed using methylated DNA, possibly due to native restriction-modification systems. However, the method still requires the presence of restriction sites within the DNA sequences, which can limit the versatility of combining heterologous genes in the plasmid and needs to be accounted for when dealing with strains that may harbor unknown restriction-modification systems.

In this work, we report a direct cloning method that leverages the Gibson assembly strategy and takes advantage of recent advances in cost-effective oligonucleotide synthesis. By doing so, we avoid the need for restriction sites and improve the feasibility of combining diverse DNA sequences to construct versatile recombinant plasmids. We demonstrate this direct cloning method in Lactiplantibacillus plantarum WCFS1, one of the commonly engineered probiotic Lactobacillus strains for which improved engineering methods are highly sought [28,29]. Furthermore, this direct cloning method is considerably quicker in comparison to indirect cloning methods requiring an intermediate host. We have characterized the efficiency and accuracy of this approach and have demonstrated the successful cloning of a gene expressing the medically relevant protein, Elafin which showed a high failure rate when being cloned through the intermediate host, E. coli. Thus, this direct cloning method will be instrumental in enabling the cloning of Lactobacilli with a wider variety of heterologous genes and with greater versatility than previously possible.

Materials and methods

Bacterial strains and growth conditions

L. plantarum WCFS1 was used as the parent strain in this study. The strain was grown in the De Man, Rogosa and Sharpe (MRS) media (Carl Roth GmbH, Germany, Art. No. X924.1). Recombinant L. plantarum WCFS1 strains were grown in MRS media supplemented with 10 μg/mL of erythromycin (Carl Roth GmbH, Art. No. 4166.2) at 37°C and 250 rpm shaking for 16 h. For the indirect cloning experiments, NEB 5-alpha Competent E. coli cells were used (New England Biolabs GmbH, Germany,Art. No. C2987). This strain was grown in Luria-Bertani (LB) medium (Carl Roth GmbH, Art. No. X968.1). Recombinant E. coli DH5α strains were grown in LB media supplemented with 200 μg/mL of erythromycin at 37°C, 250 rpm shaking conditions for 16 h.

Molecular biology

Q5 High Fidelity 2X Master Mix (New England Biolabs GmbH [NEB], Germany, No. M0492S) was used to perform DNA amplification. Amplified DNA products were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega GmbH, Germany, Art. No. A9282). Foragarose gels, 1 kb Plus DNA Ladder(Catalog Number 10787018) and Generuler 100 bp Plus DNA Ladder (Catalog Number (SM0321) was procured from ThermoFisher Scientific^TM, Germany and used for reference. Primers were synthesized by Integrated DNA Technologies (IDT) (Louvain, Belgium) and the elafin gene fragment was ordered as eBlock from IDT (Coralville, USA). All primers used in this work are shown in S1 Table in S2 File. The mCherry gene fragment was amplified by PCR from a plasmid previously generated in our lab. The genetic sequences of mCherry and elafin genes are shown in S2 Table in S2 File. The plasmid pLp3050sNuc (Addgene plasmid # 122030) [30] was used as the vector backbone in this study. The schematic for the recombinant plasmids, pLp_mCherry and pLp_elafin constructed in this study have been highlighted in S1 Fig in S2 File. The Codon Optimization tool from IDT (Choice Host Organism–L. acidophilus) was used to optimize the codon bias for mCherry coding segment. The Java Codon Adaptation Tool (JCat) [31] was used to codon-optimize the gene encoding for the human peptidase inhibitor 3, elafin (GenBank ID: AAX36874.1) using the codon optimization database for L. plantarum WCFS1. DNA assembly was performed using the HiFi Assembly Master Mix (NEB GmbH, Germany, Art. No. E5520S). For plasmid circularization, the Quick Blunting Kit (NEB GmbH, Germany, Art. No. E1201S) and the T4 DNA Ligase enzyme (NEB GmbH, Germany, Art. No. M0318S) were used.

L. plantarum WCFS1 electrocompetent cell preparation

Wild-type L. plantarum WCFS1 was cultured overnight in 5 mL of MRS media and at 37°C with shaking (250 rpm). After 16h, 1 mL of the culture (OD₆₀₀ = 2) was added to 20 mL of MRS media and 5 mL of 1% (w/v) glycine. This secondary culture was incubated at 37°C and 250 rpm until the OD₆₀₀ reached 0.8. The cells were then harvested by centrifugation at 4000 rpm (3363 X g) for 12 min at 4°C. After discarding the supernatant, the pellet was washed twice with 5 mL of ice-cold 10 mM MgCl₂. The pellet was then washed twice with ice-cold Suc/Gly solution (1 M sucrose and 10% (v/v) glycerol mixed in a 1:1 (v/v) ratio), first with 5 mL and second with 1 mL. Next, the supernatant was discarded, and the bacterial pellet was resuspended in 450 μL of ice-cold Suc/Gly solution. Finally, 60 uL aliquots were prepared and immediately used for DNA electroporation or stored at -80°C for future use.

Electroporation based transformation in L. plantarum WCFS1

For electroporation transformation, plasmids were first mixed with 60 μl of electrocompetent cells at quantities (300–1200 ng) specified in the Results section. After a short incubation on ice, the mixture was transferred to ice-cold electroporation cuvettes with a 2 mm gap (Bio-Rad Laboratories GmbH, Germany, #1652086). Electroporation was performed using the MicroPulser Electroporator (Bio-Rad Laboratories GmbH, Germany), with a single pulse (5 ms) at 1.8 kV. Immediately after the pulse, 1 mL of MRS media was added to the bacterial mixture and then transferred to a 1.5 mL Eppendorf tube for further incubation at 37°C, 250 rpm for 3 h. Following this incubation, cells were pelleted down at 4000 rpm (3363 X g) for 5 min. 800 μL of the supernatant was discarded, and the remaining 200 μL was used for cell resuspension by slow pipetting. Finally, the resuspended pellet was plated on MRS Agar plates supplemented with 10 μg/mL of Erythromycin, and plates were incubated at 37°C for 48 h for colonies to grow.

Direct cloning method in L. plantarum WCFS1

This study created and optimized a novel direct cloning method based on amplifying and circularizing in vitro-assembled gene fragments to be directly transformed in L. plantarum WCFS1 (Fig 1). For the Gibson HiFi Assembly reaction, complementary overhangs were included by PCR using a set of primers that contained the corresponding overhangs at the 5’ ends. In the Gibson HiFi assembly reaction mixture, 50 ng of the PCR-amplified linear vector with overlapping DNA fragments and 10 ng of the corresponding eBlock were mixed along with 10 μl of the HiFi DNA Assembly Master Mix (Mili-Q water was added up to 20 μl). The reaction was incubated at 50°C for 30 minutes. After that, 5 μL of the assembled product was used as template for an additional round of PCR, using a set of primers that annealed specifically to the insert region. The final volume of this PCR was 120 μl, and the amplification cycle threshold was set at 22. 5 μl of this reaction was run on an agarose gel to confirm amplification (S2 Fig in S2 File). After purifying the linear PCR product, 3500 ng of DNA were phosphorylated using the Quick Blunting Kit. This reaction was performed as suggested in the standard reaction protocol. 2.5 μl of the 10X Quick Blunting buffer and 1 μl of the Enzyme Mix were mixed with the purified DNA (3500 ng). Milli-Q water was added up to 25 μl. The reaction was incubated for 30 minutes at 25°C to allow the blunting reaction and then kept at 70°C for 10 minutes to inactivate the enzyme. In the next steps, phosphorylated DNA was ligated using the T4 ligase enzyme. This reaction was slightly modified from the standard protocol because a higher amount of DNA was added to the reaction. Per ligation, 500 ng of phosphorylated DNA (3.6 μl of the Quick Blunting reaction) were mixed with 1.5 μl of T4 Ligase enzyme and 2.5 μl of 10X T4 Ligase Buffer. Milli-Q water was added up to 25 μl. The number of ligation reactions depended on the amount of DNA intended to be circularized. The ligation reactions were incubated for 2.5 hours at 25°C and then 10 minutes at 70°C to inactivate the enzyme. Following the incubation, the ligation mixtures were combined, and the ligated dsDNA was purified using the Promega kit. In this purification, DNA was eluted 3 times with 9 μl of Milli-Q water each time to obtain the highest DNA concentration. The DNA concentration of the ligated mixture was measured (absorbance at 260 nm) using a NanoDrop Microvolume UV-Vis Spectrophotometer (ThermoFisher Scientific GmbH, Germany). The purified ligated products were then transformed into L. plantarum WCFS1 electrocompetent cells.

Fig 1 — The scheme was generated using BioRender.

The protocol described in this peer-reviewed article is published on protocols.io, https://dx.doi.org/10.17504/protocols.io.ewov1o82olr2/v1 and is included for printing as S1 File with this article.

Sequence verification was performed by PCR amplification of the target gene conducted directly from the bacterial pellet. To do so, the selected bacteria were inoculated in MRS media supplemented with 10 μg/mL of erythromycin and incubated overnight at 37°C and 250 rpm. The following day, 1 mL of the bacterial culture was collected in a 1.5 mL Eppendorf tube and centrifuged at 4°C for 3 min at 8400 X g. The supernatant was discarded, the residual pellet fraction was scratched off with a sterile pipette tip and used as a template for the PCR (100 μl as final reaction volume, 28 cycles). Alternatively, a colony grown on the MRS plate can also be used to for PCR amplification of the target gene segment. The PCR settings involved an additional initial denaturation step for 10 minutes at 98°C to ensure maximum bacterial lysis. After the PCR, 5 μl of the PCR products were assessed by agarose gel electrophoresis to confirm amplification at the expected size. Next, the PCR product was purified, and the DNA concentration was measured using the Nanodrop. Finally, 2000 ng of the purified PCR product was sent for sequencing to Eurofins Genomics GmbH (Ebersberg, Germany). An additional DNA purification step before Sanger sequencing was employed for obtaining efficient results (Additional Service: PCR Purification).

Indirect cloning via the intermediate host strain E. coli

For the indirect cloning, an additional Gibson HiFi reaction was performed (identical to the Gibson HiFi reaction set for the direct protocol). However, an additional step was done before setting this reaction, which involved the restriction enzyme digestion with DpnI (NEB GmbH, Germany, Art. No. R0176S). In this reaction, 500 ng of the purified PCR product were mixed with 1 μl of DpnI enzyme and 1 μl of rCutSmart buffer (Milli-Q water was added up to 10 μl). Incubation was performed for 30 minutes at 37°C followed by 10 minutes at 70°C. The digested product was used for the Gibson HiFi Assembly reaction. Once the reaction was done, it was transformed into NEB 5-alpha Competent cells (50 μl). In this transformation, NEB 5-alpha Competent cells were first thawed on ice for 10 minutes. After that, 8 μl of the reaction were properly mixed with the competent cells by pipetting and incubated on ice for 20 minutes Following the incubation, a 60-second heat shock was performed by placing the cells at a 42°C water bath. Next, cells were again incubated on ice for 5 minutes. After that, 950 μl of SOC media was added to the cell mixture and kept for incubation for 1 hour at 37°C. Finally, 150 μl of the culture was plated on an LB agar plate supplemented with 200 μg/mL of erythromycin and incubated at 37°C overnight.

For the pLp_mCherry cloning, the screening of positive clones was done using the Gel Documentation System Fluorchem Q (Alpha Innotech Biozym Gmbh, Germany). Bacterial colonies expressing mCherry were imaged in the Cy3 channel (Exλ/Emλ = 554 nm/568 nm) and the corresponding brightfield image was taken using the ethidium bromide channel (Exλ/Emλ = 300 nm/600 nm). One red colony was inoculated in LB media supplemented with 200 μg/mL of erythromycin and incubated at 37°C overnight. The following day, plasmid extraction was performed using the Plasmid extraction miniprep kit (Qiagen GmbH Germany, Art. No. 27104). The plasmid DNA concentration was measured using the 260 nm absorbance setting on the NanoDrop Microvolume UV-Vis Spectrophotometer. The recombinant plasmid was then transformed into L. plantarum WCFS1 electrocompetent cells.

For the pLp_elafin cloning, 20 colonies were streaked on a fresh LB agar plate supplemented with 200 μg/mL of erythromycin and incubated at 37°C overnight. The following day, positive clones were screened by PCR using a forward primer that annealed to the vector and a reverse primer that annealed to the elafin gene (100 μl as final reaction volume, 28 cycles). In the PCR, 10 minutes at 98°C were set for the initial denaturation of the samples. Three positive clones confirmed by colony PCR were inoculated in LB media supplemented with 200 μg/mL of erythromycin and incubated at 37°C overnight. The following day, the respective plasmids were extracted and sent for sequencing to Eurofins Genomics GmbH (Ebersberg, Germany).

Results and discussion

The Gibson assembly approach to combining DNA fragments requires the fragments to contain terminal overhangs that complementarily overlap by ~20 bases. The method was originally developed to stitch together the first artificial genome due to the high level of flexibility it provided compared to restriction digestion-based methods [32]. It is a single reaction assembly method that can be performed without thermal cycling and within an hour. In this work, we employ Gibson reaction to conduct in-vitro assembly of circular dsDNA constructs for direct cloning in L. plantarum WCFS1. As shown in Fig 1, our method involves PCR amplification of a vector and an insert with overlapping arms, followed by their Gibson reaction-based assembly that yields a low quantity (50–80 ng) of the assembled dsDNA. To obtain optimal concentration of dsDNA for transformation in Lactobacilli, a second amplification and recircularization step was performed, yielding >1 μg of the desired construct. This method was characterized and optimized in terms of transformation efficiency, accuracy, and capability for cloning challenging genes in Lactiplantibacillus plantarum WCFS1. Different plasmid constructs were assembled and compared with indirect cloning using E. coli as an intermediate host.

Transformation efficiency and accuracy of the direct cloning method

Transformation efficiency (TE) indicates the extent to which cells can take up DNA from the extracellular space and express the genes encoded by it [33]. While it is possible to transform L. plantarum WCFS1 with extracellular DNA, TE is typically poor [27]. To demonstrate this, a simple plasmid construct, pLp_mCherry, with gene sequences ideal for indirect cloning through E. coli was used. This plasmid consisted of a p256 replicon, an erythromycin resistance cassette and the mCherry gene driven by a strong constitutive promoter P_tlpA, [34] all of which are compatible in both E. coli and L. plantarum WCFS1. The plasmid was constructed through Gibson reaction-based assembly of the pLp-3050sNuc plasmid backbone and P_tlpA-mCherry insert and transformed in E. coli. One correctly sequenced plasmid, extracted from an E. coli DH5α clone, was transformed in L. plantarum WCFS1 at different concentrations (300, 600, 900 and 1200 ng), yielding low TE values that increased with higher DNA concentrations (20–500 cfu/μg) (Fig 2A). In the direct cloning method, the Gibson reaction based assembled dsDNA was PCR amplified using complementary primers within the insert region and the resulting linear fragments were circularized by phosphorylation and ligation to yield high amounts of plasmid DNA suitable for transformation in L. plantarum WCFS1 (> 3μg). Following the transformation of the circularized plasmid mix in L. plantarum WCFS1 at different DNA concentrations as mentioned above, TE values were found to be lower than that of the indirect method. However, despite being lower the overall TE values were within the same order of magnitude and increased drastically when the net DNA concentration used for transformation was above 1 μg (Fig 2A). The lower TE values could be due to incomplete circularization of the PCR-amplified plasmid fragments, due to which the final quantity of the circularized constructs might have been lower than the total DNA that was quantified [35]. The accuracy of clones generated from the direct cloning method, determined by their ability to express mCherry, was estimated after checking the fluorescence of 418 colonies that grew across all the DNA quantities tested. Colonies were streaked on fresh plates and the following day they were examined for the presence of fluorescent protein. Overall, 347 out of 418 colonies were red, giving an accuracy of 83% (Fig 2B). The accuracy in the indirect cloning method with the correctly sequenced recombinant plasmid isolated from E. coli DH5α was found to be above 99%, as expected.

Fig 2 — **(A)** Transformation Efficiency comparison against the total concentration of DNA transformed for the indirect and direct cloning protocol in L. *plantarum* WCFS1 (The standard deviations correspond to three independent biological replicates) **(B)** Percentage accuracy of correct recombinant clones against the total concentration of DNA transformed in L. *plantarum* WCFS1 using the direct cloning protocol (The whiskers correspond to values from three independent biological replicates). **(C)** Agarose gel showing the colony PCR product (1140 bp) corresponding to the mCherry gene of interest (GOI). A red L. *plantarum* colony obtained after direct cloning was used as the template DNA for the PCR reaction. Generuler 100 bp Plus DNA Ladder (ThermoFisher Scientific^TM) was used for the reference.

Furthermore, we wanted to prove that a DpnI digestion prior to the Gibson HiFi Assembly reaction would not have an impact on the number of positive clones obtained through the direct cloning method since we assumed that the backbone vectors used as template for the PCR would be extremely diluted during the multiple purification steps involved in the protocol. Therefore, we repeated the pLp_mCherry direct cloning with and without a DpnI digestion of the insert and vector PCR products prior to the Gibson HiFi Assembly reaction. We screened 10 red colonies for both experimental conditions by PCR amplification of a partial gene segment within the mCherry reporter. The proportion of red colonies obtained was similar in both conditions and colony PCR amplification of a part of the mCherry gene (10 red colonies from each) yielded PCR products of the same expected size from all clones. This confirmed that a DpnI digestion prior to the HiFi Assembly reaction is not necessary (S3 Fig in S2 File).

Notably, since L. plantarum WCFS1 contains 3 endogenous plasmids [36], sequencing-based verification of desired regions in the recombinant plasmid was done by PCR amplification of the entire mCherry gene of 1140 base pairs (bp) (Fig 2C). The gene segments were directly amplified from bacterial cell pellets, and the amplicon sequencing was outsourced to an external service provider, Eurofins Genomics GmbH, where their additional DNA purification option was employed. An initial purification of the PCR amplified product by us seemed to improve the quality of the sequencing chromatograms but was not absolutely necessary to get the correct results (S5A and S5B Fig in S2 File). As expected, all clones expressing mCherry yielded the correct sequences without any mutations or deletions. The whole mCherry gene was also amplified from 10 non-red colonies using the same set of primers and PCR conditions as in Fig 2C. We obtained amplification for 7 out of 10, nevertheless, the PCR product was either bigger or smaller than expected (S4 Fig in S2 File). These results suggest that mutations might have occurred during the PCR amplification steps or a minor proportion of wrongly assembled products were formed during Gibson assembly which can result in recombinant clones with the mutated gene of interest (GOI).

Time requirement for the direct and indirect cloning methods

The direct cloning method is considerably quicker and less labor-intensive than the indirect cloning method. All steps in the direct cloning method can be completed in 4 days after which PCR-amplified sequences can be sent for sequencing. In contrast, the indirect cloning method requires 5 to 6 days, depending on the time allocated for growth of bacteria on the master plate (Fig 3). Note that a master plate is needed to be made from transformed E. coli colonies in our case due to the use of erythromycin as the antibiotic resistance marker. E. coli has natural resistance to this antibiotic at low dosages which can be surpassed by supplementing the growth media with higher concentration of erythromycin. On one hand, this will prevent the growth of non-transformed cells but on the other hand it will also lead the transformed colonies to grow slowly making the colonies too small for reliable use in colony PCR analysis. In the case of L. plantarum WCFS1, a master plate was not required since colonies grown for 48 hours were large enough to handle both colony PCR analysis and inoculation in liquid cultures.

Fig 3 — The scheme was generated using BioRender.

Direct cloning of a gene incompatible with E. coli

The main advantage of the direct cloning method is demonstrated in the ability to transform genes in lactobacilli that are challenging using the indirect method. Genes encoding proteins that are toxic to E. coli, for example, often result in mutations or complete deletions of the GOI in the plasmid when transformed into E. coli [37]. We therefore tested the cloning of a plasmid containing the human peptidase inhibitor 3 gene, known as elafin, encoded downstream of a strong constitutive promoter (P_tlpA). This protease has been reported to exert anti-microbial activity with E. coli, so its constitutive expression is expected to be toxic [38,39]. Transformation of the assembled plasmid containing the constitutively expressed elafin in E. coli yielded very few colonies. The screening of positive clones were done by PCR amplification using a primer set, where one specifically annealed to the vector and the other one was complementary to the insert region. Only 3 clones showed amplification, but the amplified product was shorter than expected (524 bp) (Fig 4A). Sequencing of plasmids extracted from these clones revealed several mutations and deletions. The whole P_tlpA was deleted from all three plasmids, and two clones had the elafin coding sequence truncated (Fig 4B, S6 Fig in S2 File). On the other hand, the direct cloning method yielded over 124 colonies after transformation with 1000 ng of phosphorylated and ligated dsDNA. 10 colonies were screened by PCR, and all of them showed amplification at the expected size of 524 bp (Fig 4A). Sequencing of the gene amplified from randomly selected 3 clones revealed no mutations or deletions (Fig 4B).

Fig 4 — **(A)** The agarose gel (left) shows the colony PCR result of 10 randomly selected E. coli pLp_elafin clones. The agarose gel (right) corresponds to the colony PCR of 10 randomly selected L. plantarum WCFS1 pLp_elafin clones. All the colony PCR reactions were performed under the same conditions. The expected amplicon size for the pLp_elafin clones were 524 bp. The reference ladder used was the 1 kb Plus DNA ladder (ThermoFisher^TM) (B) Table listing the results obtained after Sanger Sequencing of the isolated pLp_elafin plasmids. The (✓) suggests a complete match to the expected sequence. Three plasmids per cloning method (direct and indirect) were sent for analysis.

Conclusions

The direct cloning method developed in this paper has proved effective in transforming circular dsDNA (plasmid) DNA into L. plantarum WCFS1 without any intermediate host requirement for plasmid amplification. We demonstrate that this method provides two major benefits for lactobacillus engineering–(i) it saves time of at least 2 days compared to commonly used indirect cloning methods involving intermediate hosts and (ii) enables the cloning of genetic constructs that might be toxic or incompatible with the intermediate host. Since this method relies on PCR-amplification based in vitro assembly of DNA fragments, it must be noted that the accuracy can be affected by mutations that occur during PCR amplification and the possible formation of unspecific assembly fragments. To minimize the risk of mutations, a high-fidelity polymerase (Q5 DNA polymerase) was used in this study [40]. To accelerate the identification of positive colonies, a visible reporter like mCherry can be included or colony PCR can be performed. Using these methods, we confirmed that the accuracy of the transformed clones was above 80%. While we have tested this direct cloning method only in L. plantarum WCFS1, we believe this strategy can also be expanded to other hard-to-transform lactobacilli, in which similar plasmids have been previously transformed using the indirect method [41]. When testing the direct cloning method on different strains, it is important to note that success will depend on whether they accept unmethylated DNA. Furthermore, if transformation is hindered by restriction-modification systems in these strains, DNA design strategies can be employed to overcome this challenge [42]. Finally, while we have used modest-sized plasmid (<4 kb) with a low copy number replicon (P256 replicon, copy number 3–5). Based on previous studies [43], it is expected that bigger plasmids with higher copy number replicons can be transformed using the direct cloning method although further investigations are definitely needed to test the effect of plasmid size on transformation efficiency and accuracy of the transformed clones.

Supporting information

S1 File. Supporting information file containing the step-by-step protocol generated in this study.

(PDF)

Click here for additional data file.^{(139.1KB, pdf)}

S2 File. Supporting information file containing all supporting information tables and figures.

(DOCX)

Click here for additional data file.^{(1.3MB, docx)}

Acknowledgments

The plasmid pLp3050sNuc was a kind gift from Prof. Geir Mathiesen.

Data Availability

All raw data including images and sequencing files related to results described in this paper have been added to the OSF data repository and can be accessed at this DOI - DOI: 10.17605/OSF.IO/C6X3D.

Funding Statement

This work was supported by a the Deutsche Forschungsgemeinschaft (DFG) Research grant [Project # 455063657 - https://gepris.dfg.de/gepris/projekt/455063657] for M.B.A., the DFG Collaborative Research Centre, SFB 1027 [Project # 200049484 - https://gepris.dfg.de/gepris/projekt/466932240] for S.S. and the Leibniz-Gemeinschaft's Leibniz Science Campus on Living Therapeutic Materials [LifeMat - https://www.lsclifemat.de/] for S.D. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0281625.r001

Decision Letter 0

Hari S Misra

6 Dec 2022

PONE-D-22-25198Gibson Assembly-based direct cloning of plasmid DNA in Lactiplantibacillus plantarum WCSF1PLOS ONE

Dear Dr. Sankaran,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Manuscript has been reviewed by 2 subject experts and both have appreciated the work that can be published. However, both have raised some important concerns and have suggested action from authors. Please go through their comments carefully and submit the revision after completely addressing the reviewer's concerns.

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Reviewers' comments:

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Reviewer #2: No

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: The manuscript by Asensio et al, is really a fantastic example on the optimization of cloning methodologies to improve both cloning and transformation efficiencies with a wide range of heterologous expression and biochemical applications. The manuscript is very clear and well written. I recommend the manuscript for publication after some minor comments have been addressed.

The introduction is well written but some more introductory information on recently developed similar cloning strategies and how this method might provide further advantages (for example, increased DNA yield, ability to skip E. coli for toxic genes etc).

The authors do not do a DpnI digestion during the direct cloning approach and it is possible for the original template DNA to make it to the beginning of their transformation step (Day 2). Although the majority of this will be lost during the subsequent purification and PCR steps, did the authors have any problems with the background empty template being transformed? Did they find empty vectors in their colony screen? Could the authors comment on the proportion of positive vs negative clones during their colony screening and subsequent verification of plasmids in the final stages of their experiments?

The schematic figure (Figure 3) is great to show an overview of both methods. Could the authors also include slightly more information such as the strain they are performing their transformation into (cloning strain/expression strain etc). This should help to simplify the advantages of one method over another.

The authors are essentially performing PCRs on PCR products and thus the higher number of cycles eventually leads to the increased chance of incorporating mutations. We use Q5 a lot and understand that it is high fidelity and that mutations are very infrequent, also that the authors performed sequencing to ensure no errors. However, I think it would be nice if an extra sentence or two was placed in the text to ensure it is clear to both the authors and future scientists performing this method are aware of the issue and how it should be handled.

I think the conclusion is a bit weak and the authors could really use this opportunity to highlight the advantages of the method over what is currently available – particularly to the great biotechnology/synthetic biology fields.

Reviewer #2: An article by Blanch-Asensio et al. “Gibson Assembly-based direct cloning of plasmid DNA in Lactiplantibacillus plantarum WCSF1” describes a modified method to deliver plasmids to L. Plantarum. This manuscript needs major revisions before it can be published.

1. The title of the paper gives off the impression that the authors successfully demonstrated in vivo DNA assembly. But the paper is just about obtaining higher plasmid copy numbers before the transformation.

2. Introduction is lacking some information/explanations.

a. Some sentences are not clear “Hence, it is desirable to be able to clone these lactobacilli without the need for an intermediate host” - deliver plasmids?

b. Why rolling circle amplification is not described? The is a great example of using this method for creating synthetic minimal cells - https://www.science.org/doi/10.1126/science.aad6253. How would this method compare with the method described in this manuscript?

3. Results and discussion

a. “As expected, all clones expressing mCherry yielded the correct sequences without any mutations or deletions” – Would you not expect some mutations since you are using PCR to amplify fragments?

b. Why there are only two biological replicates for figure 2A? Legend is a missing description – in B – how many colonies did you check?, C – what was the template to amplify this mCherry gene?

c. The last concluding paragraph is too general. It is not true that by just having more plasmid DNA it will be possible to deliver DNA to “hard-to-transform” bacteria. Restriction-modification systems should be discussed more. What are the sizes of plasmids that you could create using this method? What mutation rate would you expect?

Protocol - please make sure that all information is included: for example, steps 1/2 - how much template DNA did you use? was it plasmid, genomic DNA. What enzyme? etc.

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2023 Feb 16;18(2):e0281625. doi: 10.1371/journal.pone.0281625.r002

Author response to Decision Letter 0

18 Jan 2023

Editor's comments:

>Editor’s comment: We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

>Response: The ‘Financial Disclosure’ has been modified as follows to better match the ‘Funding Information’ section:

“This work was supported by a the Deutsche Forschungsgemeinschaft (DFG) Research grant [Project # 455063657 - https://gepris.dfg.de/gepris/projekt/455063657] for M.B.A., the DFG Collaborative Research Centre, SFB 1027 [Project # 200049484 - https://gepris.dfg.de/gepris/projekt/466932240] for S.S. and the Leibniz-Gemeinschaft's Leibniz Science Campus on Living Therapeutic Materials [LifeMat - https://www.lsclifemat.de/] for S.D. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

>Editor’s comment: Thank you for stating the following financial disclosure:

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

>Response: This statement has been added in the ‘Financial Disclosure’ section as mentioned in the answer to the previous question.

>Editor’s comment: We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

>Response: All raw data including images and sequencing files related to results described in this manuscript have been added to the OSF data repository and will be made accessible once the manuscript is accepted for publication. Till then, a view only link is provided here - https://osf.io/c6x3d/?view_only=fbb9aede3b22455799b81f23a6270c65

>Editor’s comment: PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

>Response: All raw images of gels are provided in the data repository mentioned in the previous answer

>Editor’s comment: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

>Response: The reference list has been checked and additional references after revision of the manuscript have been added and highlighted in the revised manuscript with track changes. To our knowledge we have not cited papers that were retracted. The following references have been added during the revision:

40. Potapov, V. and Ong, J.L., Examining sources of error in PCR by single-molecule sequencing. PloS one. 2017, 12(1), p.e0169774.

41. Karlskås, I.L., Maudal, K., Axelsson, L., Rud, I., Eijsink, V.G. and Mathiesen, G. Heterologous protein secretion in lactobacilli with modified pSIP vectors. PLOS one. 2014 9(3), p.e91125.

42. Johnston, C.D., Cotton, S.L., Rittling, S.R., Starr, J.R., Borisy, G.G., Dewhirst, F.E. and Lemon, K.P. Systematic evasion of the restriction-modification barrier in bacteria. Proceedings of the National Academy of Sciences, 2019 116(23), pp.11454-11459.

43. Mathiesen, G., Øverland, L., Kuczkowska, K. and Eijsink, V.G. Anchoring of heterologous proteins in multiple Lactobacillus species using anchors derived from Lactobacillus plantarum. Scientific Reports. 2020 10(1), pp.1-10.

Reviewer's comments:

Reviewer 1:

>Reviewer’s comment: The manuscript by Asensio et al, is really a fantastic example on the optimization of cloning methodologies to improve both cloning and transformation efficiencies with a wide range of heterologous expression and biochemical applications. The manuscript is very clear and well written. I recommend the manuscript for publication after some minor comments have been addressed.

>Response: We thank the reviewer for the positive remarks.

>Reviewer’s comment: The introduction is well written but some more introductory information on recently developed similar cloning strategies and how this method might provide further advantages (for example, increased DNA yield, ability to skip E. coli for toxic genes etc).

>Response: We thank the reviewer for this perspective. We tried to keep the introduction focused on the most important aspects directly related to the method we have described. We have now revised the introduction and included some more information and explanations. We expect the current introduction to have a better coverage of cloning protocols reported in literature as well as simultaneously highlighting the benefit of our work.

>Reviewer’s comment: The authors do not do a DpnI digestion during the direct cloning approach and it is possible for the original template DNA to make it to the beginning of their transformation step (Day 2). Although the majority of this will be lost during the subsequent purification and PCR steps, did the authors have any problems with the background empty template being transformed? Did they find empty vectors in their colony screen?

>Response: We acknowledge that prior to setting up the Gibson Assembly reaction in the direct cloning protocol, we did not conduct a DpnI digestion of the backbone vector template. This step is normally conducted for indirect cloning protocol mediated through E. coli DH5α in order to reduce the number of clones containing the circular vector backbone.

We assumed that the undigested circular backbone vector would be significantly diluted during the multiple purification processes in the direct cloning protocol to produce negative clones in L. plantarum. Additionally, PCR screening of randomly selected transformants showed presence of the gene of insert confirming that clones do contain the recombinant plasmid and not the empty backbone vector.

However, following the reviewer’s suggestion, we repeated the experiment for constructing the pTlpA-mCherry plasmid by conducting a DpnI digestion of the vector backbone template before the Gibson Assembly reaction. After transformation, we verified by PCR that the red colonies (producing the desired phenotype) carry the assembled plasmid after the HiFi reaction and not the vector backbone. We screened 10 colonies for this experimental setup and 10 through the non-DpnI digested experimental setup. We got amplification at the expected size (558 bp) for both conditions. This helped us confirm our assumption that DpnI digestion of the backbone vector template does not provide significant advantage against the removal of empty clones after transformation. This data can be found in Figure S3.

Concerning this experiment, we have added the following text to the manuscript:

“Furthermore, we wanted to prove that a DpnI digestion prior to the Gibson HiFi Assembly reaction would not have an impact on the number of positive clones obtained through the direct cloning method since we assumed that the backbone vectors used as template for the PCR would be extremely diluted during the multiple purification steps involved in the protocol. Therefore, we repeated the pLp_mCherry direct cloning with and without a DpnI digestion of the insert and vector PCR products prior to the Gibson HiFi Assembly reaction. We screened 10 red colonies for both experimental conditions by PCR amplification of a partial gene segment within the mCherry reporter. The proportion of red colonies obtained was similar in both conditions and colony PCR amplification of a part of the mCherry gene (10 red colonies from each) yielded PCR products of the same expected size from all clones. This confirmed that a DpnI digestion prior to the HiFi Assembly reaction is not necessary (Supporting Information Figure S3).”

>Reviewer’s comment: Could the authors comment on the proportion of positive vs negative clones during their colony screening and subsequent verification of plasmids in the final stages of their experiments?

>Response: Regarding positive and negative clones during colony screening, we found that all colonies that were red due to mCherry expression yielded positive clones on sequencing. This had been briefly mentioned in the manuscript with the following text:

“As expected, all clones expressing mCherry yielded the correct sequences without any mutations or deletions.”

On average, the number of red to non-red colonies that resulted from direct transformation was 83%. Figure 2B has now been updated in the manuscript.

Based on the reviewer’s comment, we further screened 10 non-red colonies by amplifying the whole mCherry gene by colony PCR. The expected band should be 1140 bp. We got amplification (data can be found in Figure S4) for 7 out of 10 colonies. However, for most of them the amplicon was bigger than expected, suggesting the possibility that a minor proportion of wrongly assembled products were formed during Gibson assembly or random mutations were introduced during the PCR reaction.

This is the text we have added to the manuscript addressing the screening of non-red clones.

“The whole mCherry gene was also amplified from 10 non-red colonies using the same set of primers and PCR conditions as in Figure 2C. We obtained amplification for 7 out of 10, nevertheless, the PCR product was either bigger or smaller than expected (Supporting Information Figure S4). These results suggest that mutations might have occurred during the PCR amplification steps or a minor proportion of wrongly assembled products were formed during Gibson assembly which can result in recombinant clones with the mutated gene of interest (GOI).”

>Reviewer’s comment: The schematic figure (Figure 3) is great to show an overview of both methods. Could the authors also include slightly more information such as the strain they are performing their transformation into (cloning strain/expression strain etc). This should help to simplify the advantages of one method over another.

>Response: We thank the reviewer for their suggestion. We have made modifications in the Figure 3 scheme to incorporate details about the cloning strains during the bacterial transformation steps. We have also modified the scheme of Figure 1 in order to highlight the major attributes of the cloning procedure in further detail.

>Reviewer’s comment: The authors are essentially performing PCRs on PCR products and thus the higher number of cycles eventually leads to the increased chance of incorporating mutations. We use Q5 a lot and understand that it is high fidelity and that mutations are very infrequent, also that the authors performed sequencing to ensure no errors. However, I think it would be nice if an extra sentence or two was placed in the text to ensure it is clear to both the authors and future scientists performing this method are aware of the issue and how it should be handled.

>Response: We thank the reviewer for their suggestion. We have added the following text in the newly written “Conclusions” section, highlighting the need for using a high-fidelity polymerase when following this protocol to minimize mutation risks as much as possible.

“Since this method relies on PCR-amplification based in vitro assembly of DNA fragments, it must be noted that the accuracy can be affected by mutations that occur during PCR amplification and the possible formation of unspecific assembly fragments. To minimize the risk of mutations, a high-fidelity polymerase (Q5 DNA polymerase) was used in this study [40].”

>Reviewer’s comment: I think the conclusion is a bit weak and the authors could really use this opportunity to highlight the advantages of the method over what is currently available – particularly to the great biotechnology/synthetic biology fields.

>Response: We acknowledge the need for a separate “Conclusion” subheading in the manuscript. We have used this section to highlight the advantages of this cloning methodology for different applications in the field of synthetic biology. The revised text now includes the following statements:

“The direct cloning method developed in this paper has proved effective in transforming circular dsDNA (plasmid) DNA into L. plantarum WCFS1 without any intermediate host requirement for plasmid amplification. We demonstrate that this method provides two major benefits for lactobacillus engineering – (i) it saves time of at least 2 days compared to commonly used indirect cloning methods involving intermediate hosts and (ii) enables the cloning of genetic constructs that might be toxic or incompatible with the intermediate host. Since this method relies on PCR-amplification based in vitro assembly of DNA fragments, it must be noted that the accuracy can be affected by mutations that occur during PCR amplification and the possible formation of unspecific assembly fragments. To minimize the risk of mutations, a high-fidelity polymerase (Q5 DNA polymerase) was used in this study [40]. To accelerate the identification of positive colonies, a visible reporter like mCherry can be included or colony PCR can be performed. Using these methods, we confirmed that the accuracy of the transformed clones was above 80%. While we have tested this direct cloning method only in L. plantarum WCFS1, we believe this strategy can also be expanded to other hard-to-transform lactobacilli, in which similar plasmids have been previously transformed using the indirect method [41]. When testing the direct cloning method on different strains, it is important to note that success will depend on whether they accept unmethylated DNA. Furthermore, if transformation is hindered by restriction-modification systems in these strains, DNA design strategies can be employed to overcome this challenge [42]. Finally, while we have used modest-sized plasmid (<4 kb) with a low copy number replicon (P256 replicon, copy number 3 – 5). Based on previous studies [43], it is expected that bigger plasmids with higher copy number replicons can be transformed using the direct cloning method although further investigations are definitely needed to test the effect of plasmid size on efficiency of transformation and accuracy of the transformed clones.”

Reviewer 2:

>Reviewer’s comment: An article by Blanch-Asensio et al. “Gibson Assembly-based direct cloning of plasmid DNA in Lactiplantibacillus plantarum WCSF1” describes a modified method to deliver plasmids to L. Plantarum. This manuscript needs major revisions before it can be published.

>Response: We acknowledge the suggestions of the reviewer and have made substantial revisions to the manuscript. The changes have been highlighted in the manuscript for faster identification.

>Reviewer’s comment: The title of the paper gives off the impression that the authors successfully demonstrated in vivo DNA assembly. But the paper is just about obtaining higher plasmid copy numbers before the transformation.

>Response: We agree with the concern with the reviewer regarding the title of the manuscript. Our protocol mainly focusses on the in vitro assembly of circular plasmid DNA prior to the transformation in L. plantarum competent cells and does not rely on any in vivo assembly of gene segments in the intracellular milieu of the bacteria. To avoid misleading the readers in any form we have changed the title of our manuscript to “In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1”.

>Reviewer’s comment: Introduction is lacking some information/explanations.

Apart from the introduction, we have included a dedicated Conclusions section that summarizes our results in a broader context with additional information from literature.

>Reviewer’s comment: Some sentences are not clear “Hence, it is desirable to be able to clone these lactobacilli without the need for an intermediate host” - deliver plasmids?

>Response: We agree with the reviewer that the sentence construction for the statement seems ambiguous. We have corrected the statement to convey the message more clearly:

“Hence, it is desirable to be able to directly transform circular plasmid dsDNA into the lactobacilli strains without relying on intermediate bacterial hosts like E. coli and L. lactis.”

>Reviewer’s comment: Why rolling circle amplification is not described? The is a great example of using this method for creating synthetic minimal cells -https://www.science.org/doi/10.1126/science.aad6253. How would this method compare with the method described in this manuscript?

>Response: We thank the reviewer for bringing up this complementary technique for DNA amplification. In this study, we focused on employing the most common methods used by the lactobacilli community so that they can easily adopt the method we developed. Rolling Circle Amplification is a powerful technique to greatly amplify DNA from very low template concentrations and has been used to solve complex challenges like those faced in the generation of minimal bacterial genomes. However, this method also faces several limitations like the need for specific sequences on the template DNA for the recognition of the nicking enzyme, non-specific amplification that can lead to false positives and the need for restriction enzymes to break up the long DNA fragment into individual plasmids. For these reasons, this method is not commonly used in bacterial engineering.

Accordingly, we also do not have practical experience with this method and any comparison we can make with our method would be purely speculative. Thus, we consider that the inclusion of a discussion based on RCA will be beyond the scope of this study and distract the reader from the main message of the manuscript.

>Reviewer’s comment: “As expected, all clones expressing mCherry yielded the correct sequences without any mutations or deletions” – Would you not expect some mutations since you are using PCR to amplify fragments?

>Response: We agree with the concerns of the reviewer in terms of spontaneous mutations being generated while amplifying the assembled gene fragments by PCR. In order to avoid the generation of mutations in these cases, we have chosen the high fidelity Q5 DNA Polymerase enzyme for minimizing the error rate for getting the correct amplicon. The most common form of error introduction is attributed to base substitution which has been reported to be the lowest for the Q5 polymerase (Potapov et al., 2017).

Despite that, there obviously lies a chance of generating some mutations in the gene of interest which might be passed on to the recombinant clones after successful transformation. It should be noted that sequencing was done only for red clones which provided correct-sized bands post colony PCR analysis. In these cases, it was expected that no mutations would have occurred, which is the basis of the statement mentioned by the reviewer. However, it should be noted that about 80% of the colonies were red (Figure 2B), suggesting that mutations or assembly errors might have occurred in the remaining transformants. We have now performed colony PCR with those non-red colonies and confirmed that the amplified products are bigger or smaller than what is expected (Figure S4). We have further included text to clarify the possibility of mutations or assembly errors and in the Conclusions:

“These results suggest that mutations might have occurred during the PCR amplification steps or a minor proportion of wrongly assembled products were formed during Gibson assembly.”

Conclusions – “Since this method relies on PCR-amplification based in vitro assembly of DNA fragments, it must be noted that the accuracy can be affected by mutations that occur during PCR amplification and the possible formation of unspecific assembly fragments. To minimize the risk of mutations, a high-fidelity polymerase (Q5 DNA polymerase) was used in this study [40]. To accelerate the identification of positive colonies, a visible reporter like mCherry can be included or colony PCR can be performed. Using these methods, we confirmed that accuracy of the transformed clones was above 80%.”

References:

1. Potapov, V. and Ong, J.L., 2017. Examining sources of error in PCR by single-molecule sequencing. PloS one, 12(1), p.e0169774.

>Reviewer’s comment: Why there are only two biological replicates for figure 2A? Legend is a missing description – in B – how many colonies did you check?, C – what was the template to amplify this mCherry gene?

>Response: We acknowledge the comments of the reviewer. We have performed a third replicate and updated the graphs of Figure 2A and 2B. In the accuracy graph (Figure 2B), we checked 54 colonies for the 300 ng transformations (41 were red; 75.92% accuracy), 89 for the 600 ng transformations (83 were red; 93.25% accuracy), 133 for the 900 ng transformations (105 were red; 78.94 % accuracy), and 142 for the 1200 ng transformations (118 were red; 83.09% accuracy). Overall, 347 out of 418 colonies were red, which accounted for 83.01% accuracy. We have added these details to the manuscript:

“Colonies were streaked on fresh plates and the following day they were examined for the presence of fluorescent protein. Overall, 347 out of 418 colonies were red, giving an accuracy of 83% (Figure 2B).”

For figure 2C, we used a red colony as template for the colony PCR. We have added this detail to the legend:

“Agarose gel showing the colony PCR product (1140 bp) corresponding to the mCherry gene of interest (GOI). A red L. plantarum colony obtained after direct cloning was used as the template DNA for the PCR reaction. Generuler 100 bp Plus DNA Ladder (ThermoFisher ScientificTM) was used for the reference.”

Additionally, we have added an extra figure, Figure S4. We thought that it was interesting to amplify the whole mCherry gene from non-red colonies so we could get some insight into why these clones were not red. We screened 10 non-red colonies by performing the same PCR as in Figure 2C. Out of 10 colonies, we got clear amplification for 7. However, the PCR product was either bigger or shorter than expected (Figure 2C). These results possibly point out that mutations were introduced during the PCR amplification or wrongly assembled products were formed during Gibson assembly.

Regarding this additional experiment, we have added the following text to the manuscript:

“The whole mCherry gene was also amplified from 10 non-red colonies using the same set of primers and PCR conditions as in Figure 2C. We obtained amplification for 7 out of 10, nevertheless, the PCR product was either bigger or smaller than expected (Supporting Information Figure S4). These results suggest that a minor proportion of wrongly assembled products were formed during Gibson assembly which can result in recombinant clones with the mutated gene of interest (GOI).”

>Reviewer’s comment: The last concluding paragraph is too general. It is not true that by just having more plasmid DNA it will be possible to deliver DNA to “hard-to-transform” bacteria. Restriction-modification systems should be discussed more. What are the sizes of plasmids that you could create using this method? What mutation rate would you expect?

>Response: We acknowledge that the concluding paragraph was too generalized and did not provide sufficient coverage to related aspects of bacterial transformation. We have now used the “Conclusions” section in the manuscript to provide further insights in this regard. The revised text now includes the following statements:

Reviewer’s comment: Protocol - please make sure that all information is included: for example, steps 1/2 - how much template DNA did you use? was it plasmid, genomic DNA. What enzyme? Etc.

Response: The protocol shared in the public repository has been written to provide a detailed overview of the cloning strategy. We had generalized some technical details in the protocol to provide flexibility to readers while designing individual experiments. Therefore, we had not specified the origin of the template DNA in the original version (e.g., plasmid, genomic DNA, synthetic gene).

However, we acknowledge the reviewer’s suggestions to provide further details to ensure the protocol’s reproducibility. In step 1 (Molecular Cloning Part) we have highlighted the imperative use of a high-fidelity polymerase for the reaction. In steps 1 and 2 (Molecular Cloning Part) we have also specified the amount of template DNA we recommend using (10 ng). In step 2 (Molecular Cloning Part) we have added the specific requirements pertaining to the primer overhang lengths for ensuring ideal assembly. Finally, we have also included additional details in step 6 (Molecular Cloning Part) specifying the importance of an impeccable design of primers, suggesting a thorough analysis of the primers needed for the amplification of the assembled DNA with a primer design tool.

This is the link to the updated Protocol:

https://www.protocols.io/private/CD173FD23D7711EDB7F00A58A9FEAC02

PLoS One. doi: 10.1371/journal.pone.0281625.r003

Decision Letter 1

Hari S Misra

30 Jan 2023

In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1

PONE-D-22-25198R1

Dear Dr. Sankaran,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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PLoS One. doi: 10.1371/journal.pone.0281625.r004

Acceptance letter

Hari S Misra

7 Feb 2023

PONE-D-22-25198R1

In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1

Dear Dr. Sankaran:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. Supporting information file containing the step-by-step protocol generated in this study.

(PDF)

Click here for additional data file.^{(139.1KB, pdf)}

S2 File. Supporting information file containing all supporting information tables and figures.

(DOCX)

Click here for additional data file.^{(1.3MB, docx)}

Data Availability Statement

All raw data including images and sequencing files related to results described in this paper have been added to the OSF data repository and can be accessed at this DOI - DOI: 10.17605/OSF.IO/C6X3D.

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PERMALINK

In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1

Marc Blanch-Asensio

Sourik Dey

Shrikrishnan Sankaran

Roles

Abstract

Introduction

Materials and methods

Bacterial strains and growth conditions

Molecular biology

L. plantarum WCFS1 electrocompetent cell preparation

Electroporation based transformation in L. plantarum WCFS1

Direct cloning method in L. plantarum WCFS1

Fig 1. Scheme of the PCR-based plasmid amplification in the direct cloning method.

Indirect cloning via the intermediate host strain E. coli

Results and discussion

Transformation efficiency and accuracy of the direct cloning method

Fig 2.

Time requirement for the direct and indirect cloning methods

Fig 3. Schematic representation of the steps and temporal requirements for the direct and indirect cloning methods used for L. plantarum WCFS1.

Direct cloning of a gene incompatible with E. coli

Fig 4.

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Hari S Misra

Roles

Author response to Decision Letter 0

Decision Letter 1

Hari S Misra

Roles

Acceptance letter

Hari S Misra

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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