Characterization of a novel Escherichia coli recombineering selection/counterselection cassette

doi:10.21203/rs.3.rs-2062657/v1

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Characterization of a novel Escherichia coli recombineering selection/counterselection cassette

https://doi.org/10.21203/rs.3.rs-2062657/v1

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Recombineering is a highly efficient DNA cloning and modification technique by using the recombinase-mediated homologous recombination. Selection/counterselection cassette is often used in chromosomal DNA or large episomal DNA manipulation, in which the selection marker is used for the first step cassette selection, while the counterselection marker is used for the second step replacement of the cassette by the target gene. A variety of selection/counterselection cassettes are reported, however, they suffer from the shortcomings of the requirement of pre-engineered strain or specific culture medium. Herein, we report a novel S-tetR-ptetA-ccdB-aacC1-S selection/counterselection cassette that sidesteps the shortcomings. As a proof-of-concept, one-step gene cloning (0.7 kb, 1.7 kb, and 4.2 kb) and two-step Escherichia coli chromosomal gene knock-in (0.7 kb and 4.2 kb) were performed. The gene cloning and gene knock-in efficiencies are high up to 90%. The cassette adds a powerful tool to the recombineering repertoire.

CcdB

Cloning

I-SceI

Knock-in

Recombineering

Selection/counterselection cassette

Recombineering (recombination-mediated genetic engineering), also called l Red recombineering, is a homologous recombination-based technique (Murphy 2016). Due to its high efficiency and easy manipulation, recombineering has found a variety of applications including gene cloning and genome engineering (Lobanova et al. 2022; Ma et al. 2015; Maria et al. 2021; Robertson et al. 2021; Tiwari et al. 2022). Firstly established in Escherichia coli, recombineering has extended to a large number of gram positive and gram negative bacteria. Selection/counterselection cassette is usually involved in a two-step modification of bacterial chromosome or large episomal DNA (like bacterial artificial chromosome). The first step is integration of the cassette into the target DNA via selection marker, while the second step is the replacement of the cassette with oligonucleotide or target gene, resulting in gene deletion, gene replacement or gene knock-in.

Numerous of counterselection markers were developed, such as galK, thyA, rpsL, pyrF, pheS, sacB, upp, ccdB, tetA-sacB, and tolC. However, these counterselection markers normally require pre-generated strain or specific culture medium. For example, rpsL, thyA, pyrF, upp, and tolC need corresponding gene-deficient mutant; and galK, pheS, sacB, tetA-sacB need medium containing specific (sometimes expensive) chemicals (Baas-Thomas et al. 2022; Li et al. 2013; Wang et al. 2014).

Herein, we report a new selection/counterselection cassette, S-tetR-ptetA-ccdB-aacC1-S, for E. coli recombineering. The cassette contains the gentamicin resistance gene (aacC1) as selection maker and chlortetracycline-inducible toxic gene ccdB and I-SceI as double counterselection markers. Proof-of-concept characterization of the cassette was demonstrated by one-step gene cloning (0.7 kb, 1.7 kb, and 4.2 kb) and two-step E. coli chromosomal gene knock-in (0.7 kb and 4.2 kb). Application of the cassette is straightforward, requires no pre-engineered strain or specific medium. The highly efficient selection/counterselection cassette adds a powerful tool to the recombineering toolbox.

Bacterial strains, culture conditions, and chemical regents

E. coli DH10B was used as general vector construction and chromosomal gene knock-in host. E. coli LS027 (Zhang et al. 2017) was used for R6K replicon plasmid cloning. LB (10 g/l tryptone, 5 g/l yeast extract and 10 g/l NaCl) medium and LB plate containing 1.5% agar were used for strain culture. When required, the antibiotics were supplemented as follows: Ampicillin (Ap), 50 mg/ml; chloramphenicol (Cm), 25 mg/ml; gentamicin (Gm), 25 mg/ml; kanamycin (Km), 30 mg/ml; tetracycline (Tc), 25 mg/ml. Restriction enzymes and T4 ligase were purchased form NEB (USA). Plasmid extraction kit, gel extraction kit, chlortetracycline (cTc) and other chemicals were ordered from Sangon (China). Heat-treated cTc was prepared by dissolving cTc in fresh prepared LB and autoclaved, a final concentration of 25 mg/ml (w/v) was used for the induced expression of intron-encoded homing endonuclease I-SceI.

Vector construction

Molecular biology preformation was carried out as per standard protocols. The detailed vector construction procedure is provided in Supplementary material. Strains and plasmids used and generated in this research are listed in Supplementary Table 1. The PCR primers are listed in Supplementary Table 2.

Recombineering manipulation

pLS2358 (Chen et al. 2016), initially constructed for genome engineering of Pseudomonas putida KT2440, was used for recombineering manipulation in this study. pLS2358 harbors a broad-host range of oriV replicon and the Red operon and I-SceI which were induced with 2 mM m-toluic acid and 0.2% L-rhamnose (w/v), respectively. E. coli DH10B harboring pLS2358 recombineering-proficient

electrocompetent cells preparation and electroporation were carried out as reported (Chen et al. 2016). The recombineering primers consisted of 50 bp homologous arm (HA) and 20 bp priming nucleotides. For gene cloning, one mg of PCR amplified targeting DNA and 10 ng of pLS4509 were co-electroplated with 1 mm cuvette using Bio-Rad electroporator; electroporated cells were suspended in 1 ml of LB and incubated at 37℃ for 1 h before diluted and plated on LB agar plate supplemented with Km and cTc. Randomly picked clones were duplicated on LB agar plate with Gm and Km. Gm^RKm^S clones were subsequently cultured for plasmid extraction, restriction enzyme digestions and nucleotide sequencing. For chromosomal gene knock-in, the PCR amplified HA-flanked S-araC-pBAD-ccdB-aacC1-S cassette was amplified from pLS4520 and electroporated. The cassette knock-in strain was confirmed via PCR and sequencing. The genotyping primers were designed around 100 bp away from the recombination loci. The second, gene knock-in step was conducted by electroporating the PCR amplified, HA-flanked target gene into the recombineering-proficient intermediate strain. The cTc resistant clones were duplicated on LB agar plate with and without Gm. Gm^Sclones were genotyped by PCR and sequencing. Finally, pLS2358 was eliminated from the knock-in strain by plating the growth cells on LB (without NaCl) agar plate supplemented with 10% (w/v) surose and selection of Tc^S clones.

Selection of the best selection/counterselection cassette

Both CcdB are SacB are powerful counterselecction markers; however, we still observed a large number clones under CcdB or SacB counterselection (data not shown). Homing endonuclease I-SceI recognizes and cleaves an 18 bp nucleotide sequences, causing double strand DNA break (DSB) and promotes the DSB repair. We thus assumed that double counterselection markers by combing I-SceI with either CcdB or SacB would greatly reduce or totally eliminate the background clones. Based on this assumption, we constructed four novel selection/counterselection cassettes: S-araC-pBAD-ccdB-aacC1-S, two types of S-tetR-ptetA-ccdB-aacC1-S and S-sacB-aacC1-S. Besides ccdB or sacB, I-SceI acts as the counterselection marker by cleaving the two flanked I-SceI recognition sites. The first L-arabinose-inducible ccdB expression system harbors a variant ribosome binding site (RBS) sequence that reduces the pBAD promoter activity (Chen and Zhao 2005).

We initially tried to clone the ccdB under the control of native tetR-ptetA regulatory system (Posfai et al. 1999), speculating that transformed clones would survive as tight regulation of the tetracycline derivative inducible promoter sidesteps the CcdB toxicity. Unexpectedly, only a few clones were obtained, and sequence analysis revealed that the ccdB gene was mutated in all five clones, indicating that the native ptetA promoter is not stringent for ccdB expression. We then followed the first cassette paradigm by changing the original RBS containing sequence (before start codon ATG) from GAGAAAAGTGAA to GATTGAAAACG, resulting in the first S-tetR-ptetA-ccdB-aacC1-S cassette plasmid pLS4512 with CcdB E87K mutation. We previously found that I-SceI was tightly regulated by the L-rhamnose inducible system in Pseudomonas putida KT2440 (Chen et al. 2016), therefore, we also cloned the AGGATCACATT sequence from E. coli L-rhamonose operon promoter before the start codon of ccdB, leading to the second S-tetR-ptetA-ccdB-aacC1-S cassette plasmid pLS4514 with CcdB L36R S70N mutation.

Comparison of the four selection/counterselection cassettes was carried out by electroporation of 10 ng of each plasmid into E coli DH10B. The cells were10^–³-fold diluted and spreaded on various selection plates. The cassette with least obtained clones would be the best candidate for recombineering experiments as under counterselection only recombinant plasmid could be obtained and no or very few cassette plasmid would survive. As shown in Fig. 1, while single I-SceI and CCdB counterselectionc still generated a large number of clones, combined counterselection (I-SceI with CcdB or SacB) resulted in much less transformants. And remarkably, no transformant was obtained for the two S-araC-pBAD-ccdB-aacC1-S cassettes under the combined counterselection of I-SceI and CcdB. As pLS4512 showed more clones (which means less background CcdB toxicity) in the control group than that of pLS4514, it was selected for further characterization.

(Fig. 1 inserted here)

Gene cloning

Gene cloning is a routine experiment in molecular biology. As a proof-of-concept, we first demonstrated the usage of S-araC-pBAD-ccdB-aacC1-S cassette via gene cloning with scheme shown in Fig. 2.

(Fig. 2 inserted here)

Three genes were selected: 702 bp proteinase encoding gene TEV, 1700 bp levansucrase encoding gene sacB, and 4107 bp endonuclease encoding gene cas9. The TEV gene, sacB gene and cas9 gene were PCR amplified from LS2416 (Luo et al. 2015), pKSacB (Luo et al. 2016), and pCas9 (Jiang et al. 2013), respectively. Each DNA fragment was co-transformed with pLS4509 into the Red-proficient cells of E. coli DH10B harboring pLS2358. Usually one mg of targeting DNA generated hundreds of Gm^Rclones from which 100 were duplicated and Km^RGm^Sclones were selected for plasmid validation. From one typical experiment, 87%, 84% and 79% clones were Km^RGm^Sfor TEV gene, sacB gene and cas9 gene cloning, respectively. And all the Km^RGm^Sclones were the correct recombinant plasmids. The restriction enzyme maps of the recombinant plasmids are shown in Supplementary Figure 1; the restrictive digestions of the plasmids are shown in Fig. 3A, 3B, and 3C.

(Fig. 3 inserted here)

Chromosomal gene knock-in

One main utility of recombineering is chromosomal gene knock-in. Introducing foreign gene(s) into the chromosome is essential for the construction of value-added product producing strain. Compared with plasmid-based gene expression, chromosome-based gene expression shows the advantages of cells homogeneity, no use of antibiotics, and no metabolic burden for the plasmid maintenance. As stated in “Material and methods”, we firstly knocked the S-araC-pBAD-ccdB-aacC1-S cassette into E. coli DH10B chromosomal endA1 locus. Then HA-flanked 714 bp egfp gene which was amplified from pJOE4905.1 (Motejadded and Altenbuchner 2009), and HA-flanked 4107 bp cas9 gene which was amplified form pCas9 (Jiang et al. 2013), was each electroporated into the intermediate strain. Gm^S clones were obtained and subjected to PCR screening. Nine of 10 Gm^S clones for egfp knock-in and eight of 10 Gm^S clones for cas9 knock-in ere expected knock-in clones. The PCR screening results are shown in Fig. 3D and Fig. 3E. The high knock-in efficiency makes the duplication of Gm^S procedure unnecessary. Indeed, we directly PCR genotyped the transformants and found that for both egfp and cas9 knock-in, each eight of randomly picked 10 clones were correct. The chromosomal gene knock-in scheme, including PCR genotyping of the starter strain E. coli DH10B, cassette knock-in intermediate strain, and final gene knock-in stain, is shown in Supplementary Figure 2.

In summary, a highly efficient selection/counterselection cassette was developed for E. coli recombineering. The application of the cassette requires neither pre-generated strain nor specific medium; and unlike single CcdB counterselection, the cassette circumvents the CcdA-expression plasmid (Wang et al. 2014) or chromosomal ccdB inducible elements (Zhang et al. 2017). Besides gene cloning and chromosomal gene knock-in, the novel system might also be useful for gene mutation (Wang et al. 2022) and gene replacement (Robertson et al. 2021).

Acknowledgements We thank for Dr. Josef Altenbuchner and Dr. Michael Kahn for the plasmids used in this study.

Author contributions GS conceived and designed the study. GZ, QZ, JW and JZ performed the experiments. All authors contributed to data analysis, and to writing and editing of the manuscript.

Funding This work was generously funded by National Natural Science Foundation of China (NSFC 82073742).

Conflict of interest The authors report no conflicts of interest.

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Characterization of a novel Escherichia coli recombineering selection/counterselection cassette

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Abstract

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Materials And Methods

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