1. Lec#3: Recombinant DNA technology-part 2
Dr. Shah Rukh
ASAB, NUST

2. b. Origin of Replication
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.

3. 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

4. Examples of plasmid vectors

5. 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.

6. 2. Bacteriophage (𝝀) vectors

7. 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

8. 4. BACs and YACs (Bacterial and Yeast Artificial chromosomes)
4a. BACs - Bacterial 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.

9. 4a. BAC vector oriS and oriE mediate replication
parA and parB maintain single copy number
ChloramphenicolR marker

10. 4b. Yeast artificial chromosomes YACs
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 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

11. What are clones? Clones Cloning
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

12. 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. 

13. What is Gene Cloning?
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.
The vector transport the gene in the host cell, which is usually a bacterium, although other type of cells can also be used
Within the host cell the vector multiplies, producing numerous identical copies not only itself but also the gene it carries

14. What is Gene Cloning?
When the host cell divides, copies od recombinant DNA molecule are passed to the progeny and further vector replication take place
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.

15. Gene Cloning

16. 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.

17. Selection of Recombinant Plasmid

18. 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.

19. 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.

20. 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.

21. Transformation

22. Transformation via Electroporation
To 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.

23. Transformant Selection