Seamless Cloning Research Articles

Directional, seamless, and restriction enzyme-free construction of random-primed complementary DNA libraries using phosphorothioate-modified primers

Directional cloning of complementary DNA (cDNA) primed by oligo(dT) is commonly achieved by appending a restriction site to the primer, whereas the second strand is synthesized through the combined action of RNase H and Escherichia coli DNA polymerase I (PolI). Although random primers provide more uniform and complete coverage, directional cloning with the same strategy is highly inefficient. We report that phosphorothioate linkages protect the tail sequence appended to random primers from the 5′ → 3′ exonuclease activity of PolI. We present a simple strategy for constructing a random-primed cDNA library using the efficient, size-independent, and seamless In-Fusion cloning method instead of restriction enzymes.

Interactive editing of massive imagery made simple

This article presents a simple framework for progressive processing of high-resolution images with minimal resources. We demonstrate this framework's effectiveness by implementing an adaptive, multi-resolution solver for gradient-based image processing that, for the first time, is capable of handling gigapixel imagery in real time. With our system, artists can use commodity hardware to interactively edit massive imagery and apply complex operators, such as seamless cloning, panorama stitching, and tone mapping. We introduce a progressive Poisson solver that processes images in a purely coarse-to-fine manner, providing near instantaneous global approximations for interactive display (see Figure 1). We also allow for data-driven adaptive refinements to locally emulate the effects of a global solution. These techniques, combined with a fast, cache-friendly data access mechanism, allow the user to interactively explore and edit massive imagery, with the illusion of having a full solution at hand. In particular, we demonstrate the interactive modification of gigapixel panoramas that previously required extensive offline processing. Even with massive satellite images surpassing a hundred gigapixels in size, we enable repeated interactive editing in a dynamically changing environment. Images at these scales are significantly beyond the purview of previous methods yet are processed interactively using our techniques. Finally our system provides a robust and scalable out-of-core solver that consistently offers high-quality solutions while maintaining strict control over system resources.

A novel approach for the construction of plant amiRNA expression vectors

Artificial microRNA (amiRNA) technology has been applied in Arabidopsis thaliana and other plants to efficiently silence target genes of interest. Here we described a novel approach to construct plant amiRNA expression vectors with seamless enzyme-free cloning (SEFC) and mating-assisted genetically integrated cloning (MAGIC). Two pairs of primers were designed when the loop of amiRNA precursor was longer than 60 bp while three oligonucleotides were used to amplify the linearized vector containing the amiRNA precursor whose loop was smaller than 60 bp. The PCR products were transformed into Escherichia coli to generate the donor plasmid containing the amiRNA expression cassette through homologous recombination in vivo. The amiRNA expression cassette was then transferred to the recipient plasmid via MAGIC and an amiRNA expression plasmid was created. More than 200 amiRNA expression vectors were generated with this approach, three of which have been transformed into A. thaliana and successfully silence the target genes. Given its low-cost and simplicity, this novel approach of plant amiRNA expression vectors construction will benefit the study of individual gene function and establishment of plant amiRNA libraries.

Harmonic coordinates for real-time image cloning

Traditional gradient domain seamless image cloning is a time consuming task, requiring the solving of Poisson’s equations whenever the shape or position of the cloned region changes. Recently, a more efficient alternative, the mean-value coordinates (MVCs) based approach, was proposed to interpolate interior pixels by a weighted combination of values along the boundary. However, this approach cannot faithfully preserve the gradient in the cloning region. In this paper, we introduce harmonic cloning, which uses harmonic coordinates (HCs) instead of MVCs in image cloning. Benefiting from the non-negativity and interior locality of HCs, our interpolation generates a more accurate harmonic field across the cloned region, to preserve the results with as high a quality as with Poisson cloning. Furthermore, with optimizations and implementation on a graphic processing unit (GPU), we demonstrate that, compared with the method using MVCs, our harmonic cloning gains better quality while retaining real-time performance.

Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection

BackgroundMolecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products.ResultsOur vectors apply positive selection for the insert or negative selection against empty vector molecules and drive strong expression of target proteins in E.coli cells. Variable tags are available both in N-terminal or C-terminal position. A newly developed β-lactamase (ΔW290) selection cassette contains a segment inside the β-lactamase open reading frame encoding a stretch of hydrophilic amino acids that result in a T7 promoter when back-translated. This position of the promoter permits positive selection and attenuated expression of fusion proteins with C-terminal tags. We have tested eight vectors by inserting six target sequences of variable length, provenience and function. The target proteins were cloned, expressed and detected using an automated Tecan Freedom Evo II liquid handling work station. Only two colonies had to be picked to score with 85% correct inserts while 80% of those were positive in expression tests.ConclusionsOur results establish co-transformation and positive/negative selection cloning in conjunction with the provided vectors and selection cassettes as an automatable alternative to commercialized high-throughput cloning systems like Gateway® or ligase-independent cloning (LIC) .

Recursive Directional Ligation by Plasmid Reconstruction Allows Rapid and Seamless Cloning of Oligomeric Genes

This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.

A GPU Laplacian solver for diffusion curves and Poisson image editing

We present a new Laplacian solver for minimal surfaces---surfaces having a mean curvature of zero everywhere except at some fixed (Dirichlet) boundary conditions. Our solution has two main contributions: First, we provide a robust rasterization technique to transform continuous boundary values (diffusion curves) to a discrete domain. Second, we define a variable stencil size diffusion solver that solves the minimal surface problem. We prove that the solver converges to the right solution, and demonstrate that it is at least as fast as commonly proposed multigrid solvers, but much simpler to implement. It also works for arbitrary image resolutions, as well as 8 bit data. We show examples of robust diffusion curve rendering where our curve rasterization and diffusion solver eliminate the strobing artifacts present in previous methods. We also show results for real-time seamless cloning and stitching of large image panoramas.

High-throughput cloning of human liver complete open reading frames using homologous recombination in Escherichia coli

In this article, we describe a high-throughput cloning method, seamless enzyme-free cloning (SEFC), which allows one-step assembly of DNA fragments in vivo via homologous recombination in Escherichia coli. In the method, the desired open reading frame (ORF) is amplified by use of ORF-specific primers with flanking sequences identical to the two ends of a linearized vector. The polymerase chain reaction (PCR) product and the linearized vector are then cotransformed into E. coli cells, where the ORF is incorporated into the vector in vivo. SEFC is a simple, reliable, and inexpensive method of cloning in which PCR fragments are fused into expression vectors without unwanted amino acids or extra in vitro manipulations apart from the single PCR amplification step. Using this method, we successfully cloned human liver complete ORFs into the yeast AD and DB vectors and generated a clone resource of 4964 AD–ORFs and 4676 DB–ORFs in 3months. This approach will be useful for daily DNA cloning and for creating proteome-scale clone resources.

Coordinates for instant image cloning

Seamless cloning of a source image patch into a target image is an important and useful image editing operation, which has received considerable research attention in recent years. This operation is typically carried out by solving a Poisson equation with Dirichlet boundary conditions, which smoothly interpolates the discrepancies between the boundary of the source patch and the target across the entire cloned area. In this paper we introduce an alternative, coordinate-based approach, where rather than solving a large linear system to perform the aforementioned interpolation, the value of the interpolant at each interior pixel is given by a weighted combination of values along the boundary. More specifically, our approach is based on Mean-Value Coordinates (MVC). The use of coordinates is advantageous in terms of speed, ease of implementation, small memory footprint, and parallelizability, enabling real-time cloning of large regions, and interactive cloning of video streams. We demonstrate a number of applications and extensions of the coordinate-based framework.

Positive-Selection Vector for Direct Protein Expression

We describe the development of a novel positive-selection vector, RHP-AmpS, that is suitable for seamless cloning and high-level protein expression in Escherichia coli. In this vector, beta-lactamase (Bla) was rendered nonfunctional by replacing the codon for the C-terminal amino acid of the beta-lactamase gene (bla) with a stop codon. Insertion of a target gene in the correct orientation (tail to tail) results in the reconstruction of the C-terminal codon (W290) of bla. This restores the function of the gene and allows the selection of positive recombinants on agar plates containing ampicillin. To allow a high level of protein expression, this selection cassette was inserted into the T7 polymerase-based expression cassette of the Novagen pET28a expression vector. To our knowledge, this is the first example of true positive-selection cloning and direct, high-level expression from a single vector.

Seamless cloning and domain swapping of synthetic and complex DNA

The use of synthetic DNA can avoid problems that are sometimes encountered with conventional molecular biology techniques using DNA with high GC content, strong secondary structures, or repeat sequences. However, very complex DNA may still resist PCR and synthesis of DNA from oligonucleotides. In the method described here, separately synthesized DNA segments were seamlessly joined independently of the presence of restriction sites in the target DNA. This method allowed the reconstruction of complex DNA by concatenation of easily synthesized segments and permitted repeated swapping of segments, from a few nucleotides to large fragments of complex DNA for phenotypic analysis.

Molecular Cloning of Protein-Based Polymers

Protein-based biopolymers have become a promising class of materials for both biomedical and pharmaceutical applications, as they have well-defined molecular weights, monomer compositions, as well as tunable chemical, biological, and mechanical properties. Using standard molecular biology tools, it is possible to design and construct genes encoding artificial proteins or protein-based polymers containing multiple repeats of amino acid sequences. This article reviews some of the traditional methods used for constructing DNA duplexes encoding these repeat-containing genes, including monomer generation, concatemerization, iterative oligomerization, and seamless cloning. A facile and versatile method, called modules of degenerate codons (MDC), which uses PCR and codon degeneracy to overcome some of the disadvantages of traditional methods, is introduced. Re-engineering of the random coil spacer domain of a bioactive protein, WPT2-3R, is used to demonstrate the utility of the MDC method. MDC re-constructed coding sequences facilitate further manipulations, such as insertion, deletion, and swapping of various sequence modules. A summary of some promising emerging techniques for synthesizing repetitive sequence-containing artificial proteins is also provided.

A Novel Variational Image Model: Towards a Unified Approach to Image Editing

In this paper we propose a unified variational image editing model. It interprets image editing as a variational problem concerning the adaptive adjustments to the zero- and first-derivatives of the images which correspond to the color and gradient items. By varying the definition domain of each of the two items as well as applying diverse operators, the new model is capable of tackling a variety of image editing tasks. It achieves visually better seamless image cloning effects than existing approaches. It also induces a new and efficient solution to adjusting the color of an image interactively and locally. Other image editing tasks such as stylized processing, local illumination enhancement and image sharpening, can be accomplished within the unified variational framework. Experimental results verify the high flexibility and efficiency of the proposed model.

Biosynthesis of an Elastin‐Mimetic Polypeptide with Two Different Chemical Functional Groups within the Repetitive Elastin Fragment

A new protein engineering strategy was utilized to synthesize an elastin-mimetic polypeptide. The primary structure represents an elastic motif composed of thirty amino acids with one lysine and one glutamic acid per repeat unit EMM = (VPGVG VPGKG VGPVG VPGVG VPGEG VPGIG). The gene was constructed using a Seamless Cloning method by generating three DNA cassettes which all encoded the EMM repeat unit, but with different flanking restriction recognition sites. The DNA cassettes were assembled to yield a gene that could be directly cloned into the multiple cloning site of pBluescript II SK+. The resulting gene (EMM)(7) with approximately 650 base pairs in length was further cloned into the expression vector pET-28b. Protein biosynthesis in E. coli strain BLR(DE3) resulted in the 21.5 kDa repeating polypeptide His(6)-(EMM)(7) yielding up to 50 mg . L(-1) of cell culture. Secondary structure analysis by far UV circular dichroism revealed a minimum at 197 nm and a shoulder at 218 nm indicative for a random coil with some type II beta-turn conformation content. Lower critical solution temperature (LCST) behavior strongly depends on salt and polypeptide concentration. Importantly, first cross-linking experiments indicate successful hydrogel formation with a surface structure reminiscent to natural elastin as visualized by SEM micrographs.

An Escherichia coli expression vector that allows recovery of proteins with native N-termini from purified calmodulin-binding peptide fusions.

We describe a T7-based Escherichia coli expression vector in which protein coding sequence is seamlessly fused to the N-terminal calmodulin-binding peptide (CBP) purification tag. We combined the use of the site-specific protease enterokinase (EK) and the type IIs restriction enzyme Eam1104 I, which cleave outside their respective (amino acid and nucleotide) target sequences, such that any amino acid sequence may be fused directly C-terminal to the EK cleavage site without codon constraints conferred by the cloning method. PCR products are cloned using ligation-dependent or ligation-independent methods with high cloning efficiencies (>10(6) cfu/microg vector), allowing production of insert quantities sufficient for several cloning experiments with a limited number of PCR cycles, resulting in a significant time-savings and reduced likelihood of accumulating PCR-derived mutations. CBP fusion proteins are expressed to high levels when the CBP peptide is positioned at the N-terminus. CBP binds to calmodulin with nanomolar affinity, and fusion proteins are purified to near homogeneity from crude extracts with one pass through calmodulin affinity resin using gentle binding and elution conditions. We show high efficiency seamless cloning of three inserts into the pCAL-n-EK vector, including one encoding the protein c-Jun N-terminal kinase (JNK). CBP-EK-JNK fusion protein was synthesized to 10-20 mg/liter culture and purified to near homogeneity in one step with calmodulin affinity resin. The fusion tag was efficiently removed with EK to yield active JNK with native N-terminal amino acid sequence.

Rapid Assembly of Synthetic Genes Encoding Protein Polymers

A general method is described for the rapid assembly of synthetic genes encoding protein polymers based on the Seamless cloning technique. Genes encoding repeats of the elastin-mimetic polypeptides [(Val-Pro-Gly-Val-Gly)4(Val-Pro-Gly-Xaa-Gly)] (Xaa = Lys 1; Ile, 2) were constructed using this technique. The use of this method eliminates the dependence of the concatamerization on a limited pool of nonpalindromic restriction endonucleases and reduces the number of subcloning steps. A synthetic gene of approximately 3000 base pairs in length was isolated that encoded a protein polymer based on repeat sequence 1. An inducible expression of this gene in bacterial cultures of recombinant E. coli afforded a 90 kDa protein polymer of 1 in high yield (64 mg/L of bacterial culture). The protein was purified to homogeneity using immobilized metal affinity chromatography. The sequence of the protein polymer was confirmed by N-terminal amino acid sequence analysis and MALDI-TOF mass spectrometry of site-specific proteolytic cleavage fragments.