Restriction-free Cloning Research Articles

Application of Restriction Free (RF) Cloning in Circular Permutation.

The restriction free (RF) cloning has emerged as one of the highly efficient techniques in the area of genetic engineering. RF cloning has wide range of applications in plasmid DNA manipulation including cloning of a single gene, simultaneous assembly of multiple DNA fragments, and mutagenesis from single to multiple simultaneous alterations of a target DNA. Recently, we have developed a new technique of circular permutation using RF cloning. Circular permutation is widely used to investigate the mechanisms of protein folding and function. Previously, restriction enzyme based cloning was used to introduce circular permutation. Our RF cloning method made the protocol faster and more cost-effective. In this chapter, we describe a step-by-step protocol for generating circular permutants using RF methodology.

Restriction-free cloning for molecular manipulation and augmented expression of banana bunchy top viral coat protein

The online version contains supplementary material available at 10.1007/s13205-021-03017-x.

Production of Multiprotein Complexes Using the Baculovirus Expression System: Homology-Based and Restriction-Free Cloning Strategies for Construct Design.

Most cellular processes are mediated by multi-subunit protein complexes which have attracted major interest in both academia and industry. Recombinant production of such entities in quantity and quality sufficient for functional and structural investigations may be extremely challenging and necessitate specific technologies. The baculovirus expression vector system is widely used for the production of eukaryotic multiprotein complexes, and a variety of strategies are available to assemble transfer vectors for the generation of recombinant baculoviruses. Here we detail applications of homology-based cloning techniques for one-step construction of dual promoter baculovirus transfer plasmids and of restriction-free (RF) cloning for the modification of existing constructs.

ABC cloning: An efficient, simple, and rapid restriction/ligase-free method

DNA cloning remains the primary step before the further investigation of gene function. Restriction enzyme-based cloning methods are still widely used and numerous restriction-free cloning techniques are available as alternatives. Here we describe a PCR-based cloning method named ABC cloning. This method uses PCR to combine three overlapping DNA fragments into a recombinant vector that can be immediately transformed into competent cells. This technique uses only a thermostable DNA polymerase and is more rapid and efficient than previously described methods.

The benefits of foliar inoculation with Azospirillum brasilense in soybean are explained by an auxin signaling model

Azospirillum sp. is one of the most studied genera of plant growth-promoting rhizobacteria (PGPR). The ability of Azospirillum sp. to promote plant growth has been associated with its ability to produce several phytohormones, such as auxins, gibberellins and cytokinins, but mainly indole-3-acetic acid (IAA). It has been propoosed that the production of IAA explains the positive effects of co-inoculation with Azospirillum sp. on the rhizobia-legume symbiosis. In this study, we constructed an IAA-deficient mutant of A. brasilense Az39 (ipdC − ) by using a restriction-free cloning method. We inoculated soybean seeds with 1·106 cfu·seed−1 of Bradyrhizobium japonicum E109 and co-inoculating leaves at the V3 stage with 1·108 cfu.plant−1 of A. brasilense Az39 wt or ipdC − or inoculated leaves with 20 μg.plant−1 synthetic IAA. The results confirmed soybean growth promotion as there was increased total plant and root length, aerial and root dry weight, number of nodules on the primary root, and an increase in the symbiosis established with B. japonicum E109. Nodule weight also increased after foliar co-inoculation with the IAA- producer A. brasilense Az39. The exogenous application of IAA decreased aerial and root length, as well as the number of nodules on primary roots in comparison with the Az39 wt strain. These results allow us to propose a biological model of response to foliar co-inoculation of soybean with IAA-producing rhizobacteria. This model clearly shows that both the presence of microorganism as part of the colonization process and the production of IAA in situ are co-responsible, via plant signaling molecules, for the positive effects on plant growth and symbiosis establishment.

Development of live attenuated bacterial vaccines targeting Escherichia coli heat-labile and heat-stable enterotoxins

Enterotoxigenic Escherichia coli (ETEC), defined by the production of heat labile (LT) and/or heat stable (ST) toxins, are major causes of diarrhea in animals, children in developing countries and to travelers. No broadly protective ETEC vaccine is available, largely because of the difficulty in inducing immunity to the small ST molecule. To take advantage of the demonstration (Liu et al., 2011; Zhang et al., 2013, 2010) that genetically produced fusions of mutant ST with LT subunits can induce effective immunity against both toxins, we engineered a live attenuated vaccine vector strain of E. coli (ZCR533), expressing the immunogenic LT-ST fusions. To present the LT-ST fusions to the mucosal immune system, we used restriction-free cloning to incorporate them into the passenger domain of the autotransporter protein (EspP) expressed on a medium copy number plasmid. This versatile system permits expression of incorporated antigens in either surface-bound or secreted forms by the ZCR533 vector, for delivery to the mucosal inductive sites. Incorporation of the fusions into EspP plasmids was confirmed by PCR and DNA sequencing. Protein expression was confirmed by Western blot of whole cell lysates and culture supernatents using polyclonal antisera to LT. Expression of the surface-targeted fusion on the surface of ZCR533 was confirmed by immuno-fluorescent staining. These studies show that antigenic LT-ST fusions can be surface-expressed or secreted, by our attenuated E. coli ZCR533 vaccine vector via the EspP autotransporter. These constructs could serve as broadly protective vaccine candidates to protect against both LT- and ST-producing ETEC.

Simultaneous Removal of Multiple DNA Segments by Polymerase Chain Reactions.

Precise DNA manipulation is a key enabling technology for synthetic biology. Approaches based on restriction digestion are often limited by the presence of certain restriction enzyme recognition sites. Recent development of restriction-free cloning approaches has greatly enhanced the flexibility and speed of molecular cloning. Most restriction-free cloning methods focus on DNA assembly. Much less work has been dedicated towards DNA removal. Here we introduce a protocol that allows simultaneous removal of multiple DNA segments from a plasmid using polymerase chain reactions (PCR). Our approach will be beneficial to applications in multiple sites mutagenesis, DNA library construction, genetic and protein engineering, and synthetic biology.

Polymerase chain reaction-based gene removal from plasmids.

This data article contains supplementary figures and methods to the research article entitled, “Multiplex gene removal by two-step polymerase chain reactions” (Krishnamurthy et al., Anal. Biochem., 2015, doi:http://dx.doi.org/10.1016/j.ab.2015.03.033), which presents a restriction-enzyme free method to remove multiple DNA segments from plasmids. Restriction-free cloning methods have dramatically improved the flexibility and speed of genetic manipulation compared to conventional assays based on restriction enzyme digestion (Lale and Valla, 2014. DNA Cloning and Assembly Methods, vol. 1116). Here, we show the basic scheme and characterize the success rate for single and multiplex gene removal from plasmids. In addition, we optimize experimental conditions, including the amount of template, multiple primers mixing, and buffers for DpnI treatment, used in the one-pot reaction for multiplex gene removal.

Multiplex gene removal by two-step polymerase chain reactions

Precise DNA manipulation is critical for molecular biotechnology. Restriction enzyme-based approaches are limited by their requirement of specific enzyme sites. Restriction-free cloning has greatly improved the flexibility and speed of precise DNA assembly. Most of these approaches focus on DNA assembly rather than gene removal. Here we present a polymerase chain reaction (PCR)-based cloning method that allows removal of multiple gene segments from plasmids without using restriction enzymes and thermostable ligase. We demonstrate simultaneous removal of three gene segments from a plasmid. This approach could be beneficial to DNA library construction, genetic and protein engineering, and synthetic biology.

An efficient strategy for heterologous expression and purification of active peptide hainantoxin-IV.

Hainantoxin-IV (HNTX-IV) from the venom of the spider Selenocosmia hainana is a potent antagonist that specifically inhibits the tetrodotoxin-sensitive (TTX-S) sodium channels. The toxin peptide consists of 35 amino acids and adopts a typical inhibitory cystine knot (ICK) motif. To obtain adequate HNTX-IV peptides for further insight into the structure-activity relationships of the toxin, a novel strategy including cloning, expression and purification was developed in an E. coli expression system. For this purpose, a seamless restriction-free (RF) cloning method was employed for the construction of an expression vector to avoid introducing unwanted sequences into the target gene. Furthermore, the solubility of recombinant HNTX-IV could be promoted efficiently by the combination of a glutathione S-transferase (GST) tag and a small ubiquitin-related modifier (SUMO) tag. Finally, an affinity-chromatography-free purification strategy was developed by cut-off dialysis tubing combined with trichloroacetic acid (TCA) extraction. Further HPLC purification yielded recombinant, tag-free HNTX-IV with high yield and purity. The molecular weight of recombinant HNTX-IV (rHNTX-IV) is identical to its theoretical value according to Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) analysis. The recombinant toxin has similar activity (IC50 value of 120 nM) on the tetrodotoxin-sensitive (TTX-S) sodium channels in adult rat dorsal root ganglion (DRG) neurons to native toxins. In the report, an efficient and cost-effective strategy for producing rHNTX-IV was developed, which paved the way for the further study of structure-activity relationships of rHNTX-IV and its pharmaceutical applications.

Mo1949 Rapamycin Protects Intestinal Ischemia/Reperfusion-Induced Remote Lung Injury in Mice

Background: Enterotoxigenic Escherichia coli (ETEC), defined by their production of heat labile (LT) and/or heat stable (ST) toxins, are major causes of infantile diarrhea in developing countries and of travelers' diarrhea in visitors to the developing world. Despite our understanding of ETEC pathogenesis, no broadly protective ETEC vaccine is available. This is largely a result of the difficulty in inducing immunity to the small ST molecule. Our goal is to develop a broadly protective live attenuated vaccine against ETEC by taking advantage of the recent demonstration (Zhang et al.,2013,PLoSONe 8 (10):e77386) that genetically produced fusions of mutant ST with LT components can induce effective immunity against both toxins. Methods: To achieve this aim, we engineered an attenuated strain of E. coli, which we have developed as a vaccine vector for mucosal antigen delivery, to express immunogenic fusion constructs of LT and mutant ST. Our vaccine vector strain ZCR533, is a derivative of O157:H7 enterohemorrhagic E. coli (EHEC) strain 86-24 attenuated by deleting its genes for Shiga toxin production and truncating its major adhesin, intimin, such that it can no longer adhere intimately, but can still induce anti-intimin antibody. To express LT-ST fusions in immunogenic form in our vector strain, we used restriction-free cloning technology to incorporate them into the passenger domain of an autotransporter protein (EspP) expressed on a medium copy number plasmid. This system is designed to express the toxin antigens in either surface-bound or secreted forms, and to provide effective delivery to the mucosal immune system as we have done for other toxin antigens. Results: The incorporation of the LT-ST fusions into the passenger domain of the EspP autotransporter plasmids was confirmed by PCR and DNA sequencing. Protein expression by ZCR533 containing the autotransporter plasmids was confirmed by SDS-PAGE, and Western blot of whole cell lysates and culture supernates, using poly-clonal antisera to the LT component. Moreover, appropriate expression of the LT-ST fusions on the bacterial surface of ZCR533 was confirmed by immuno-fluorescent staining with the specific antisera. Conclusions: These studies show that immunogenic fusions of LT and mutant ST can be expressed on the surface, and in the culture supernates, of our attenuated E. coli vaccine vector ZCR533 via the EspP autotransporter. Since our recent studies using the similarly-sized Shiga toxin B subunit antigen have indicated the validity of this approach to mucosal immunization against toxin antigens, these studies could lead to the development of broadly protective vaccines directed against both LTand ST-producing ETEC, as well as against attaching/ effacing E. coli strains expressing intimin.

Mo1948 Toward a Broadly Protective Vaccine Against Enterotoxigenic E.coli (ETEC) Infections

Background: Enterotoxigenic Escherichia coli (ETEC), defined by their production of heat labile (LT) and/or heat stable (ST) toxins, are major causes of infantile diarrhea in developing countries and of travelers' diarrhea in visitors to the developing world. Despite our understanding of ETEC pathogenesis, no broadly protective ETEC vaccine is available. This is largely a result of the difficulty in inducing immunity to the small ST molecule. Our goal is to develop a broadly protective live attenuated vaccine against ETEC by taking advantage of the recent demonstration (Zhang et al.,2013,PLoSONe 8 (10):e77386) that genetically produced fusions of mutant ST with LT components can induce effective immunity against both toxins. Methods: To achieve this aim, we engineered an attenuated strain of E. coli, which we have developed as a vaccine vector for mucosal antigen delivery, to express immunogenic fusion constructs of LT and mutant ST. Our vaccine vector strain ZCR533, is a derivative of O157:H7 enterohemorrhagic E. coli (EHEC) strain 86-24 attenuated by deleting its genes for Shiga toxin production and truncating its major adhesin, intimin, such that it can no longer adhere intimately, but can still induce anti-intimin antibody. To express LT-ST fusions in immunogenic form in our vector strain, we used restriction-free cloning technology to incorporate them into the passenger domain of an autotransporter protein (EspP) expressed on a medium copy number plasmid. This system is designed to express the toxin antigens in either surface-bound or secreted forms, and to provide effective delivery to the mucosal immune system as we have done for other toxin antigens. Results: The incorporation of the LT-ST fusions into the passenger domain of the EspP autotransporter plasmids was confirmed by PCR and DNA sequencing. Protein expression by ZCR533 containing the autotransporter plasmids was confirmed by SDS-PAGE, and Western blot of whole cell lysates and culture supernates, using poly-clonal antisera to the LT component. Moreover, appropriate expression of the LT-ST fusions on the bacterial surface of ZCR533 was confirmed by immuno-fluorescent staining with the specific antisera. Conclusions: These studies show that immunogenic fusions of LT and mutant ST can be expressed on the surface, and in the culture supernates, of our attenuated E. coli vaccine vector ZCR533 via the EspP autotransporter. Since our recent studies using the similarly-sized Shiga toxin B subunit antigen have indicated the validity of this approach to mucosal immunization against toxin antigens, these studies could lead to the development of broadly protective vaccines directed against both LTand ST-producing ETEC, as well as against attaching/ effacing E. coli strains expressing intimin.

A high-throughput, restriction-free cloning and screening strategy based on ccd B-gene replacement

BackgroundIn high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein. Here, we address recently developed restriction-free methods in cloning, and present a more cost-efficient protocol that has been optimized to improve both cloning and clone screening.ResultsIn our case study, three homologous β-lactamase genes were successfully cloned using these restriction-free protocols. To clone the genes, we chose a gene replacement strategy, where the recombinant genes contained overhangs that targeted a region of the expression vector including a cytotoxin-encoding ccdB-gene.ConclusionWe provide further evidence that gene replacement can be applied with high-throughput cloning protocols. Targeting a replacement of the ccdB-gene was found to be very successful for counterselection using these protocols. This eliminated the need for treatment with the restriction enzyme DpnI that has so far been the preferred clone selection approach. We thus present an optimized cloning protocol using a restriction-free ccdB-gene replacement strategy, which allows for parallel cloning at a high-throughput level.

Recombination-assisted megaprimer (RAM) cloning

No molecular cloning technique is considered universally reliable, and many suffer from being too laborious, complex, or expensive. Restriction-free cloning is among the simplest, most rapid, and cost-effective methods, but does not always provide successful results. We modified this method to enhance its success rate through the use of exponential amplification coupled with homologous end-joining. This new method, recombination-assisted megaprimer (RAM) cloning, significantly extends the application of restriction-free cloning, and allows efficient vector construction with much less time and effort when restriction-free cloning fails to provide satisfactory results. The following modifications were made to the protocol:•Limited number of PCR cycles for both megaprimer synthesis and the cloning reaction to reduce error propagation.•Elimination of phosphorylation and ligation steps previously reported for cloning methods that used exponential amplification, through the inclusion of a reverse primer in the cloning reaction with a 20 base pair region of homology to the forward primer.•The inclusion of 1M betaine to enhance both reaction specificity and yield.

Exponential megapriming PCR (EMP) cloning--seamless DNA insertion into any target plasmid without sequence constraints.

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.

Bacillus anthracis Thioredoxin Systems, Characterization and Role as Electron Donors for Ribonucleotide Reductase

Bacillus anthracis is the causative agent of anthrax, which is associated with a high mortality rate. Like several medically important bacteria, B. anthracis lacks glutathione but encodes many genes annotated as thioredoxins, thioredoxin reductases, and glutaredoxin-like proteins. We have cloned, expressed, and characterized three potential thioredoxins, two potential thioredoxin reductases, and three glutaredoxin-like proteins. Of these, thioredoxin 1 (Trx1) and NrdH reduced insulin, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and the manganese-containing type Ib ribonucleotide reductase (RNR) from B. anthracis in the presence of NADPH and thioredoxin reductase 1 (TR1), whereas thioredoxin 2 (Trx2) could only reduce DTNB. Potential TR2 was verified as an FAD-containing protein reducible by dithiothreitol but not by NAD(P)H. The recently discovered monothiol bacillithiol did not work as a reductant for RNR, either directly or via any of the redoxins. The catalytic efficiency of Trx1 was 3 and 20 times higher than that of Trx2 and NrdH, respectively, as substrates for TR1. Additionally, the catalytic efficiency of Trx1 as an electron donor for RNR was 7-fold higher than that of NrdH. In extracts of B. anthracis, Trx1 was responsible for almost all of the disulfide reductase activity, whereas Western blots showed that the level of Trx1 was 15 and 60 times higher than that of Trx2 and NrdH, respectively. Our findings demonstrate that the most important general disulfide reductase system in B. anthracis is TR1/Trx1 and that Trx1 is the physiologically relevant electron donor for RNR. This information may provide a basis for the development of novel antimicrobial therapies targeting this severe pathogen.

RF-Cloning.org: an online tool for the design of restriction-free cloning projects

Restriction-free cloning (RF-cloning) is a PCR-based technology that expands on the QuikChange™ mutagenesis process originally popularized by Stratagene in the mid-1990s, and allows the insertion of essentially any sequence into any plasmid at any location. While RF-cloning is a powerful tool for the design of custom plasmids when restriction sites are not conveniently situated, manually designing the requisite primers can be tedious and error prone. We present here a web-service that automates the primer design process, along with a user interface that includes a number of useful tools for managing both the input sequences and the resulting outputs. RF-Cloning is free and open to all users, and can be accessed at http://www.rf-cloning.org.

Efficient gene disruption in Saccharomyces cerevisiae using marker cassettes with long homologous arms prepared by the restriction-free cloning strategy

Here we report an improved method for targeted gene disruption with high efficiency in S. cerevisiae, where the selection markers with long homologous arms are defined by the choice of the primer binding sites at the target locus and the disruption cassettes are constructed by restriction-free (RF) cloning strategy. Three genes, SAM1, IDH1 and IDH2, were disrupted with this method and the disruption efficiencies of SAM1 was improved several folds with much lower false-positive rates compared to the conventional one-step PCR-based gene disruption method. This approach for gene disruption cassettes construction with long flanking homologous arms may be readily applicable to facilitate targeted gene disruption in other non-conventional yeasts and fungi.

Construction and application of a built-in dual luciferase reporter for microRNA functional analysis

Background: As key gene regulators, microRNAs post-transcriptionally modulate gene expression via binding to partially complementary sequence in the 3' UTR of target mRNA. An accurate, rapid and quantitative tool for sensing and validation of miRNA targets is of crucial significance to decipher the functional implications of miRNAs in cellular pathways. Results: Taking advantage of an improved restriction-free cloning method, we engineered a novel built-in dual luciferase reporter plasmid where Firefly and Renilla luciferase genes were assembled in a single plasmid named pFila. This design eliminates the transfection of a separate control plasmid and thus minimizes the time and labor required for miRNA-target sensing assays. pFila consistently produces Firefly and Renilla luciferase activities when transfected into human-, monkey- and mouse-derived mammalian cell systems. Moreover, pFila is capable of recapitulating the interaction of miR-16 and its known target CCNE1 in Hela cells. Additionally, pFila is shown to be a sensitive miR-biosensor by evaluating the inhibition efficiency of endogenous miRNA. Conclusions: pFila would facilitate miRNA target identification and verification in a rapid and simplified manner. Also, pFila is a sensitive biosensor for active miRNA profiling in vivo .

Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression

Molecular manipulations, including DNA cloning and mutagenesis are basic tools used on a routine basis in all life-science disciplines. Over the last decade new methodologies have emerged that facilitated and expanded the applications for DNA cloning and mutagenesis. Ligation-Independent Cloning (LIC) techniques were developed and replaced the classical Ligation Dependent Cloning (LDC) platform. Restriction Free (RF) cloning was originally developed for introduction of foreign DNA into a plasmid at any predetermined position. RF cloning is based on PCR amplification of a DNA fragment, which serves as a mega-primer for the linear amplification of the vector and insert. Here we present several novel applications of the Restriction Free (RF) cloning platform for DNA cloning and mutagenesis. The new applications include simultaneous cloning of several DNA fragments into distinct positions within an expression vector, simultaneous multi-component assembly, and parallel cloning of the same PCR product into a series of different vectors. In addition, we have expanded the application of the RF cloning platform for multiple alterations of the target DNA, including simultaneous multiple-site mutagenesis and simultaneous introduction of deletions and insertions at different positions. We further demonstrate the robustness of the new applications for facilitating recombinant protein expression in the Escherichia coli system.