生物技术专业英语第4课.doc

Lesson 4 LIGATION, TRANSFORMATION AND ANALYSIS OF RECOMBINANTSDNA ligation: T4 DNA ligase repairs breaks in a dsDNA DNA backbone and can covalently rejoin annealed cohesive ends in the reverse of a restriction enzyme酶 reaction, to create new DNA molecules.DNA 连接：T4 DNA连接酶在双链DNA的分水岭处修复破损的地方，然后在反向限制酶反应中，能共价地再加入退火的粘性末端，从而形成新的DNA分子。Recombinant DNA molecules: The use of a restriction enzyme, followed by DNA ligase, can create recombinant plasmids, with a target DNA fragment inserted into a vector plasmid.重组DNA分子：限制性内切酶的应用，紧随DNA连接酶，能通过将目标片段插入一个人载体质粒形成重组质粒。Alkaline phosphatase: Treatment of the linear vector molecule with alkaline phosphatase will remove the 5-phosphates and render the vector unable to ligate into a circle without an inserted target, so reducing the proportion of recreated vector in the mixture.碱性磷酸酶：碱性磷酸酶对线状载体分子的处理是移除5的磷酸键和会使没带出入目标基因的载体连接成一个环，从而混合物中减少重新形成的载体的比率。ransformation: Transformation is the process of take-up of foreign DNA, normally plasmids, by bacteria. Plasmids are cloned by transfer into strains of E. coli with defined genetic properties. The E. coli cells can be made competent to take up plasmid DNA by treatment with Ca2+. The cells are plated out on agar and grown to yield single colonies, or clones.转换：转换是提取外来DNA的过程，一般是在细菌里的质粒。带有明确遗传特性的质粒是通过转移到大肠杆菌菌株才被克隆的。大肠杆菌细胞能通过Ca2+ 的处理提取质粒DNA。细胞被平铺在琼脂上，然后逐渐产出单一的菌落或是克隆体。Selection: Bacteria which have taken up a plasmid are selected by growth on a plate containing an antibiotic to which the plasmid vector encodes resistance.筛选：已经提取了质粒的细菌是通过含有看抗生素的生长培养基选出来的，而这种抗生素是阻碍质粒编码的。Transformation efficiency: The efficiency of the transformation step is given by the number of anti-biotic-resistant colonies per microgram of input plasmid DNA.转换效率：转换步骤的效率是通过输出质粒DNA每微克抗生素抗性菌落的数量所给定的。Screening transformants: In many cases, such as when using DNA libraries, plasmid and other vectors have been designed to facilitate the screening of transformants for recombinant plasmids. In the case of a simple subcloning experiment, transformants are screened most easily by digesting消化 the DNA from minipreparations of the transformants, followed by analysis on an agarose gel.筛选转化株：在许多情况里，如利用DNA文库、质粒和其他载体是已经设计好促进筛选重组质粒的转换株的。在亚克隆实验情况里，转换株通过消化来自转换株的小量准备的DNA最容易被筛选出来，它是紧随琼脂糖凝胶分析的。Growth and storage of transformants: Single colonies from a transformation plate are grown in liquid medium, maintaining the antibiotic selection for the plasmid, and a portion of the culture is stored for later use as a frozen glycerol stock.转换株的生长和储存：来自转换平板的单一菌落是在一体培养基中生长的，继续抗生素对质粒的选择，也是被储存到冰冻的丙三醇库存储存起来为以后使用的培养菌的一部分。Gel analysis: Recombinant plasmids can be distinguished from vectors by size on an agarose gel and by excising the inserted fragment with the same restriction enzyme(s) used to insert it.凝胶分析：重组质粒子能在琼脂糖凝胶上通过大小来区别于载体和通过相同的用来插入到载体的限制酶切除插入的片段。Fragment orientation: The orientation of the insert in the vector may be determined using an agarose gel by digestion of the plasmid with a restriction enzyme known to cut asymmetrically within the insert sequence.片段定位：载体的插入物的定位通过在插入序列里不对称地切割质粒的消化利用琼脂糖凝胶可以准确测定。D NA ligation:To insert a target DNA fragment into a vector, a method for the covalent joining of DNA molecules is essential.DNA连接：将目标DNA片段插入到载体中必须需要DNA分子的共价连接方法。DNA ligase enzymes perform just this function; they will repair (ligate) a break in one strand of a dsDNA DNA molecule (see Topic E2), provided the 5-end has a phosphate group.只在这种作用下DNA连接酶才表现；它们会在一条双链DNA分子的一条带修复破损的位置，给予带有磷酸基团的5末端。They require an adenylating agent to activate the phosphate group for attack by the 3-OH; the E. coli enzyme uses NAD+, and the more commonly used enzyme from bacteriophage T4 uses ATP.为了来自于3-OH的制止，它们需要一种腺苷化剂去激活磷酸基团；大肠杆菌酶利用NAD+ ，而通常大多数来自于噬菌体T4的适应酶利用ATP。Ligases are efficient at sealing the broken phosphodiester bonds in an annealed pair of cohesive ends (see Topic G2), essentially the reverse of a restriction enzyme reaction, and T4 ligase can even ligate one blunt end to another, albeit with rather lower efficiency.在一个粘性末端的退火碱基处，连接酶对确定破损的磷酸二酯键是有效的，基本上是限制内切酶的反转防御，以及T4连接酶甚至能连接一个迟钝的末端到另个上面唔；即使是更低的效率。Recombinant DNA molecules:We can now envisage an experiment in which a DNA fragment containing a gene (X) of interest (the target DNA) is inserted into a plasmid vector (Fig. 1).重组DNA分子：现在我们可以设想一个这样的实验，含有目标基因的片段被插入到一个智力载体当中（图1）。The target DNA may be a single fragment isolated from an agarose gel (see Topic G3), or a mixture of many fragments from, for example, genomic DNA (see Topic Il).目标DNA可能是来自于琼脂糖凝胶分离出来的单一片段，或者是如来自于基因组许多片段的混合物。If the target has been prepared by digestion with EcoRI, then the fragment can be ligated with vector DNA cut with the same enzyme (Fig. 1).如果目标基因已经由EcoRI消化准备好，然后通过切割DNA相同的酶的作用连被连接。In practice, the vector should have only one site for cleavage with the relevant enzyme, since otherwise, the correct product could only be formed by the ligation of three or more fragments, which would be very inefficient.事实上，载体应该只有一个与相关酶的切割位点，否则，正确的产物只能够形成于3个或更多片段的连接，这样的话将非常没效率。There are many possible products from this ligation reaction, and the outcome will depend on the relative concentrations of the fragments as well as the conditions, but the products of interest will be circular molecules with the target fragment inserted at the EcoRI site of the vector molecule (with either orientation), to form a recombinant molecule (Fig. 1).来自于连接反应有许多可能的产物 ，结果依靠不同相对浓度的片段和条件，但将于插入在载体分子的EcoRI位点目标产物的目标片段形成环状的分子。The recreation of the original vector plasmid, by circularization of the linear vector alone, is a competing side reaction which can make the identification of recombinant products problematic.通过线状的单一载体的通函询证，原始在体质粒的反应是一个竞争的副反应，这个反应可以识别不确定的重组的产物。One solution is to prepare both the target and the vector using a pair of distinct restriction enzymes, such that they have noncompatible cohesive ends at either end. The likelihood of ligating the vector into a circle is then much reduced.一个方法是利用一对不同的限制性内切酶制备目标和载体，如他们在另外的末端有不兼容的粘性末端。连接在环里的载体有很大可能被分解掉。Alkaline phosphatase:If it is inconvenient to use two restriction enzymes, then the linear ector fragment may be treated with the enzyme alkaline phosphatase after restriction enzyme digestion.碱性磷酸酶：如果不能使用两种限制性内切酶，可以在限制性内切酶消化后用碱性磷酸酶处理线性载体片段。Alkaline phosphatase removes phosphate groups from the 5-ends of DNA molecules.碱性磷酸酶移走位于DNA分子5端的磷酸基团。The linear vector will hence be unable to ligate into a circle, since no phosphates are available for the ligation reaction (Fig. 2).因此线性载体将不能连接在环里，因为没有磷酸基团不能进行连接反应。A ligation with a target DNA insert can still proceed, since one phosphate is present to ligate one strand at each cut site (Fig. 2).一个连接目标DNA插入物仍然可以前进，因为每个磷酸基团在每个位点用来连接每条带的。The remaining nicks in the other strands will be repaired by cellular mechanisms after transformation (see following).在转换之后，剩余的在其他带的割伤能通过细胞机理被修补好。Transformation:The components of the mixture of recombinant and other plasmidz molecules formed by ligation (Fig. I) must now be isolated from one another and replicated (doned) by transfer into a host organism. By far the most common hosts for simple cloning experiments are strains of E. coli which have specific genetic properties.通过连接形成的重组或其他质粒分子混合物的成分现在必须从另外一个中被分离出来，然后转移到宿主有机体中进行复制。到目前为止用于简单的克隆实验的大多数普通的宿主是大肠杆菌菌株，因为其有特殊的遗传特性。One obvious requirement, for example, is that they must not express a restriction-modification system (see Topic G3).例如，一个明显的必要条件是它们必须不能表达限制修饰系统。It was discovered that E. coli cells treated with solutions containing Ca2+ ions were rendered susceptible to take up exogenous DNA, a process known as transformation.被含有Ca2+ 的溶液处理的大肠杆菌细胞易被影响利用用外生的DNA，这个过程叫转换。Cells pre-treated with Ca2+ (and sometimes more exotic metal ions such as Rb+ and Mn2+), in order to render them able to take up DNA, are known as competent cells.利用Ca2+ 处理细胞是为了能让它们能够利用好DNA。 In transformation of E. coli, a solution of a plasmid molecule, or a mixture of molecules formed in a ligation reaction, is combined with a suspension of competent cells for a period, to allow the DNA to be taken up.大肠杆菌的转换，一种质粒分子溶液或者是形成于连接反应的分子混合物跟一个周期的有能力的细胞的悬浮液结合，从而使DNA能够被利用。The precise mechanism of the transfer of DNA into the cells is obscure.将DNA转移到细胞的精确机理是模糊的。The mixture is then heat-shocked at 42 for 1-2 min.然后混合物在42热休克1-2分钟。This induces enzymes involved in the repair of DNA and other cellular components, which allow the cells to recover from the unusual conditions of the transformation process, and increases the efficiency.参与DNA修复的诱导酶和其他细胞成分允许细胞转换过程从转换过程与众不同的条件恢复，并提高效率。The cells are then incubated in a growth medium and finally spread on an agar plate and incubated until single colonies of bacteria grow (Fig. 3).细胞接着在成长培养基中逐渐形成，最后在琼脂平板扩散开来，直到单一的细菌菌落逐渐地长出来。All the cells within a colony originate from division of a single individual. Thus, all the cells will have the same genotype, barring spontaneous mutations (see Topic FI), including the presence of any plasmid introduced in the transformation step (in other words, they will be clones).所有细胞的菌落来源于单一个体的分离。因此所有的细胞将拥有相同的基因型，出来自发突变，包括在转换步骤中设计的每一个质粒。Selection:If all the competent cells present in a transformation reaction were allowed to grow on an agar plate, then many thousands of millions of colonies would result.如果所有在转换反应出现的有能力的细胞被允许在琼脂平板上成长，那么成千上万的菌落将产生。Furthermore, transformation is an inefficient process; most of the resultant colonies would not contain a plasmid molecule and it would not be obvious which did.而且，转换是一个没效率的过程大多数合成的菌落将不含有质粒分子以及它将表现地不明显。A method for the selection of clones containing a plasmid is required.需要筛选含有质粒菌落的方法。This is almost always provided by the presence of an antibiotic resistance gene on the plasmid vector, for example the -lactamase gene (arnpr)conferring resistance to ampicillin (see Topic G2).这种方法几乎总是使用在出现抗生素抗性基因的质粒载体，例如内酰胺酶赋予对氨比西林的阻力。If the transformed cells are grown on plates containing ampicillin, only those cells which are expressing -1actamase due to the presence of a transformed plasmid will survive and grow.如果转换的细胞在含有氨比西林的平板上生长，那么只有那些因含有转换质粒而能够表达内酰胺酶的细胞才能够存活以及生长。We can therefore be sure that colonies formed on an ampicillin plate after transformation have grown from single cells which contained a plasmid with an intact-1actamase gene.因此，我们能够确定，在转换后形成于氨比西林平板的菌落已经从单一细胞生长出来，是含有一个完整无缺内酰胺酶质粒的。If a ligation mixture had been used for the transformation, we would not know at this stage which clones contain recombinant plasmids with a target fragment incorporated (Fig. 1).如果一个连接混合物已经被用于转换，那么我们将不知道眼下那一个菌落是包含有目标片段的重组质粒。The quality of a given preparation of competent cells may be measured by determining the transformation efficiency, defined as the number of colonies formed (on a selective plate) per microgram of input DNA, where that DNA is a pure plasmid, most commonly, the vector to be used in a cloning experiment.转换效率：一个给定的目标细胞的制备的特性可以通过确定转换效率而被测量，规定为每微克输入DNA形成的菌落数量，DNA是一个纯的质粒，最常见的是，载体将被用于一个克隆实验中。Transformation efficiencies can range from 103 per g for crude transformation protocols, which would only be appropriate for transferring an intact plasmid to a new host strain, to more than 108 per g for very carefully prepared competent cells to be used for the generation of libraries (see Topic I2).转换效率能够对未加工的转换协议在103 每微克内变动，这只适合于将完整无缺的质粒转移到新的宿主菌株中，对于非常谨慎地准备用于文库代数的目的细胞需要非常谨慎地准备超过108每微克。A transformation efficiency of around l0s perg would be adequate for a simple cloning experiment of the kind outlined here.大约l0s每微克的转换效率对于这里概述中的简单克隆实验是足够的。Screening transformants: Once a set of transformant clones has been produced-in a cloning experiment, the first requirement is to know which clones contain a recombinant plasmid, with inserted target fragment. Plasmids have been designed to facilitate this process, and are described in Topic H1.筛选转换株：一旦一套转换株的无性繁殖个体已经在一个克隆实验被生产，第一个需要是了解哪一个无性繁殖个体是含有插入目标片段的重组质粒。质粒已经被设计来促进这一过程，并且在Topic H1有描述。In many cases, such as the screening of a DNA library (see Topic I3), it will then be necessary to identify the clone of interest from amongst thousands or even hundreds of thousands of others.在许多情况下，如一个DNA文库的筛选有必要在上千甚至成百上千的其他目标克隆中鉴别出所需的目标克隆。This can be the most time-consuming part of the process, and it is discussed in Topic I3. In the case of a simple subcloning experiment, the design of the experiment can maximize the production of recombinant clones, for example by alkaline phosphatase treatment of the vector.这是过程中最费时的部分，在Topic I3有讲。至于一个简单的亚克隆实验，实验的设计能后最大化地得到重组克隆的产量，如通过碱性磷酸酶对载体的处理。In this case, the normal method of screening is to prepare the plasmid DNA from a number of许多 clones and analyze it by agarose gel electrophoresis.在这种情况下，筛选上的正常方法是从许多克隆中午制备质粒DNA以及通过琼脂糖凝胶电泳分析。Growth and storage of transformants: Single colonies from a transformation plate are transferred to culture broth and grown overnight to stationary phase.转换株的成长和储备：来自于 转换平板的单一菌落被转移到液体培养基，接着隔夜成长到平稳期。The broth must include the antibiotic used to select the transformants on the original plate to maintain the selection for the presence of the plasmid.液体培养基在原始平板上必须含有用于选择转换株的抗生素，以保持对存在之力的筛选。Some plasmids may be lost from their host strains during prolonged growth without selection, since the plasmid-bearing bacteria may be out-competed by those which accidentally lose the plasmid, enabling them to replicate with less energy cost.在没有筛选的持续增长的期间内一些之力可能从他们的宿主菌株中丢失，因为质粒轴承细菌可能被那些意外失去质粒的所击败，使它们用较少的能量消耗复制。The plasmids are then prepared from the cultures by the minipreparation technique (see Topic G2). It is normal practice to prepare a stock of each culture at this stage, by freezing a portion of the culture in the presence of glycerol, to protect the cells from ice crystal formation (a glycerol stock). The stock will enable the same strain/plasmid to be grown and prepared again if and when it is required.接着质粒通过小量制备技术从培养基中被制备出来。先要准备每种储存的培养基是正常实际的，通过冰冻有甘油存在的培养基的一部分，以保护来自冰晶形态的细胞。当有需要的时候，备有的将是相同的菌株或者质粒再次成长或者制备。Gel analysis :Recombinant plasmids can usually be simply distinguished from recreated vectors by the relative sizes of the plasmids, and further by the pattern of restriction digests凝胶分析：通过相关质粒大小，重组质粒经常可以简单区别于再创造的载体，接着更进一步通过限制性消化的模式。.Figure 4 shows a hypothetical gel representing the analysis of the plasmids in Fig. 1. Tracks corresp