Novel Strategies to Construct Complex Synthetic Vectors to Produce DNA Molecular Weight Standards

DNA molecular weight standards (DNA markers, nucleic acid ladders) are commonly used in molecular biology laboratories as references to estimate the size of various DNA samples in electrophoresis process. One method of DNA marker production is digestion of synthetic vectors harboring multiple DNA fragments of known sizes by restriction enzymes. In this article, we described three novel strategies—sequential DNA fragment ligation, screening of ligation products by polymerase chain reaction (PCR) with end primers, and “small fragment accumulation”—for constructing complex synthetic vectors and minimizing the mass differences between DNA fragments produced from restrictive digestion of synthetic vectors. The strategy could be applied to construct various complex synthetic vectors to produce any type of low-range DNA markers, usually available commercially. In addition, the strategy is useful for single-step ligation of multiple DNA fragments for construction of complex synthetic vectors and other applications in molecular biology field. Zhe Chen and Jianbing Wu contributed to this work equally.     

Multi-fragment site-directed mutagenic overlap extension polymerase chain reaction as a competitive alternative to the enzymatic assembly method

Methods for introducing multiple site-directed mutations are important experimental tools in molecular biology. Research areas that use these methods include the investigation of various protein modifications in cellular processes, modifying proteins for efficient recombinant expression, and the stabilization of mRNAs to allow for increased protein expression. Introducing multiple site-directed mutations is also an important tool in the field of synthetic biology. There are two main methods used in the assembling of fragments generated by mutagenic primers: enzymatic assembly and overlap extension polymerase chain reaction (OE–PCR). In this article, we present an improved OE–PCR method that can be used for the generation of large DNA fragments (up to 7.4 kb) where at least 13 changes can be introduced using a genomic template. The improved method is faster (due to fewer reaction steps) and more accurate (due to fewer PCR cycles), meaning that it can effectively compete with the enzymatic assembly method. Data presented here show that the site-directed mutations can be introduced anywhere between 50 and 1800 bp from each other. The method is highly reliable and predicted to be applicable to most DNA engineering when the introduction of multiple changes in a DNA sequence is required.     

基于抗性恢复的高效克隆超短片段重组质粒的构建

分子克隆作为一种常规技术被广泛应用于DNA及蛋白质的研究。在传统的分子克隆中,主要通过限制性内切酶先分别消化目的 DNA片段及载体,再纯化回收,然后用DNA连接酶将二者连接。而对一些超短基因片段(300 bp),通过酶切及切胶纯化后,回收率极低,导致插入表达载体比较困难。文中介绍了一种新的利用质粒抗性恢复进行克隆的方法,大大提高了克隆效率,为短基因片段的分子克隆提供了一种高效的方法。     

Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector

Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named p GF(plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid p CC1 BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome(YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction(PCR) from the plasmid p BS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination(TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using p GF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction.     

Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC

We describe a new cloning method, sequence and ligation-independent cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing. SLIC mimics in vivo homologous recombination by relying on exonuclease-generated ssDNA overhangs in insert and vector fragments, and the assembly of these fragments by recombination in vitro. SLIC inserts can also be prepared by incomplete PCR (iPCR) or mixed PCR. SLIC allows efficient and reproducible assembly of recombinant DNA with as many as 5 and 10 fragments simultaneously. SLIC circumvents the sequence requirements of traditional methods and functions much more efficiently at very low DNA concentrations when combined with RecA to catalyze homologous recombination. This flexibility allows much greater versatility in the generation of recombinant DNA for the purposes of synthetic biology.     

Engineering of a genome-reduced host: practical application of synthetic biology in the overproduction of desired secondary metabolites

Synthetic biology aims to design and build new biological systems with desirable properties, providing the foundation for the biosynthesis of secondary metabolites. The most prominent representation of synthetic biology has been used in microbial engineering by recombinant DNA technology. However, there are advantages of using a deleted host, and therefore an increasing number of biotechnology studies follow similar strategies to dissect cellular networks and construct genome-reduced microbes. This review will give an overview of the strategies used for constructing and engineering reduced-genome factories by synthetic biology to improve production of secondary metabolites.     

High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing

The construction of synthetic biological systems involving millions of nucleotides is limited by the lack of high-quality synthetic DNA. Consequently, the field requires advances in the accuracy and scale of chemical DNA synthesis and in the processing of longer DNA assembled from short fragments. Here we describe a highly parallel and miniaturized method, called megacloning, for obtaining high-quality DNA by using next-generation sequencing (NGS) technology as a preparative tool. We demonstrate our method by processing both chemically synthesized and microarray-derived DNA oligonucleotides with a robotic system for imaging and picking beads directly off of a high-throughput pyrosequencing platform. The method can reduce error rates by a factor of 500 compared to the starting oligonucleotide pool generated by microarray. We use DNA obtained by megacloning to assemble synthetic genes. In principle, millions of DNA fragments can be sequenced, characterized and sorted in a single megacloner run, enabling constructive biology up to the megabase scale.     

Synthetic biology of minimal systems

“Synthetic biology” is a concept that has developed together with, or slightly after, “systems biology”. But while systems biology aims at the full understanding of large systems by integrating more and more details into their models, synthetic biology phrases different questions, namely: what particular biological function could be obtained with a certain known subsystem of reduced complexity; can this function be manipulated or engineered in artificial environments or genetically modified organisms; and if so, how? The most prominent representation of synthetic biology has so far been microbial engineering by recombinant DNA technology, employing modular concepts known from information technology. However, there are an increasing number of biophysical groups who follow similar strategies of dissecting cellular processes and networks, trying to identify functional minimal modules that could then be combined in a bottom-up approach towards biology. These modules are so far not as particularly defined by their impact on DNA processing, but rather influenced by core fields of biophysics, such as cell mechanics and membrane dynamics. This review will give an overview of some classical and some quite new biophysical strategies for constructing minimal systems of certain cellular modules. We will show that with recent advances in understanding of cytoskeletal and membrane elements, the time might have come to experimentally challenge the concept of a minimal cell.     

平末端DNA随机连接构建重组质粒

目的:拼接DNA片段并克隆。方法:用T4DNA连接酶将DNA片段以平末端随机连接,随后用限制性内切酶切割,琼脂糖电泳分离酶切产物,挑选特定片段纯化回收,与线性化的载体质粒连接,转化大肠杆菌感受态细胞。结果:通过以上步骤,成功拼接了不同DNA片段,构建了含有目的拼接片段的重组质粒。结论:该方法简便、易行、可靠,可作为拼接、克隆DNA的备选方案,在分子生物学研究和基因工程中应用。     

从琼脂糖凝胶中高效回收DNA技术的探讨

用两只离心管制成的凝胶过滤装置,从电泳后的琼脂糖凝胶中回收DNA片段的简易方法。它依次包括以下步骤：凝胶过滤装置的制作、凝胶切割、凝胶低温冷冻、低温高速离心、ddH20洗胶、DNA纯化和回收效果检测等。用此方法回收的DNA片段产率高、质量纯,可直接用于分子生物学实验的后续操作,如载体连接、PCR模板获得、DNA探针制备、基因测序等。其优点是：DNA片段的回收率高（90％以上）,质量好;操作简便,耗时短;回收装置简单,成本低廉,可进行商品化开发。     

哺乳动物抑制型转录因子及启动子对的全新设计与构建

转录因子及启动子是基因回路的基础。相较于原核启动子,真核启动子作用机制复杂,增加了全新设计与改造的难度。目前有限数量的真核转录因子及启动子成为在哺乳动物细胞中设计并实现复杂基因回路以满足各类临床或工业应用需求的瓶颈。文中介绍了基于能够结合特定DNA序列的DNA结合结构域,通过柔性连接肽连接到转录抑制模块KRAB,构建抑制型转录因子以及通过在SV40启动子下游插入结合序列构建对应启动子的方法。而后,在哺乳动物细胞系中通过流式细胞术对其抑制转录的强度、不同转录因子及启动子对之间的正交性进行了测定。文中提供了一套标准化的、可调节的转录因子及启动子的全新设计与构建方案。基于该方案所构建的5对抑制型转录因子及启动子对能够在哺乳动物细胞中起到不同程度的抑制效果且相互正交。文中构建的哺乳动物转录因子及启动子对扩充了哺乳动物生物元件库,为构建复杂真核基因回路打下了基础;运用该设计方法能够根据需求构建更多正交的人工转录因子及启动子对。     

Available methods for assembling expression cassettes for synthetic biology

Studies in the structural biology of the multicomponent protein complex, metabolic engineering, and synthetic biology frequently rely on the efficient over-expression of these subunits or enzymes in the same cell. As a first step, constructing the multiple expression cassettes will be a complicated and time-consuming job if the classic and conventional digestion and ligation based cloning method is used. Some more efficient methods have been developed, including (1) the employment of a multiple compatible plasmid expression system, (2) the rare-cutter-based design of vectors, (3) in vitro recombination (sequence and ligation independent cloning, the isothermally enzymatic assembly of DNA molecules in a single reaction), and (4) in vivo recombination using recombination-efficient yeast (in vivo assembly of overlapping fragments, reiterative recombination for the chromosome integration of foreign expression cassettes). In this review, we systematically introduce these available methods.     

Construction of DNA Marker Plasmids Based on Taq Tailing Activity and Selective Recovery of Ligation Products

DNA fragments with standard molecular weights (DNA markers, which are usually commercial products) are routinely electrophoresed in conjunction with DNA samples in molecular biology labs to serve as references for DNA molecular weight; this is done by referencing their relative molecular weights. In this study, we present a new technical strategy for constructing super-plasmids for homemade DNA marker production with single restriction enzyme digestion. In this study, two super-plasmids for DNA marker production have been developed, based on tailing activity of Taq polymerase and selective recovery of ligation products following agarose gel electrophoresis.     

DNA组装技术

DNA组装是合成生物学研究的核心技术。随着合成生物学的发展,研究者开发了依赖于DNA聚合酶或DNA连接酶的不同DNA组装技术;为了降低组装成本和便于实现DNA组装的自动化,也发展了一些非酶依赖的DNA组装技术;而几百kb到Mb的大片段DNA的组装则多数依赖于微生物体内重组。文中主要综述了酶依赖、非酶依赖和体内同源重组三类DNA组装技术及其发展情况。     

Advances in molecular cloning

“Molecular cloning” meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful tools showing exponential growth in applications and sophistication of recombinant DNA technology. Cloning genes has become simple what led to an explosion in the understanding of gene function by seamlessly stitching together multiple DNA fragments or by the use of swappable gene cassettes, maximizing swiftness and litheness. A novel archetype might materialize in the near future with synthetic biology techniques that will facilitate quicker assembly and iteration of DNA clones, accelerating the progress of gene therapy vectors, recombinant protein production processes and new vaccines by in vitro chemical synthesis of any in silico-specified DNA construct. The advent of innovative cloning techniques has opened the door to more refined applications such as identification and mapping of epigenetic modifications and high-throughput assembly of combinatorial libraries. In this review, we will examine the major breakthroughs in cloning techniques and their applications in various areas of biological research that have evolved mainly due to easy construction of novel expression systems.     

Rapid and Robust PCR-Based All-Recombinant Cloning Methodology

We report here a PCR-based cloning methodology that requires no post-PCR modifications such as restriction digestion and phosphorylation of the amplified DNA. The advantage of the present method is that it yields only recombinant clones thus eliminating the need for screening. Two DNA amplification reactions by PCR are performed wherein the first reaction amplifies the gene of interest from a source template, and the second reaction fuses it with the designed expression vector fragments. These vector fragments carry the essential elements that are required for the fusion product selection. The entire process can be completed in less than 8 hours. Furthermore, ligation of the amplified DNA by a DNA ligase is not required before transformation, although the procedure yields more number of colonies upon transformation if ligation is carried out. As a proof-of-concept, we show the cloning and expression of GFP, adh, and rho genes. Using GFP production as an example, we further demonstrate that the E. coli T7 express strain can directly be used in our methodology for the protein expression immediately after PCR. The expressed protein is without or with 6xHistidine tag at either terminus, depending upon the chosen vector fragments. We believe that our method will find tremendous use in molecular and structural biology.     

Construction of recombinant DNA by exonuclease recession.

We describe a new exonuclease-based method for joining and/or constructing two or more DNA molecules. DNA fragments containing ends complementary to those of a vector or another independent molecules were generated by the polymerase chain reaction. The 3' ends of these molecules as well as the vector DNA were then recessed by exonuclease activity and annealed in an orientation-determined manner via their complementary single-stranded regions. This recombinant DNA can be transformed directly into bacteria without a further ligase-dependent reaction. Using this approach, we have constructed recombinant DNA molecules rapidly, efficiently and directionally. This method can effectively replace conventional protocols for PCR cloning, PCR SOEing, DNA subcloning and site-directed mutagenesis.     

Positive selection improves the efficiency of DNA assembly

With the advent of synthetic biology and cell engineering, the demand for large synthetic DNA fragments has been steadily increasing. Consequently, a number of multi-fragment cloning technologies optimized for the assembly of sizable DNA constructs have been developed. Still, screening for the right clone can be tedious because the high incidence of illegitimate assembly results in a relatively large proportion of missing or shuffled DNA elements. To mitigate this risk, we have developed a strategy that reduces the rate of fragment mis-assembly and is compatible with a variety of cloning methodologies. The approach is based on the positive selection of truncated plasmid markers, which are rendered active by providing their missing sequences during the assembly process. The method has been successfully validated in the context of complex in vivo and in vitro homologous recombination workflows, but it could be readily adapted to other cloning strategies, including those based on restriction endonucleases.     

DNA组装新方法的研究进展

2010年,蕈状支原体Mycoplasma mycoides的人工合成,迎来了合成生物学的崭新时代.这种突破性的进展主要得益于酵母自身强大的DNA体内重组能力.近几年来,除了利用体内重组的DNA大片段拼接技术,基于连接或聚合思想的不同尺度的DNA体外组装方法也相继出现,如Biobrick\Bglbrick、SLIC与Gibson等温一步法等,这些方法的应用加快了合成生物学功能元件库、生物合成途径乃至微生物染色体的人工构建.事实上,目前所建立的各种DNA组装方法,均是由DNA分子拼接理念(包括两分子衔接思想与多片段组装模式)衍生而来.文中将在介绍DNA组装基本理念的基础上,对体内、体外主要的DNA组装方法进行简要梳理,希望为不同类型的合成生物学功能器件及生物合成途径的构造提供参考与借鉴.