Many bacteria contain an extrachromosomal element of DNA, termed a plasmid, which is a relatively small, covalently closed circular molecule, carrying genes for antibiotic resistance, conjugation or the metabolism of ‘unusual’ substrates. Some plasmids are replicated at a high rate by bacteria such as E. coli and so are excellent potential vectors. In the early 1970s, a number of natural plasmids were artificially modified and constructed as cloning vectors, by a complex series of digestion and ligation reactions. One of the most notable plasmids, termed pBR322 after its developers Bolivar and Rodriguez (pBR), was widely adopted and illustrates the desirable features of a cloning vector, as indicated below (Figure 1):

• The plasmid is much smaller than a natural plasmid, which makes it more resistant to damage by shearing, and increases the efficiency of uptake by bacteria, a process termed transformation.

• A bacterial origin of DNA replication (ori) ensures that the plasmid will be replicated by the host cell. Some replication origins display stringent regulation of replication, in which rounds of replication are initiated at the same frequency as cell division. Most plasmids, including pBR322, have a relaxed origin of replication, whose activity is not tightly linked to cell division, and so plasmid replication will be initiated far more frequently than chromosomal replication. Hence a large number of plasmid molecules will be produced per cell.

• Two genes coding for resistance to antibiotics have been introduced. One of these allows the selection of cells that contain plasmid: if cells are plated on medium containing an appropriate antibiotic, only those that contain plasmid will grow to form colonies. The other resistance gene can be used, as described below, for detection of those plasmids that contain inserted DNA.

• There are single recognition sites for a number of restriction enzymes at various points around the plasmid, which can be used to open or linearise the circular plasmid. Linearising a plasmid allows a fragment of DNA to be inserted and the circle closed. The variety of sites not only makes it easier to find a restriction enzyme that is suitable for both the vector and the foreign DNA to be inserted, but, since some of the sites are placed within an antibiotic resistance gene, the presence of an insert can be detected by loss of resistance to that antibiotic. This is termed insertional inactivation.

Fig1. Map and important features of pBR322.

Insertional inactivation is a useful selection method for identifying recombinant vectors with inserts. For example, a fragment of chromosomal DNA digested with Bam HI is isolated and purified. The plasmid pBR322 is also digested at a single site using Bam HI, and both samples are then subjected to conditions that denature all proteins (e.g. thermal denaturation), thus inactivating the restriction enzyme. Bam HI cleaves to give sticky ends, and so it is possible to obtain ligation between the plasmid and digested DNA fragments in the presence of T4 DNA ligase. The products of this ligation will include plasmids containing a single fragment of the DNA as an insert, but there will also be unwanted products, such as plasmid that has recircularised without an insert, dimers of plasmid, fragments joined to each other, and plasmid with an insert composed of more than one fragment. Most of these unwanted molecules can be eliminated during subsequent steps. The products of such reactions are usually identified by agarose gel electrophoresis.

The ligated DNA must now be used to transform E. coli. Bacteria do not normally take up DNA from their surroundings, but can be induced to do so by prior treatment with Ca 2+ at 4 °C; they are then termed competent, since DNA added to the suspension of competent cells will be taken up during a brief increase in temperature termed heat shock . Small, circular molecules are taken up most efficiently, whereas long, linear molecules will not enter the bacteria.

After a brief incubation to allow expression of the antibiotic resistance genes, the cells are plated onto medium containing the antibiotic, e.g. ampicillin. Colonies that grow on these plates must be derived from cells that contain plasmid, since this carries the gene for resistance to ampicillin. It is not, at this stage, possible to distinguish between those colonies containing plasmids with inserts and those that simply con tainrecircularised plasmids. To do this, the colonies are replica plated, using a sterile velvet pad, onto plates containing tetracycline in their medium. Since the Bam HI site lies within the tetracycline resistance gene, this gene will be inactivated by the presence of an insert, but will be intact in those plasmids that have merely recircularised (Figure 2). Thus colonies that grow on ampicillin but not on tetracycline must contain plasmids with inserts. Since replica plating gives an identical pattern of colonies on both sets of plates, it is straightforward to recognise the colonies with inserts, and to recover them from the ampicillin plate for further growth. This illustrates the importance of a second gene for antibiotic resistance in a vector.

Fig2. Replica plating to detect recombinant plasmids. A sterile velvet pad is pressed onto the surface of an agar plate, picking up some cells from each colony growing on that plate. The pad is then pressed on to a fresh agar plate, thus inoculating it with cells in a pattern identical to that of the original colonies. Clones of cells that fail to grow on the second plate (e.g. owing to the loss of antibiotic resistance) can be recovered from their corresponding colonies on the first plate.

Although recircularised plasmid can be selected against, its presence decreases the yield of recombinant plasmids containing inserts. If the digested plasmids are treated with the enzyme alkaline phosphatase prior to ligation, recircularisation will be pre vented, since this enzyme removes the 5′-phosphate groups that are essential for ligation. Links can still be made between the 5′-phosphate of insert and the 3′-hydroxyl of the plasmid, so only recombinant plasmids and chains of linked DNA fragments will be formed. It does not matter that only one strand of the recombinant DNA is ligated, since the nick will be repaired by bacteria transformed with these molecules.

The valuable features of pBR322 have been enhanced by the construction of a series of plasmids termed pUC (produced at the University of California) (Figure 3). There is an antibiotic resistance gene for tetracycline and origin of replication for E. coli . In addition, the most popular restriction sites are concentrated into a region termed the multiple cloning site ( MCS). In addition, the MCS is part of a gene in its own right and codes for a portion of a polypeptide called β-galactosidase. When the pUC plasmid has been used to transform the host cell E. coli, the gene may be switched on by adding the inducer IPTG (isopropyl-β- D -thiogalactopyranoside). Its presence causes the enzyme β-galactosidase to be produced. The functional enzyme is able to hydrolyse a colourless substance called X-gal (5-bromo-4-chloro-3-indolyl-β-galactopyranoside) into a blue insoluble material (5,5′-dibromo-4,4′–dichloro indigo) (Figure 4). However, if the gene is disrupted by the insertion of a foreign fragment of DNA, a non-functional enzyme results, which is unable to carry out hydrolysis of X-gal. Thus, a recombinant pUC plasmid can be easily detected since it is white or colourless in the presence of X-gal, whereas an intact non-recombinant pUC plasmid will be blue, since its gene is fully functional and not disrupted. This elegant system, termed blue/white selection, allows the initial identification of recombinants to be undertaken very quickly and has been included in a number of subsequent vector systems. This selection method and insertional inactivation of antibiotic resistance genes do not, however, provide any information on the character of the DNA insert, just the status of the vector.

Fig3. Map and important features of pUC18.

Fig4. Principle of blue/white selection for the detection of recombinant vectors.

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قسم صناعة الشبابيك يواصل أعمال الإدامة لأحد أبواب حرم مرقد أبي الفضل العباس (عليه السلام)

ضمن فعاليات ملتقى النورين.. المجمع العلمي يقيم ندوة فكرية في بابل

المجمع العلمي يحيي ليلة القدر الأولى في مقام الإمام المهدي (عجّل الله فرجه)

مركز السيد جعفر الحلي الطبي يقدم الاستشارات والخدمات الصحية في ليالي القدر المباركة

المزيد

الآخبار الصحية

خمسة مؤشرات صحية مهملة قد تنقذ حياتك

متى تكون الحمى طبيعية؟

التدخين الإلكتروني يهدد قلبك.. دراسة جديدة تكشف مخاطر ارتفاع ضغط الدم

دراسة تكشف فائدة بعض أنواع الشاي في محاربة الاكتئاب

المزيد

الآخبار التكنلوجية

الجهاز المناعي يظهر دورا غير متوقع في صحة العظام

ابتكار من جامعة الشارقة منخفض التكلفة للحماية من الزلازل

اكتشاف تأثير غير متوقع للكاكاو على وظائف الدماغ

باحثون يرصدون أثر تغيّر المناخ في دم البشر

المزيد

مواضيع ذات صلة

Cloning Vectors : Cosmid-Based Vectors

Cloning Vectors : Phagemid-Based Vectors

Cloning Vectors : Virus-Based Vectors

The TATA Box, Initiators, and CpG Islands Function as Promoters in Eukaryotic DNA

Chromosome Painting and DNA Sequencing Reveal the Evolution of Chromosomes

During Metaphase, Chromosomes Can Be Distinguished by Banding Patterns and Chromosome Painting

Chromosome Number, Size, and Shape at Metaphase Are Species-Specific

Origins and Types of Mutations

Regulation of Gene Expression

The Human Genome

The Polymerase Chain Reaction (PCR)

Additional Nonhistone Proteins Regulate Transcription and Replication