Vectors

Cloning vectors Allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level. 1 Plasmid vectors 2 Bacteriophage vectors 3 Cosmids 4 BACs & YACs

Plasmid vectors Small, easy to handle Plasmid vectors are double-stranded, circular, self-replicating, extra-chromosomal DNA molecules. Advantages: Small, easy to handle Straightforward selection strategies Useful for cloning small DNA fragments (< 10kbp) Disadvantages: Less useful for cloning large DNA fragments (> 10kbp)

Plasmid vectors Plasmids are circular DNA molecules present in the cytoplasm of the bacteria Capable of autonomous replication Can transfer genes from one cell to other Act as vectors in genetic engineering. Can also present in Yeasts

Plasmid vectors may encode genetic information for properties 1 Resitance to Antibiotics 2 Bacteriocins production 3 Enterotoxin production 4 Enhanced pathogen city 5 Reduced Sensitivity to mutagens 6 Degrade complex organic molecules

Plasmid vector for cloning Contains an origin of replication, allowing for replication independent of host’s genome. Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistance blue-white screening Contains a multiple cloning site (MCS) Easy to be isolated from the host cell.

Plasmids are widely used as cloning vehicles Basic properties. Plasmids are replicons which are stably inherited in an extrachromosomal state. Most plasmids exist as double-stranded circular DNA molecules. If both strands of DNA are intact circles the molecules are described as covalently closed circles or CCC DNA If only one strand is intact, then the molecules are described as open circles or OC DNA If both strands of DNA are intact circles the molecules are described as covalently closed circles or CCC DNA

Not all plasmids exist as circular molecules Not all plasmids exist as circular molecules. Linear plasmids have been found in a variety of bacteria, e.g. Streptomyces sp. and Borrelia burgdorferi. To prevent nuclease digestion, the ends of linear plasmids need to be protected, and two general mechanisms have evolved. Either there are repeated sequences ending in a terminal DNA hairpin loop (Borrelia) or the ends are protected by covalent attachment of a protein (Streptomyces). Plasmids are widely distributed throughout the prokaryotes, vary in size from less than 1 × 106 daltons to greater than 200 × 106, and are generally dispensable.

Plasmids can be categorized into one of two major types – conjugative or non-conjugative – depending upon whether or not they carry a set of transfer genes, called the tra genes, which promote bacterial conjugation. Plasmids can also be categorized on the basis of their being maintained as multiple copies per cell (relaxed plasmids) or as a limited number of copies per cell (stringent plasmids). Generally, con- jugative plasmids are of relatively high molecular weight and are present as one to three copies per chromosome, whereas non-conjugative plasmids are of low molecular weight and are present as multi- ple copies per chromosome (Table 4.2).

Good plasmid cloning vehicles share a number of desirable features An ideal cloning vehicle would have the following three properties: • low molecular weight; • ability to confer readily selectable phenotypic traits on host cells; • single sites for a large number of restriction endo- nucleases, preferably in genes with a readily scorable phenotype .

CLONING VECTORS Cloning vectors are DNA molecules that are used to "transport" cloned sequences between biological hosts and the test tube. Cloning vectors share four common properties: 1. Ability to promote autonomous replication. 2. Contain a genetic marker (usually dominant) for selection. 3. Unique restriction sites to facilitate cloning of insert DNA. 4. Minimum amount of nonessential DNA to optimize cloning.

PLASMIDS Bacterial cells may contain extra-chromosomal DNA called plasmids. Plasmids are usually represented by small, circular DNA. Some plasmids are present in multiple copies in the cell

2.1.3 Plasmid classification The most useful classification of naturally occurring plasmids is based on the main characteristic coded by the plasmid genes. The five major types of plasmid according to this classification are as follows: l. Fertility or F plasmids carry only tra genes and have no characteristic beyond the ability to promote conjugal transfer of plasmids. A well-known example is the F plasmid of E. coli. 2. Resistance or R plasmids carry genes conferring on the host bacterium resistance to one or more antibacterial agents, such as chloramphenicol, ampicillin, and mercury. R plasmids are very important in clinical microbiology as their spread through natural populations can have profound consequences in the treatment of bacterial infections. An example is RP4, which is commonly found in Pseudomonas, but also occurs in many other bacteria. Col plasmids code for colicins, proteins that kill other bacteria. An example is ColE1 of E. coli. l Degradative plasmids allow the host bacterium to metabolize unusual molecules such as toluene and salicylic acid, an example being TOL of Pseudomonas putida. l Virulence plasmids confer pathogenicity on the host bacterium; these include the Ti plasmids of Agrobacterium tumefaciens, which induce crown gall disease on dicotyledonous plants

PLASMID VECTORS Plasmid vectors are ≈1.2–3kb and contain: replication origin (ORI) sequence a gene that permits selection, Here the selective gene is ampr; it encodes the enzyme b-lactamase, which inactivates ampicillin. Exogenous DNA can be inserted into the bracketed region .

SELECTIVE MARKER Selective marker is required for maintenance of plasmid in the cell. Because of the presence of the selective marker the plasmid becomes useful for the cell. Under the selective conditions, only cells that contain plasmids with selectable marker can survive Genes that confer resistance to various antibiotics are used. Genes that make cells resistant to ampicillin, neomycin, or chloramphenicol are used

ORIGIN OF REPLICATION Origin of replication is a DNA segment recognized by the cellular DNA-replication enzymes. Without replication origin, DNA cannot be replicated in the cell.

MULTIPLE CLONING SITE Many cloning vectors contain a multiple cloning site or polylinker: a DNA segment with several unique sites for restriction endo- nucleases located next to each other Restriction sites of the polylinker are not present anywhere else in the plasmid. Cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector

MULTIPLE CLONING SITE Gene to be cloned can be introduced into the cloning vector at one of the restriction sites present in the polylinker

TYPES OF CLONING VECTORS

CLONING VECTORS Different types of cloning vectors are used for different types of cloning experiments. The vector is chosen according to the size and type of DNA to be cloned

PLASMID VECTORS Plasmid vectors are used to clone DNA ranging in size from several base pairs to several thousands of base pairs (100bp -10kb). ColE1 based, pUC vehicles commercially available ones, eg pGEM3, pBlueScript

Disadvantages using plasmids Cannot accept large fragments Sizes range from 0- 10 kb Standard methods of transformation are inefficient

Plasmid vectors

pBR322 The nomenclature of plasmid cloning vectors The name “pBR322” conforms with the standard rules for vector nomenclature: “p” indicates that this is indeed a plasmid. “BR” identifies the laboratory in which the vector was originally constructed (BR stands for Bolivar and Rodriguez, the two researchers who developed pBR322). “322” distinguishes this plasmid from others developed in the same laboratory (there are also plasmids called pBR325, pBR327, pBR328, etc.).

Gene cloning by T. A. Brown

The useful properties of pBR322 The genetic and physical map of pBR322 (Figure 6.1) gives an indication of why this plasmid was such a popular cloning vector. The first useful feature of pBR322 is its size. it was stated that a cloning vector ought to be less than 10 kb in size, to avoid problems such as DNA breakdown during purification pBR322 is 4363 bp, which means that not only can the vector itself be purified with ease, but so can recombinant DNA molecules constructed with it. Even with 6 kb of additional DNA, a recombinant pBR322 molecule is still a manageable size

it carries two sets of antibiotic resistance genes it carries two sets of antibiotic resistance genes. Either ampicillin or tetracycline resistance can be used as a selectable marker for cells containing the plasmid, and each marker gene includes unique restriction sites that can be used in cloning experiments. Insertion of new DNA into pBR322 that has been restricted with PstI, PvuI, or ScaI inactivates the ampR gene, and insertion using any one of eight restriction endonucleases (notably BamHI and HindIII) inactivates tetracycline resistance. This great variety of restriction sites that can be used for insertional inactivation means that pBR322 can be used to clone DNA fragments with any of the sticky ends A third advantage of pBR322 is that it has a reasonably high copy number. Generally there are about 15 molecules present in a transformed E. coli cell, but this number can be increased, up to 1000–3000, by plasmid amplification in the presence of a protein synthesis inhibitor such as chloramphenicol . An E. coli culture therefore provides a good yield of recombinant pBR322 molecules.

The Tn3 transposon is a 4957 base pair mobile genetic element, found in prokaryotes. It encodes three proteins: β-lactamase, an enzyme that confers resistance to β-lactam antibiotics (and is encoded by the gene Bla). Tn3 transposase (encoded by gene tnpA) Tn3 resolvase (encoded by gene tnpR) A transposon (or transposable element) is a small piece of DNA that inserts itself into another place in the genome Col E1 – Colcin producing Plasmid Col Plasmids code for colcinins, proteins that kill other bacteria. An example is ColE1 of E.coli