A useful feature of any cloning vector is the amount of DNA it may accept or have inserted before it becomes non-viable. Inserts greater than 5 kb increase plasmid size to the point at which efficient transformation of bacterial cells decreases markedly, and so bacteriophages (bacterial viruses) have been adapted as vectors in order to propagate larger fragments of DNA in bacterial cells. Cloning vectors derived from λ bacteriophage are commonly used, since they offer an approximately 16-fold advantage in cloning efficiency in comparison with the most efficient plasmid cloning vectors.

Phage λ is a linear double-stranded phage approximately 49 kb in length (Figure 1). It infects E. coli with great efficiency by injecting its DNA through the cell membrane. In the wild-type phage λ, the DNA follows one of two possible modes of replication. Firstly, the DNA may either become stably integrated into the E. coli chromosome where it lies dormant until a signal triggers its excision; this is termed the lysogenic life cycle. Alternatively, it may follow a lytic life cycle where the DNA is replicated upon entry to the cell, phage head and tail proteins are synthesised rapidly and new functional phage assembled. The phage are subsequently released from the cell by lysing the cell membrane to infect further E. coli cells nearby. At the extreme ends of phage λ are 12 bp sequences termed cos (cohesive) sites. Although they are asymmetric, they are similar to restriction sites and allow the phage DNA to be circularised. Phage may be replicated very efficiently in this way, the result of which are concatemers of many phage genomes, which are cleaved at the cos sites and inserted into newly formed phage protein heads.

Fig1. The lysogenic and lytic cycles of bacteriophage λ.

Much use of phage λ has been made in the production of gene libraries, mainly because of its efficient entry into the E. coli cell and the fact that larger fragments of DNA may be stably integrated. For the cloning of long DNA fragments, up to approximately 25 kb, much of the non-essential λ DNA that codes for the lysogenic life cycle is removed and replaced by the foreign DNA insert. The recombinant phage is then assembled into pre-formed viral protein particles, a process termed in vitro packaging. These newly formed phage are used to infect bacterial cells that have been plated out on agar (Figure 2).

Fig2. Two strategies for producing in vitro packaging extracts for bacteriophage λ.

Once inside the host cells, the recombinant viral DNA is replicated. All the genes needed for normal lytic growth are still present in the phage DNA, and so multiplication of the virus takes place by cycles of cell lysis and infection of surrounding cells, giving rise to plaques of lysed cells on a background, or lawn , of bacterial cells. The viral DNA, including the cloned foreign DNA, can be recovered from the viruses in these plaques and analysed further by restriction mapping and agarose gel electrophoresis.

In general, two types of λ phage vectors have been developed, λ insertion vectors and λ replacement vectors (Figure 3). The λ insertion vectors accept less DNA than the replacement type since the foreign DNA is merely inserted into a region of the phage genome with appropriate restriction sites; common examples are λgt10 and λcharon 16A. With a replacement vector, a central region of DNA not essential for lytic growth is removed (a stuffer fragment) by a double digestion with, for example, Eco RI and Bam HI. This leaves two DNA fragments termed right and left arms. The central stuffer fragment is replaced by inserting foreign DNA between the arms to form a functional recombinant λ phage. The most notable examples of λ replacement vectors are λEMBL and λZap.

Fig3. General schemes used for cloning into λ insertion and λ replacement vectors. C l857 is a temperature-sensitive mutation that promotes lysis at 42 °C after incubation at 37 °C.

λZap is a commercially produced cloning vector that includes unique cloning sites clustered into a multiple cloning site (MCS) (Figure 4). Furthermore, the MCS is located within a lacZ region providing a blue/white screening system based on insertional inactivation. It is also possible to express foreign cloned DNA from this vector. This is a very useful feature of some λ vectors since it is then possible to screen for protein product rather than the DNA inserted into the vector. The screening is therefore undertaken with antibody probes directed against the protein of interest. Other features that make this a useful cloning vector are the ability to produce RNA transcripts termed cRNA or riboprobes . This is possible because two promoters for RNA polymerase enzymes exist in the vector, a T7 and a T3 promoter, which flank the MCS.

Fig4. General map of λZap cloning vector, indicating important areas of the vector. The multiple cloning site is based on the lacZ gene, providing blue/white selection based on the β-galactosidase gene. In between the initiator (I) site and the terminator (T) site lie sequences encoding the phagemid Bluescript.

One of the most useful features of λZap is that it has been designed to allow automatic excision in vivo of a small 2.9 kb colony-producing vector termed pBluescript SK, a phagemid. This technique is sometimes termed single-stranded DNA rescue and occurs as the result of a process termed superinfection , where helper phage are added to the cells, which are then grown for an additional period of approximately 4 hours (Figure 5).

Fig5. Single-stranded DNA rescue of phagemid from λZap. The single-stranded phagemid pBluescript SK may be excised from λZap by addition of helper phage. This provides the necessary proteins and factors for transcription between the I and T sites in the parent phage to produce the phagemid with the DNA cloned into the parent vector.

The helper phage displaces a strand within the λZap that contains the foreign DNA insert. This is circularised and packaged as a filamentous phage similar to M13. The packaged phagemid is secreted from the E. coli cell and may be recovered from the supernatant. The λZap vector therefore allows a number of diverse manipulations to be undertaken without the necessity of recloning or subcloning foreign DNA fragments. The process of subcloning is sometimes necessary when the manipulation of a gene fragment cloned in a general purpose vector needs to be inserted into a more specialised vector for the application of techniques such as in vitro mutagenesis or protein production.

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

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

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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

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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 : Plasmids

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