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The problem of transfection of recombinant molecules constructed in vitro was overcome by the successful in vitro assembly of viable and infectious phage particles. Two types of in vitro packaging systems have been developed so far, i.e., two-strain packaging and single-strain packaging.

Two-strain packaging.

The basis of the two-strain in vitrop ackaging system is the complementation of two amber mutations. Two lambda lysogens, each carrying a single amber mutation in a distinctly different gene, are induced and grown separately so that they can synthesize the necessary proteins. Neither of the lysogens alone is capable of packaging the phage DNA. The role of various phage products in DNA packaging has been studied in detail[3]. The E protein is the major component of the bacteriophage head, and in its absence all the viral capsid components accumulate. The D protein is involved in the coupled process of insertion of bacteriophage DNA into the prehead precursor and the subsequent maturation of the head. The A protein is required for the cleavage of the concatenated precursor DNA at the cos sites. Two phage lysogens carrying A and E or D and E mutations in the phage genome are induced separately, and cell extracts are prepared. Neither of the extracts can produce infectious phage particles. However, when the extracts are mixed, mature phage particles are produced by complementation.

The major drawback of the two-strain system is the competition of native phage DNA with recombinant molecules. In both the cell extracts, native phage DNA is also present and can be packaged with an efficiency equal to that of the chimeric DNA. This reduces the proportion of recombinants obtained in a library. The problem of regeneration of endogenous phages obtained in the library was partially overcome by the use of b2-deleted prophages, which poorly excise out of the host chromosome or by UV irradiation of packaging extracts.

Single-strain packaging.

Rosenberg have successfully developed a single-strain packaging system by introducing deletion in the cos region of prophage, rendering the prophage DNA unpackagable because cos is the packaging origin. Induction of the lysogen results in the intracellular accumulation of all protein components needed for packaging.

However, packaging of phage DNA is prevented by the lack of cos sites on the prophage DNA. On the other hand, exogenous DNA with cos sites is packaged efficiently to produce an infectious bacteriophage particle. The single-strain system is superior to two-strain system in having a lower background of parental phages. In addition, it uses E. coli C, which lacks the EcoK restriction system, as the host for the lysogen.

7.3 Biological Containment

The biological containment of recombinant phages is an important aspect from the point of view of ethics and eventual biohazards. It is desirable that cloning vectors and recombinants have poor survival in the natural environment and require special laboratory conditions for their replication and survival. According to Blattner, the lytic phages offer a natural advantage in this respect since the phage and the sensitive bacteria coexist only briefly. A newly inserted segment may not be compatible with E. coli metabolism for extended periods. To make the phage vectors more safe, three amber mutations were introduced in its genome. The new vector Xgt WES XC is safer because an amber suppressor host strain is a very rare occurrence in the natural environment. Many vectors carry one of the amber mutations on the genome so that they can be propagated only on an appropriate suppressor host.

2. Phage vectors

Many phage vectors have been constructed in the recent past, each with its own special features. There is no universal lambda vector which can fulfill all the desired objectives of the cloning experiments.

The choice of a vector depends mainly on

• the size of a DNA fragment to be inserted,

• the restriction enzymes to be used,

• the necessity for expression of the cloned fragment,

• the method of screening to be used to select the desired clones.

Bacteriophage lambda vectors can be broadly classified into two types:

1. replacement vectors ,

2. insertion vectors.

Figure 7. Lambda Phage genome

8.1 Replacement Vectors

Taking advantage of the maximum and minimum genome size essential for efficient packaging and the presence of the nonessential central fragment, it is possible to remove the stuffer fragment and replace it with a foreign DNA fragment in the desired size range. This forms the basis of lambdaderived replacement vectors.

Cloning of a foreign DNA in these vectors involves

• preparation of left and right arms by physical elimination of the nonessential region,

• ligation of the foreign DNA fragment between the arms,

• in vitro packaging and infection.

The replacement vectors contain a pair of restriction sites to excise the central stuffer fragments, which can be replaced by a desired DNA sequence with compatible ends. The presence of identical sites within the stuffer fragment but not in the arms facilitates the separation of the arms and the stuffer on density gradient centrifugation. In many vectors, sets of such sites are provided on attached polylinkers so that an insert can be easily excised. Two purified arms cannot be packaged despite their being ligated to each other, because they fall short of the minimum length required for packaging. This provides positive selection of recombinants. The replacement vectors are convenient for cloning of large (in some cases up to 24 kbp) DNA fragments and are useful in the construction of genomic libraries of higher eukaryotes. Charon and EMBL are among the popular replacement vectors.

8.2 Insertion Vectors

Because the maximum packagable size of lambda genome is 53 kb, small DNA fragments can be introduced without removal of the nonessential (stuffer) fragment. These vectors are therefore called insertion vectors. Cloning of foreign DNA in these vectors exploits the insertional inactivation of the biological function, which differentiates recombinants from nonrecombinants. Insertion vectors are particularly useful in cloning of small DNA fragments such as cDNA.

AgtlO and Agtll are examples of this type of vector. In recent years a multitude of lambda vectors have been constructed. Many innovative approaches have been used to introduce desired properties into the vectors. The following section deals with the strategies adopted for the construction of some of the commonly used vectors and their salient features, utilities, and limitations.

8.3 Storage of Lambda Stocks

Most of the lambda strains are stable for several years when stored at 4°C in SM buffer containing 0.3% freshly distilled chloroform (94). The master stocks of bacteriophage lambda are kept in 0.7% (vol/vol) dimethyl sulfoxide at -70°C for long-term storage. Klinman and Cohen have developed a method for storage of a phage library at -70°C by using top agar containing 30% glycerol.

Conclusion

In my work I determined investigations in Molecular cloning, familiarized with vectors for molecular cloning, summarized the received information and made consequences of scientists researches, defined the main tasks of molecular cloning, and made such conclusions:

1. sequences that permit the propagation of itself in bacteria (or in yeast for YACs) .

2. a cloning site to insert foreign DNA; the most versatile vectors contain a site that can be cut by many restriction enzymes .

3. a method of selecting for bacteria (or yeast for YACs) containing a vector with foreign DNA; uually accomplished by selectable markers for drug resistance .

Cloning vector - a DNA molecule that carries foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell and produces many copies of itself and the foreign DNA .

General Steps of Cloning with Any Vector :

1. prepare the vector and DNA to be cloned by digestion with restriction enzymes to generate complementary ends ;

2. ligate the foreign DNA into the vector with the enzyme DNA ligase;

3. introduce the DNA into bacterial cells (or yeast cells for YACs) by transformation ;

4. select cells containing foreign DNA by screening for selectable markers (usually drug resistance);

Literature

1. Finbar Hayes The Function and Organization of Plasmids// E. coli Plasmid Vectors Methods and Applications.- 2007.- vol.235 – pp. 1-18.

2. Mallory J. A. White and Wade A. Nichols Cosmid Packaging and Infection of E. coli// E. coli Plasmid Vectors Methods and Applications.- 2007.- vol.235 – pp. 67-70

3. Tim S. Poulsen and Hans E. Johnsen BAC End Sequencing // Bacterial Artificial Chromosomes Volume 1: Library Construction, Physical Mapping, and Sequencing.- 2007. – vol.255 - pp.157-162.

4. Andrew Preston Choosing a Cloning Vector// E. coli Plasmid Vectors Methods and Applications.- 2007.- vol.235 – pp. 19-22.

5. Sambrook, J., Fritsch, E. F., and Maniatis, T. (eds.) (1989) Bacteriophage λvectors, in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 2.3–2.125.

6. Srividya Swaminathan and Shyam K. Sharan Bacterial Artificial Chromosome Engineering// Bacterial Artificial Chromosomes Volume 2 :Functional Studies.- 2007. – vol.256 – pp. 089-106

7. www.Microbiologybytes.com

8. www.wikigenes.org

9. http:// plasmid.hms.harvard.edu

10. www. Bookrags.com/YAC

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