Efficient Cloning Research Articles

A novel series of high-efficiency vectors for TA cloning and blunt-end cloning of PCR products

An efficient PCR cloning method is indispensable in modern molecular biology, as it can greatly improve the efficiency of DNA cloning processes. Here, I describe the development of three vectors for TA cloning and blunt-end cloning. Specifically, pCRT and pCRZeroT were designed to improve the efficiency of TA cloning. pCRZeroT can also be used with pCRZero to facilitate blunt-end cloning using the ccdB gene. Using pCRZero and pCRZeroT and applying the Golden Gate reaction, I developed a direct PCR cloning protocol with non-digested circular vectors and PCR products. This direct PCR cloning protocol yielded colony-formation rates and cloning efficiencies that are comparable with those obtained by conventional PCR cloning with pre-digested vectors and PCR products. The three plasmids I designed are available from Addgene (https://www.addgene.org/).

A new vector coupling ligation-independent cloning with sortase a fusion for efficient cloning and one-step purification of tag-free recombinant proteins

We have developed a new ligation independent cloning (LIC) vector – pSrtA9, which can be utilized for one-step purification of recombinant proteins. The new LIC site in the pSrtA9 vector, hosts a DNA sequence centered on a SfoI restriction site and integrates a coding sequence for sortase A (SrtA) recognition. Preceding the LIC site, pSrtA9 incorporates an N-terminal 6xHis-tag and the catalytic core of SrtA from Staphylococcus aureus (SrtAΔ59). Thus, after cloning and protein expression in Escherichia coli, the resultant fusion protein comprises an N-terminal 6xHis-tag, SrtAΔ59, an L-P-E-T-G linker and the protein of interest at the C-terminus. The fusion protein can be captured onto immobilized Ni-NTA resin and any unwanted proteolysis activity of SrtA is suppressed during the purification by optimisation of solution conditions. Upon addition of Ca2+ and triglycine (Gly3), the immobilized fusion protein undergoes on-column SrtA-mediated cleavage at the T-G bond of LPETG linker to selectively release 90% of the protein of interest within 3 h when incubated at room temperature. This new pSrtA9 vector, thus, offers an efficient method for LIC of genes and a one-step purification procedure to obtain a tag-free recombinant protein, and is therefore suitable for the high-throughput proteins production.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation.

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Fast and efficient cloning of human IgE, IgG1 and IgG4 antibodies specific for beta-lactoglobulin from cow milk by Polymerase Incomplete Primer Extension (PIPE)

Fast and efficient cloning of human IgE, IgG1 and IgG4 antibodies specific for beta-lactoglobulin from cow milk by Polymerase Incomplete Primer Extension (PIPE)

Selection of Heterologous Protein-Producing Strains in Yarrowia lipolytica.

Yarrowia lipolytica has emerged as an alternative expression system for heterologous protein production and enzyme evolution. Several different expression systems dedicated for this species have been developed, ranging from the simple cloning of expression vectors to recently developed high-throughput methodologies using efficient cloning and assembly such as Gateway and Golden Gate strategies. The latter strategies, due to their modular character, enable multiple vector construction and the construction of expression cassettes containing different genes or a gene under different promoters of various strengths.Here, we present the Golden Gate cloning strategy for the construction of multiple expression cassettes, the transformation into Y. lipolytica, and the selection of efficient enzyme-producing strains using an insect alpha-amylase as a reporter detected via athermal cycler-based microassay.

Overview of High-Throughput Cloning Methods for the Post-genomic Era.

The advent of new DNA sequencing technologies leads to a dramatic increase in the number of available genome sequences and therefore of target genes with potential for functional analysis. The insertion of these sequences into proper expression vectors requires a simple an efficient cloning method. In addition, when expressing a target protein, quite often it is necessary to evaluate different DNA constructs to achieve a soluble and homogeneous expression of the target with satisfactory yields. The development of new molecular methods made possible the cloning of a huge number of DNA sequences in a high-throughput manner, necessary for meeting the increasing demands for soluble protein expression and characterization. In this chapter several molecular methods suitable for high-throughput cloning are reviewed.

T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis.

The assembly of DNA fragments with homologous arms is becoming popular in routine cloning. For an in vitro assembly reaction, a DNA polymerase is often used either alone for its 3′-5′ exonuclease activity or together with a 5′-3′ exonuclease for its DNA polymerase activity. Here, we present a ‘T5 exonuclease DNA assembly’ (TEDA) method that only uses a 5′-3′ exonuclease. DNA fragments with short homologous ends were treated by T5 exonuclease and then transformed into Escherichia coli to produce clone colonies. The cloning efficiency was similar to that of the commercial In-Fusion method employing a proprietary DNA polymerase, but higher than that of the Gibson method utilizing T5 exonuclease, Phusion DNA polymerase, and DNA ligase. It also assembled multiple DNA fragments and did simultaneous site-directed mutagenesis at multiple sites. The reaction mixture was simple, and each reaction used 0.04 U of T5 exonuclease that cost 0.25 US cents. The simplicity, cost effectiveness, and cloning efficiency should promote its routine use, especially for labs with a budget constraint. TEDA may trigger further development of DNA assembly methods that employ single exonucleases.

Direct Cloning of Bacterial Surface Polysaccharide Gene Cluster for One-Step Production of Glycoconjugate Vaccine.

Bacterial pathogen infections are fast-growing public health threats and worldwide problems. Glycoconjugate vaccines are among the most effective means in combating such infections. Recent advances in bacterial protein glycan coupling technology (PGCT) have revolutionized the production of glycoconjugate vaccines and drawn enormous attention from both researchers and pharmaceutical companies. Cloning of bacterial surface polysaccharide gene cluster is a prerequisite for the application of PGCT. In this study, we applied the RecET direct cloning strategy for rapid and efficient cloning of O-antigen polysaccharide gene clusters from Escherichia coli serotypes O25b, O26, and O55 in a high-fidelity manner. Then, these gene clusters were applied in PGCT to produce corresponding glycoconjugates. Subsequent immunological studies verified the abilities of glycoconjugate vaccine candidates O25-maltose-binding protein (MBP), O26-MBP, and O55-MBP to generate serotype-specific antibodies and confer protection against E.coli infections. The combination of RecET direct cloning and PGCT makes the rapid production of glycoconjugate vaccines against fast-expanding bacterial pathogens possible.

Keeping pace with the increasing demand for high quality drug candidates in pharmaceutical research: Development of a new two-step preparative tandem high performance chromatographic system for the purification of antibodies

In the area of biological drug development, high throughput (HT) technologies are key to identifying the most promising therapeutic candidate in a time-efficient and market-competitive manner. While efficient cloning and expression methods exist, HT downstream processing mainly relies on liquid handling workstations applying miniaturized chromatography columns or resin-based 96-well plates to shorten process development time. However, there is still a lack of generic, preparative chromatographic methods devoid of aggregates and endotoxins with sufficient throughput. The only truly generic antibody purification strategy including an efficient dimer removal consists of Protein A capture followed by size exclusion chromatography (SEC) as a polishing step. Other polishing methods, including IEX, HIC, and CHT, require an antibody-specific fine tuning. However, standard preparative SEC setups tend to be rather time-consuming, and so limit throughput.In this work, we devised a unique chromatography setup enabling an unattended two-step purification of IgGs on the milligram scale directly from 35 mL clarified cell supernatants, processing up to 48 samples in 44.0 h. By introducing a silica-based SEC column, preparative SEC could be accelerated. By further developing an HT two-step preparative Protein A/alternating column regeneration SEC system using Agilent 1260 Infinity LC components, mAbs can be purified generically by two chromatographic steps in 55 min. In this way, by using a 2-position/10-port valve and two quaternary pumps, two SEC columns can be run in parallel, excluding the cleaning and equilibrating phase from the actual cycle time. By further applying a third pump, the Protein A step can be run independently, resulting in a time-optimized process nesting. By introducing a CETAC ASX-520 autosampler, 48 samples can be run automatically without any user intervention over two working days. The developed system is highly reproducible for all tested human IgG1 antibodies, easily generating milligram scale material sufficient for full characterization of the antibodies and for their use in in vitro and in vivo activity assessments.

An improved micropropagation and ex vitro rooting of a commercially important crop Henna (Lawsonia inermis L.).

An improved micropropagation protocol has been developed for a cosmetically important, dye yielding crop, henna (Lawsonia inermis). Quality of henna product is governed by naphthoquinone based pigment lawsone, thus in vitro multiplication of superior healthy plant to achieve enhanced productivity in terms of dye content and biomass deserve due attention. In the present study, nodal explants collected from an elite plant screened on the basis of superiority in lawsone content was cultured on MS medium supplemented with 0.5μM benzyl adenine (BA) gave significantly (p < 0.05) high number of shoots (24.33). The explants placed on MS medium augmented with 0.5μM BA and 2-isopentenyladenine (2-iP) resulted in the formation of maximum number of shoots (43.67) and was elongated (12.57cm) within 4weeks of culture period. Enhanced axillary bud proliferation and production of mass number of micro shoots was achieved by the continuous subculture in MS medium containing 0.5μM BA and 2-iP. In vitro raised micro shoots were dipped in 0.44mM NAA for 5min followed by planting in polyethylene pots containing a soil: vermiculite (1:1 v/v) mixture produced rooted plantlets (100%). Different auxin types and its concentrations had significant role rooting of L. inermis. Rooting response of various size shoots of L. inermis treated with 0.44mM NAA showed 100% rooting in 4.1-5cm size class shoots. After two months of potting, survived (95%) plants were successfully transferred to medicinal plant garden of the Department. The lawsone content of one-year-old micropropagated plants (23.04mg/gdw) growing in normal environmental conditions and elite mother plant (22.84mg/gdw) was almost similar. Through the present study, efficient cloning of superior germplasm of L. inermis was established.

High-throughput, one-step screening, cloning and expression based on the lethality of DpnI in Escherichia coli

The manipulation of recombinant DNA has been an integral step in molecular biology to date. A number of strategies have been developed over the years, as traditional cloning methods are time consuming, have high backgrounds and low efficiency and are often limited by the number of suitable restriction sites available. Here, we constructed a series of new positive-selection-based cloning vectors that overcome most of the above mentioned drawbacks and can be applied in both eukaryotic and prokaryotic systems. This strategy is based on the extreme toxicity of DpnI in wild-type E. coli and the inactivation of this lethality by the introduction of target gene within multiple cloning sites. There are no rapid approaches for identifying soluble proteins for high-throughput screening. In this study, we combined this highly efficient cloning strategy with rapid identification of soluble proteins to construct vectors with multiple fusion tags, such as MBP, GST, CBD, NusA, and Sumo, to generate enzymes with potential diagnostic, industrial or therapeutic applications. Thus, this versatile positive-selection-based technology is appropriate for routine cloning, DNA library construction, and high-throughput screening for the expression of proteins of interest.

Loci and natural alleles underlying robust roots and adaptive domestication of upland ecotype rice in aerobic conditions.

A robust (long and thick) root system is characteristic of upland japonica rice adapted to drought conditions. Using deep sequencing and large scale phenotyping data of 795 rice accessions and an integrated strategy combining results from high resolution mapping by GWAS and linkage mapping, comprehensive analyses of genomic, transcriptomic and haplotype data, we identified large numbers of QTLs affecting rice root length and thickness (RL and RT) and shortlisted relatively few candidate genes for many of the identified small-effect QTLs. Forty four and 97 QTL candidate genes for RL and RT were identified, and five of the RL QTL candidates were validated by T-DNA insertional mutation; all have diverse functions and are involved in root development. This work demonstrated a powerful strategy for highly efficient cloning of moderate- and small-effect QTLs that is difficult using the classical map-based cloning approach. Population analyses of the 795 accessions, 202 additional upland landraces, and 446 wild rice accessions based on random SNPs and SNPs within robust loci suggested that there could be much less diversity in robust-root candidate genes among upland japonica accessions than in other ecotypes. Further analysis of nucleotide diversity and allele frequency in the robust loci among different ecotypes and wild rice accessions showed that almost all alleles could be detected in wild rice, and pyramiding of robust-root alleles could be an important genetic characteristic of upland japonica. Given that geographical distribution of upland landraces, we suggest that during domestication of upland japonica, the strongest pyramiding of robust-root alleles makes it a unique ecotype adapted to aerobic conditions.

T7 Expression Systems for Inducible Production of Proteins from Cloned Genes in E. coli.

Inducible T7 expression systems are capable of producing a wide range of proteins in E. coli. Improvements over common practice include: (1) preventing unintended induction by establishing and maintaining expression strains in non-inducing growth media composed entirely of purified components instead of complex growth media that may variably induce target proteins on approach to saturation; and (2) expressing many target proteins in parallel by convenient and productive auto-induction in BL21(DE3) and other suitable hosts, instead of IPTG induction. From the earliest days, basal expression prevented establishment of inducible strains for producing proteins that are stressful to the host. Newly developed pAL vectors now reduce basal expression to levels where coding sequences for even the most stressful proteins can be maintained and induced. Asymmetric ligation allows simple and efficient cloning of individual coding sequences or simultaneous cloning of two or three coding sequences for co-expression from a single pAL vector. © 2018 by John Wiley & Sons, Inc.

PXST, a novel vector for TA cloning and blunt-end cloning

BackgroundWith the rapid development of sequencing technologies, increasing amount of genomic information has been accumulated. To clone genes for further functional studies in large scale, a cheap, fast and efficient cloning vector is desired.ResultsA bifunctional vector pXST has been constructed. The pXST vector harbors a XcmI-ccdB-XcmI cassette and restriction site SmaI. Digestion the vector with XcmI generates a single thymidine (T) overhang at 3′ end which facilitates TA cloning, and SmaI gives blunt end that enables the blunt-end ligation. Multiple products with various sizes were amplified from cassava genome by PCR and each PCR fragment was separately cloned into a pXST using TA cloning and blunt-end ligation methods. In general, the TA cloning gave higher transformation efficiency than blunt-end ligation for inserts with all different sizes, and the transformation efficiency significantly decreased with increasing size of inserts. The highest transformation efficiency (8.6 × 106 transformants/μg) was achieved when cloning 517 bp DNA fragment using TA cloning. No significant difference observed in the positive cloning efficiency between two ligation methods and the positive cloning efficiency could reach as high as 100% especially for small inserts (e.g. 517 and 957 base pairs).ConclusionsWe describe a simple and general method to construct a novel pXST vector. We confirm the feasibility of using pXST vector to clone PCR products amplified from cassava genome with both TA cloning and blunt-end ligation methods. The pXST plasmid has several advantages over many currently available vectors in that (1) it possesses XcmI-ccdB-XcmI cassette and restriction site SmaI, enabling both TA cloning and blunt-end ligation. (2) it allows direct selection of positive recombinant plasmids in Escherichia coli through disruption of the ccdB gene. (3) it improves positive cloning efficiency by introducing the ccdB gene, reducing the possibility of self-ligation from insufficient digested plasmids. (4) it could be used by high performance and cost-effective cloning methods. Therefore, this dual function vector would offer flexible alternatives for gene cloning experiments to researchers.

Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method.

Homologous alignment cloning (HAC) is a rapid method of molecular cloning that facilitates low-cost, highly efficient cloning of polymerase chain reaction products into any plasmid vector in approximately 2 min. HAC facilitates insert integration due to a sequence alignment strategy, by way of short, vector-specific homology tails appended to insert during amplification. Simultaneous exposure of single-stranded fragment ends, utilising the 3′→5′ exonuclease activity of T4 DNA polymerase, creates overlapping homologous DNA on each molecule. The exonuclease activity of T4 polymerase is quenched simply by the addition of EDTA and a simple annealing step ensures high yield and high fidelity vector formation. The resultant recombinant plasmids are transformed into standard E. coli cloning strains and screened via established methods as necessary. HAC exploits reagents commonly found in molecular research laboratories and achieves efficiencies that exceed conventional cloning methods, including another ligation-independent method we tested. HAC is also suitable for combining multiple fragments in a single reaction, thus extending its flexibility.

Multi-Compartment and Multi-Host Vector Suite for Recombinant Protein Expression and Purification.

Recombinant protein expression has become an invaluable tool in basic and applied research. The accumulated knowledge in this field allowed the expression of thousands of protein targets in a soluble, pure, and homogeneous state, essential for biochemical and structural analyses. A lot of progress has been achieved in the last decades, where challenging proteins were expressed in a soluble manner after evaluating different parameters such as host, strain, and fusion partner or promoter strength, among others. In this regard, we have previously developed a vector suite that allows the evaluation of different promoters and solubility enhancer-proteins, through an easy and efficient cloning strategy. Nonetheless, the proper expression of many targets remains elusive, requiring, for example, the addition of complex post-translation modifications and/or passage through specialized compartments. In order to overcome the limitations found when working with a single subcellular localization and a single host type, we herein expanded our previously developed vector suite to include the evaluation of recombinant protein expression in different cell compartments and cell hosts. In addition, these vectors also allow the assessment of alternative purification strategies for the improvement of target protein yields.

A uniform cloning platform for mycobacterial genetics and protein production

Molecular research on mycobacteria relies on a multitude of tools for the genetic manipulation of these clinically important bacteria. However, a uniform set of vectors allowing for standardized cloning procedures is not available. Here, we developed a versatile series of mycobacterial vectors for gene deletion, complementation and protein production and purification. The vectors are compatible with fragment exchange (FX) cloning, a recently developed high-throughput cloning principle taking advantage of the type IIS restriction enzyme SapI and its capacity to generate sticky trinucleotide ends outside of its recognition sequence. FX cloning allows for the efficient cloning into an entry vector and the facile transfer of the sequenced insert into a variety of destination vectors. We generated a set of mycobacterial expression vectors spanning a wide range of expression strengths, tagging variants and selection markers to rapidly screen for the optimal expression construct in order to purify membrane proteins from the model organism Mycobacterium smegmatis. Further, we generated a series of suicide vectors containing two counterselection markers and used them to delete twenty genes encoding for potential drug efflux pumps in M. smegmatis. The vectors will further facilitate genetic and biochemical research on various mycobacterial species.

Development of Versatile Vectors for Heterologous Expression in Bacillus.

The discovery of new enzymes for industrial application relies on a robust discovery pipeline. Such a pipeline should facilitate efficient molecular cloning, recombinant expression and functional screening procedures. Previously, we have developed a vector set for heterologous expression in Escherichia coli. Here, we supplement the catalogue with vectors for expression in Bacillus. The vectors are made compatible with a versatile cloning procedure based on type IIS restriction enzymes and T4 DNA ligase, and encompass an effective counter-selection procedure and complement the set of vectors with options for secreted expression. We validate the system with expression of recombinant subtilisins, which are generally challenging to express in a heterologous system. The complementarity of the E. coli and Bacillus systems allows rapid switching between the two commonly used hosts without comprehensive intermediate cloning steps. The vectors described are not limited to the expression of certain enzymes, but could also be applied for the expression of other enzymes for more generalized enzyme discovery or development.

Versatile Cas9-Driven Subpopulation Selection Toolbox for Lactococcus lactis.

CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis The Cas9 expression vector pLABTarget, encoding the Streptocccus pyogenes Cas9 under the control of a constitutive promoter, was constructed, allowing plug and play introduction of short guide RNA (sgRNA) sequences to target specific genetic loci. Introduction of a pepN-targeting derivative of pLABTarget into L. lactis strain MG1363 led to a strong reduction in the number of transformants obtained, which did not occur in a pepN deletion derivative of the same strain, demonstrating the specificity and lethality of the Cas9-mediated double-strand breaks in the lactococcal chromosome. Moreover, the same pLABTarget derivative allowed the selection of a pepN deletion subpopulation from its corresponding single-crossover plasmid integrant precursor, accelerating the construction and selection of gene-specific deletion derivatives in L. lactis Finally, pLABTarget, which contained sgRNAs designed to target mobile genetic elements, allowed the effective curing of plasmids, prophages, and integrative conjugative elements (ICEs). These results establish that pLABTarget enables the effective exploitation of Cas9 targeting in L. lactis, while the broad-host-range vector used suggests that this toolbox could readily be expanded to other Gram-positive bacteria.IMPORTANCE Mobile genetic elements in Lactococcus lactis and other lactic acid bacteria (LAB) play an important role in dairy fermentation, having both positive and detrimental effects during the production of fermented dairy products. The pLABTarget vector offers an efficient cloning platform for Cas9 application in lactic acid bacteria. Targeting Cas9 toward mobile genetic elements enabled their effective curing, which is of particular interest in the context of potentially problematic prophages present in a strain. Moreover, Cas9 targeting of other mobile genetic elements enables the deciphering of their contribution to dairy fermentation processes and further establishment of their importance for product characteristics.

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.

The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.