Solid phase ligation of synthetic DNA fragments

A synthetic oligodeoxyribonucleotide (oligo) covalently bound by an internucleotide linkage to the succinylated Sephacryl S-500 support through 1.9-diaminononane spacer was used as starting compound to assemble the E. coli rec A promoter DNA fragment from synthetic oligos by means of T4 DNA ligase. The solid-phase assembly of the designed DNA was performed by two ways: stepwise ligation of two pairs of oligos (2 dyads) or simultaneous ligation of four oligos (tetrad). Both ways gave equal results with some preference in the tetrad case. The reliability of E. coli promoter DNA fragment assembly was demonstrated by cloning it in a plasmid vector and sequencing the cloned DNA by the solid-phase Maxam--Gilbert technique.     

Automated solid-phase subcloning based on beads brought into proximity by magnetic force

In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.     

几种新型植物基因表达载体的构建方法

利用基因工程技术手段研究基因功能过程中,构建基因表达载体处于转基因植物的主导地位,采用合适的构建方法会使实验效果事半功倍。植物基因表达载体的构建方法除了传统构建法、Gateway技术、三段T-DNA法、一步克隆法等,还有近年来出现的几种新型的载体构建方法:基于竞争性连接原理快速构建小片段基因表达载体;MicroRNA前体PCR置换法适用于构建小分子RNA表达载体;重组融合PCR法特别适用于插入片段中含有较多限制性酶切位点的载体构建;利用In-Fusion试剂盒可以将任何目的片段插入一个线性化载体的某个区域;构建多片段复杂载体可采用不依赖序列和连接的克隆方法(Sequence and ligation-independent cloning,SLIC)法;Gibson等温拼接法;Golden Gate拼接法。本文将在总结分析前人工作的基础上,结合自己工作的体会和经验分析这7种新方法的特点,期望通过这几种新的方法给植物基因工程表达载体的构建提供新的思路。     

A simple strategy for generation of gene knockdown constructs with convergent H1 and U6 promoters

RNA interference (RNAi) is a powerful tool for functional genetic studies in model organisms and mammalian cells. To facilitate rapid construction of gene knockdown constructs and RNAi libraries for known genes of mammalian cells, a new and simple strategy to produce small interfering RNA (siRNA) expression vectors with two opposing polymerase III promoters was developed. The design involved a one-step PCR amplification and single cloning procedure to construct a dual promoter siRNA expression vector. The forward primer is identical for all PCR reactions, only a single reverse primer that contains the siRNA targeting sequence has to be synthesized in the construction of each individual vector. This single primer design is cost-effective and it reduces the risk of sequence errors during synthesis of long oligos. Sense and antisense strands of siRNA duplexes were transcribed from the same template and this eliminated the need to synthesize long hairpin-forming oligonucleotides. Our study demonstrated that this vector design could mediate potent inhibition of expression of both exogenous and endogenous genes in mammalian cells.     

An alternative approach in gene synthesis: use of long selfpriming oligodeoxynucleotides for the construction of double-stranded DNA

A novel approach for the synthesis of double-stranded DNA fragments from only one long oligodeoxynucleotide (oligo) is presented. The basic strategy is to use oligos which possess a short inverted repeat at their 3' end resulting in the formation of a hairpin structure. The 3' end of this hairpin then serves as a primer in the Klenow (large) fragment of E. coli DNA polymerase I-mediated synthesis of the second DNA strand. Removal of the loop structure as well as generation of sticky ends for subsequent cloning is achieved by digestion with restriction enzymes. Several oligos ranging in size from 130 to 147 nt were synthesized and successfully used in the cloning of gene fragments of up to 120 bp in length. Furthermore, a strategy for the simultaneous cloning of two synthetic DNA fragments is outlined yielding even larger gene fragments. By sequential cloning of these gene fragments the methodology presented here will allow the synthesis of genes of any size. The proposed methodology should also be useful for site-directed mutagenesis as well as saturation mutagenesis.     

A new yeast artificial chromosome vector designed for gene transfer into mammalian cells

This report describes the construction of a new yeast artificial chromosome (YAC) vector designed for gene transfer into mammalian cells. For ease of use, the two arms of the vector were cloned separately. The vector harbours the Neo and Hyg genes for dominant selection in mammalian cells, a putative human origin of replication, a synthetic matrix attachment region and two loxP sites (one on each arm). The cloning ability of the vector was demonstrated by successful propagation of the cDNA of the cystic fibrosis gene, CFTR, as a YAC in Saccharomyces cerevisiae. A YAC containing the entire CFTR gene was also constructed by retrofitting the two arms of a pre-existing clone (37AB12) with the two arms of the novel vector. Both the cDNA and entire gene containing YACs were circularized in yeast by inducible expression of the Cre recombinase. Recombination occurred very specifically at the loxP sequences present on the two arms of the YAC. Applications of the vector to gene transfer are discussed.     

Error removal in microchip-synthesized DNA using immobilized MutS

The development of economical de novo gene synthesis methods using microchip-synthesized oligonucleotides has been limited by their high error rates. In this study, a low-cost, effective and improved-throughput (up to 32 oligos per run) error-removal method using an immobilized cellulose column containing the mismatch binding protein MutS was produced to generate high-quality DNA from oligos, particularly microchip-synthesized oligonucleotides. Error-containing DNA in the initial material was specifically retained on the MutS-immobilized cellulose column (MICC), and error-depleted DNA in the eluate was collected for downstream gene assembly. Significantly, this method improved a population of synthetic enhanced green fluorescent protein (720 bp) clones from 0.93% to 83.22%, corresponding to a decrease in the error frequency of synthetic gene from 11.44/kb to 0.46/kb. In addition, a parallel multiplex MICC error-removal strategy was also evaluated in assembling 11 genes encoding ∼21 kb of DNA from 893 oligos. The error frequency was reduced by 21.59-fold (from 14.25/kb to 0.66/kb), resulting in a 24.48-fold increase in the percentage of error-free assembled fragments (from 3.23% to 79.07%). Furthermore, the standard MICC error-removal process could be completed within 1.5 h at a cost as low as $0.374 per MICC.     

Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways

High-throughput genomics and the emerging field of synthetic biology demand ever more convenient, economical, and efficient technologies to assemble and clone genes, gene libraries and synthetic pathways. Here, we describe the development of a novel and extremely simple cloning method, circular polymerase extension cloning (CPEC). This method uses a single polymerase to assemble and clone multiple inserts with any vector in a one-step reaction in vitro. No restriction digestion, ligation, or single-stranded homologous recombination is required. In this study, we elucidate the CPEC reaction mechanism and demonstrate its usage in demanding synthetic biology applications such as one-step assembly and cloning of complex combinatorial libraries and multi-component pathways.     

A Series of TA-Based and Zero-Background Vectors for Plant Functional Genomics

With the sequencing of genomes from many organisms now complete and the development of high-throughput sequencing, life science research has entered the functional post-genome era. Therefore, deciphering the function of genes and how they interact is in greater demand. To study an unknown gene, the basic methods are either overexpression or gene knockout by creating transgenic plants, and gene construction is usually the first step. Although traditional cloning techniques using restriction enzymes or a site-specific recombination system (Gateway or Clontech cloning technology) are highly useful for efficiently transferring DNA fragments into destination plasmids, the process is time consuming and expensive. To facilitate the procedure of gene construction, we designed a TA-based cloning system in which only one step was needed to subclone a DNA fragment into vectors. Such a cloning system was developed from the pGreen binary vector, which has a minimal size and facilitates construction manipulation, combined with the negative selection marker gene ccdB, which has the advantages of eliminating the self-ligation background and directly enabling high-efficiency TA cloning technology. We previously developed a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, subcellular localization analysis and promoter activity detection. Our results show that such a system is highly efficient and serves as a high-throughput platform for transient or stable transformation in plants for functional genome research.     

A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique – USER cloning – to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.     

Improved assembly of multimeric genes for the biosynthetic production of protein polymers

We report a general method for the construction of highly repetitive synthetic genes and their use in the biosynthetic production of artificial protein polymers. Through the application of improved recombinant DNA techniques and high-throughput screening methods, we have developed a facile approach to rapid gene assembly and cloning which is widely applicable in the biosynthesis of novel protein polymers. Using this technique, synthetic genes encoding tandem repeats of the beta-sheet forming amino acid sequence AEAEAKAK were constructed and subsequently cloned into a bacterial expression host for inducible protein production. A 17-kDa fusion protein, poly-EAK9, was isolated from Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. The amino acid sequence and molecular weight were confirmed by amino acid analysis, N-terminal sequencing, and MALDI-TOF mass spectrometry. Circular dichroism studies on the artificial protein poly-EAK9 demonstrate the formation of a beta-sheet structure in aqueous solution.     

High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system

The success of structural genomics and proteomics initiatives is dependent on the availability of target genes in vectors suitable for protein production. Here, we compare two high-throughput methods for producing expression vectors from plasmid-derived cDNA fragments. Expression vectors were constructed for compatibility with the Gateway recombination cloning system and the Flexi Vector restriction-based cloning system. Cloning protocols for each system were conducted in parallel for 96 different target genes from PCR through the production of sequence-verified expression clones. The short nucleotide sequences required to prepare the target open reading frames for Flexi Vector cloning allowed a single-step PCR protocol, resulting in fewer mutations relative to the Gateway protocol. Furthermore, through initial cloning of the target open reading frames directly into an expression vector, the Flexi Vector system gave time and cost savings compared to the protocol required for the Gateway system. Within the Flexi Vector system, genes were transferred between four different expression vectors. The efficiency of gene transfer between Flexi Vectors depended on including a region of sequence identity adjacent to one of the restriction sites. With the proper construction in the flanking sequence of the vector, gene transfer efficiencies of 95-98% were demonstrated.     

Construction of a sequenced Clostridium perfringens-Escherichia coli shuttle plasmid.

A new Clostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from the C. perfringens replicon pIP404 and the E. coli vector pUC18. The multiple cloning site and lacZ' gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants in E. coli. Both chloramphenicol and erythromycin resistance can be selected in C. perfringens and E. coli since pJIR418 carries the C. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes from C. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.     

Streamlined cell-free protein synthesis from sequence information

In this study, we describe a cell-free protein synthesis consolidated with polymerase chain reaction (PCR)-based synthetic gene assembly that allows for streamlined translation of genetic information. In silico-designed fragments of target genes were PCR-assembled and directly expressed in a cell-free synthesis system to generate functional proteins. This method bypasses the procedures required in conventional cell-based gene expression methods, integrates gene synthesis and cell-free protein synthesis, shortens the time to protein production, and allows for facile regulation of gene expression by manipulating the oligomer sequences used for gene synthesis. The strategy proposed herein expands the flexibility and throughput of the protein synthesis process, a fundamental component in the construction of synthetic biological systems.     

cDNA文库的构建策略及其应用

cDNA文库在基因分离和克隆中具有重要的作用。从cDNA文库中能筛选出所需要的目的基因,并直接用于该目的基因的表达。cDNA文库是发现新基因和研究基因功能的基础工具。随着分子生物学技术的发展。cDNA文库构建方法有了很大改进和提高,就cDNA文库的构建方法及其应用进行综述。     

DNA克隆和组装技术研究进展

DNA克隆和组装技术是重要的分子生物学工具。近年来,随着合成生物学的飞速发展,对大片段DNA元件的快速有效组装就显得尤为关键。同时,各种DNA克隆和组装技术也竞相发展起来。通过对基于非典型酶切连接、PCR、同源重组、单链退火拼接等原理发展起来的各种DNA克隆和组装技术进行综述,为合成生物学的进一步发展提供有效的操作工具。     

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.     

Rapid gene cloning using terminator primers and modular vectors

We seek to create useful biological diversity by exploiting the modular nature of genetic information. In this report we describe experiments that focus on the modular nature of plasmid cloning vectors. Bacterial plasmids are modular entities composed of origins of replication, selectable markers and other components. We describe a new ligation-independent cloning method that allows for rapid and seamless assembly of vectors from component modules. We further demonstrate that gene cloning can be accomplished simultaneously with assembly of a modular vector. This approach provides considerable flexibility as it allows for ‘menu driven’ cloning of genes into custom assembled modular vectors.