Lec#3: Recombinant DNA technology-part 2

Dr. Shah Rukh ASAB, NUST

a. 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. For example genes that make cells resistant to ampicillin, neomycin, or chloramphenicol are used.

b. 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.

c. Multiple Cloning Sites• 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

Examples of plasmid vectors

2. Bacteriophage (𝝀) vectors• Phage lambda is a bacteriophage or phage, i.e. bacterial virus,

that uses E. coli as host.• Its structure is that of a typical phage: head, tail, tail fibres.• Lambda viral genome: 48.5 kb linear DNA with a 12 base ssDNA

"sticky end" at both ends; these ends are complementary in sequence and can hybridize to each other (this is the cos site: cohesive ends).

• Infection: lambda tail fibres adsorb to a cell surface receptor, the tail contracts, and the DNA is injected.

• The DNA circularizes at the cos site, and lambda begins its life cycle in the E. coli host.

Left arm: Head and tail proteins

Right arm: DNA synthesis, regulation and host lysis.

2. Bacteriophage (𝝀) vectors

3. Cosmid vectors• Combine the properties of plasmid vectors

with the useful properties of the l cos site.• Clone large inserts of DNA: size ~ 45 kb.• Presence of the Cos site permits in vitro

packaging of cosmid DNA into Lambda particles.

• Infection process rather than transformation for entry of chimeric DNA into E. coli host

• Maintain Cosmids as phage particles in solution

• But Cosmids are Plasmids:Thus do NOT form plaques but rather cloning proceeds via E. coli colony formation

4. BACs and YACs (Bacterial and Yeast Artificial chromosomes)

These chimeric DNA molecules use a naturally-occurring low-copy number bacterial plasmid origin of replication, such as that of F-plasmid in E. coli. Can be cloned as a plasmid in a bacterial host, and its natural stability generally permits cloning of large pieces of insert DNA, i.e. up to a few hundred kb of DNA.

4a. BACs - Bacterial Artificial Chromosomes

4a. BAC vector

• oriS and oriE mediate replication

• parA and parB maintain single copy number

• ChloramphenicolR marker

• YAC are special linear DNA vectors that resemble normal yeast chromosome. YACs contain telomers that stabalise chromosome ends, centromer, that ensures chromosome partitioning between the two daughter cells and a selective marker gene.

• Capable of carrying inserts of 200 - 2000 kbp in yeast.• Cloning vehicles that propogate in eukaryotic cell hosts as eukaryotic

Chromosomes• YAC cloning vehicles are plasmids

Final chimeric DNA is a linear DNA molecule with telomeric ends: Artificial Chromosome.

• Often have a selection for an insert• YAC cloning vehicles often have a bacterial origin of DNA replication (ori) and a

selection marker for propogation of the YAC through bacteria.• The YAC can use both yeast and bacteria as a host

4b. Yeast artificial chromosomes YACs

What are clones?

• Clones – Genetically identical molecules, cells, or organisms

all derived from a single ancestor

• Cloning– The production of identical copies of molecules,

cells, or organisms from a single ancestor

What is DNA cloning?

• DNA cloning is a technique for reproducing DNA fragments.

• It can be achieved by two different approaches:

▪ cell based ▪ using polymerase chain reaction (PCR) • a vector is required to carry the DNA fragment

of interest into the host cell.

What is Gene Cloning?

1. A fragment of DNA, containing the gene to be cloned, is inserted into a circular DNA molecule called a vector, to produce a recombinant DNA molecule.

2. The vector transport the gene in the host cell, which is usually a bacterium, although other type of cells can also be used

3. Within the host cell the vector multiplies, producing numerous identical copies not only itself but also the gene it carries

What is Gene Cloning?

4. When the host cell divides, copies od recombinant DNA molecule are passed to the progeny and further vector replication take place

5. After a large number of cell divisions, a clony or clone, of identical host cell is produced. Each cell in the clone contains one or more copies of the recombinant DNA molecule; the gene carried by recombinant DNA molecule is said to be cloned.

Gene Cloning

Selection of Recombinant Plasmid

• Insertion of a piece of DNA into the plasmid cloning vector pUC19 to produce a recombinant DNA molecule.

• The vector pUC19 contains several unique restriction enzyme sites localized in a polylinker that are convenient for constructing recombinant DNA molecules.

• The insertion of a DNA fragment into the polylinker disrupts part of the -galactosidase (lacZ+) gene, leading to nonfunctional -galactosidase in E. coli.

• The blue–white color selection test can be used to select for vectors with or without inserts.

Selection of Recombinant Plasmid

LacZ, White Blue Selection

• Colonies with recombinant plasmids are white, and colonies with nonrecombinant plasmids are blue.

• Resistant to ampicillin, has (ampr gene)• Contains portion of the lac operon which

codes for beta-galactosidase.• X-gal is a substrate of beta-galactosidase

and turns blue in the presence of functional beta-galactosidase is added to the medium.

• Insertion of foreign DNA into the polylinker disrupts the lac operon, beta-galactosidase becomes non-functional and the colonies fail to turn blue, but appear white.

X-gal and Beta-galactosidase• The presence of an active β-galactosidase

can be detected by X-gal, a colourless analog of lactose that may be cleaved by β-galactosidase to form 5-bromo-4-chloro-indoxyl, which then spontaneously dimerizes and oxidizes to form a bright blue insoluble pigment 5,5'-dibromo-4,4'-dichloro-indigo. This results in a characteristic blue colour in cells containing a functional β-galactosidase.

• Isopropyl β-D-1-thiogalactopyranoside (IPTG), which functions as the inducer of the lac operon, may be used in the media to enhance the production of LacZ.

Transformation

• The process of transferring exogenous DNA into cells is call “transformation”

• There are basically two general methods for transforming bacteria. • The first is a chemical method utilizing CaCl2 and heat shock to promote

DNA entry into cells.• A second method is called electroporation based on a short pulse of

electric charge to facilitate DNA uptake.

Transformation

Transformation via ElectroporationTo electroporate DNA into cells, washed E. coli are mixed with the DNA to be transformed and then pipetted into a plastic cuvette containing electrodes. A short electric pulse, about 2400 volts/cm, is applied to the cells causing smalls holes in the membrane through which the DNA enters.

Transformant Selection