1. Cloning Vectors

2. DNA Technology I. Amplification of DNA II. Detection of DNA
DNA Technology is the application of our learned properties of DNA
I. Amplification of DNA
Cloning (Amplification / expression)
PCR
II. Detection of DNA
Gel electrophoresis
Sequencing
Hybridization

3. Amplification of DNA
DNA amplification to generate multiple copies of a specific gene or DNA segment, a small fraction of chromosomal DNA, mitochondrial DNA, or plasmid DNA.
Cloning: amplification by replication inside cells
PCR : amplification by DNA polymerase enzymes outside cells (artificial process)

4. Bacterial plasmid vector
Molecular Cloning
MCS
Bacterial plasmid vector
Origin of replication
Multiply

5. Gene Cloning
Isolation and amplification of an individual gene sequence by insertion of that sequence into a cells where it can be replicated
Involves the construction of novel DNA molecules by joining DNA from different sources
Product is Recombinant DNA (rDNA)

6. Basic Events in Gene Cloning
Isolation and amplification of gene of interest
Incorporate gene into a vector (small replicating
DNA molecule, usually circular)
Introduce recombinant vector into host cell via
transformation
Select for the cells that have acquired the
recombinant DNA molecule
Multiply recombinant vector within host cell to
produce a number of identical copies of the
cloned gene
Extract the vector to obtain the copy of the gene

7. Components of Gene Cloning
Vectors (cloning vehicles)
Enzymes for cutting and joining the DNA fragments
The DNA fragments (Target DNA)
Selection process

8. Vectors
Must contain a replicon that enables it to replicate in host cells (region of DNA that is amplified, i.e.: has origin of replication)
Small enough and unlikely to degrade during purification.
Several marker genes
Unique cleavage site(s)
For expression, must contain control elements, such as promoters, terminators, ribosome binding sites, etc…

9. Types of Vectors Plasmids Cosmids Fosmids Phages
Yeast Artificial Chromosomes (YACs)
Transposons
Bacterial Artificial Chromosomes (BACs)
Viruses
retroviruses
adenoviruses
adeno-associated viruses
herpes simplex virus
rhinoviruses
Human Immunodeficiency Virus (HIV)

10. Vectors for Bacterial Cells

11. Plasmid Vectors The most widely used vectors for bacterial cells.
These vectors have their origin from extra-chromosomal circular DNA (plasmid) found in certain bacterial cells.
Typically less than 5 kb.
Large DNA molecules are difficult to handle and often subject to degradation.
The efficiency of transformation decreases with increasing size of the plasmid

12. Plasmid Vectors …….. cont
Double stranded, circular DNA which exist in bacteria.
May exist as single copy per cell or multi-copy per cell (10-20 genomes/cell), or even under relaxed replication control where up to 1000 copies/cell can be maintained
Size of rDNA insertions limited to ~10kb

13. Plasmid vector’s structural elements.
Replication origin
Cloning sites (multiple cloning sites=MCS)
Selectable markers: These are usually antibiotic resistance genes
Expression vector: contain a promoter upstream of MCS.
Optional but popular feature: polyhistidine sequence (e.g. 5'-CACCACCACCACCACCAC encoding 6 histidines)

14. Purification of his-tagged protein on an affinity column

15. Characteristics of Plasmids as Cloning Vectors
Natural vectors
Useful cloning vectors
Small
Easy to isolate and purify
Independently replicating
Multiply copy number
Presence of selectable markers
Antibiotic resistance genes

17. High and Low Copy Plasmids.
Plasmids can be grouped into:
high copy (≥100 copies/cell); ex: pUC
low copy plasmids (1 -25 copies/cell); ex: pBR322
High copy:
Good for yield
Not good if it has adverse effect
Copy number is depend on:
Origin or replication
Size of plasmid and associated insert

18. Structure of Plasmid pBR322
Antibiotic
resistance
Restriction enzyme cut site

19. Advantages of Using pBR322
Small
Relative stable in E. coli with 20 – 30 copies/cell
Can be amplified to 1000 copies/cell
Up to 10 kbp can be inserted
Complete sequence known
Single cut restriction sites
Amp and tet resistance as tags

20. Cloning Genes with pBR322

21. pUC Plasmids The β-lactamase gene (ampicillin resistance, AmpR
The lac operon in pUC contains a truncated lacZ (β-galactosidase) gene
MCS is inserted into the lacZ' region
The pUC plasmids are expression vectors, because the lac operon is active when isopropyl-P-D-thiogalactopyranoside (IPTG) is supplied.

22. The ampicillin resistance gene is a selectable marker.

23. Promoters and RNA Polymerases.
The bacteriophage T3, T7, and SP6 promoters are also used in the construction of bacterial expression vectors
These promoters are only recognized specifically by their respective RNA polymerases, and not by the E. coli RNA polymerases.

24. Topoisomerase-based Cloning.
Topoisomerase is to cleave and rejoin DNA during replication
Binding and cleavage occur at a pentameric motif 5'- (C or T)CCTT in duplex DNA.
Both sticky end and blunt end ligations can be achieved

25. Bacteriophage Vectors
Viruses that attack specific bacteria
Must first deactivate lysogenic growth component of phage (phage DNA inserts into host DNA, creating prophage)
Allow lytic growth – cell death after infection and replication. Cell death revealed as plaques
Insert rDNA into phage (usu. up to 25kb)
Infect bacteria with phage
Infected bacteria form plaques
Advantage: Transformation, selection very easy

26. Bacteriophages

27. Bacteriophage λ Life Cycle.

28. Bacteriophage λ Vectors
Designed to facilitate:
DNA insertion,
Screening for recombinants
Gene expression.
Contains a lacZ gene and a unique EcoRl restriction site at the 5' end of the gene
Insertion of a DNA segment or a gene at the unique restriction site interrupts the lacZ gene sequence.
The cloned DNA or gene sequence is expressed as a fusion protein with β-galactosidase --- It can also be screened by immunodetection methods

29. Bacteriophage λ Vectors ….. cont
The genes related to integration are deleted, and thus no induction is required to switch from lysogenic to the lytic mode
A region containing the terminator for RNA synthesis is deleted
Nonsense mutations are introduced in the genes required for lytic growth A reversion of this effect of mutation can be achieved by suppression in the anticodon of the tRNA carried out by the host strain (Ex: specific E. coli)

30. Genetic map of bacteriophage λ and λgt11 vector

31. M13 Bacteriophages life cycle
RF: replicative form
(double stranded DNA)

32. M13 Bacteriophages M13 is a filamentus bacteriophage of male E. coli.
Contains single-stranded circular DNA: (+) strand
RF is replicated and amplified to copies/cell
The (+) strand continues to be synthesized, and the (-) strand is prevented from replication. The accumulated (+) strands are packaged with the viral proteins to generate phage particles
The plagues appear turbid, because M13 is non-lytic (no dissolution of the bacterial cell wall)

33. M13 Vectors A lacI'OPZ' operon
A multiple cloning site constructed in the lacZ' region
The M13 phage DNA is not infectious, but bacterial cells can pick up both ss and RF forms with CaCl2 treatment in the same way as plasmids.
The E. coli host strain must contain the F' episome (specialized plasmids containing an F factor that encodes sex pili in the male E. coli cells)
The genotype of the M13 host strain is lacIqZ∆MI5
Inserted with biosynthesis proline genes
Inserted with mutated LacZ --- LacZ∆Mi5
Inserted with mutated LacI --- LacIq

34. M13 Vector

35. λ Replacement vector λ insertion vector R R R R R R R λ genome New DNA
Inserted R R R R

36. Cosmids Plasmid vectors that contain a bacteriophage lamda cos site
The cos site results in efficient packaging of lamda DNA into virus particles
In the normal life cycle the λDNA molecules produced in replication are joined by the cohesive ends (cos site) to form a concatamer (long chains of DNA molecules)
With the cos site, larger DNA inserts are possible (up to ~40 kb)

37. Cosmid replicates like a plasmid and is packaged like phage λDNA

38. Phagemids Combine features of filamentous phage and plasmid.
Allow the propagation of cloned DNA as conventional plasmids.
When the vector-containing cells are infected with a helper phage, the mode of replication is changed to that of a phage in that copies of ssDNA are produced.

39. Phagemids
contains a bacterial plasmid origin of replication and a selectable marker
A filamentous phage origin of replication enables the production ssDNA under the infection with a helper (filamentous) phage.
The ssDNA produced
Circularized
Packaged
Released.
MCS inserted into the lacZ a peptide sequence

40. Yeast Cloning Vectors

41. Yeast Artificial Chromosome (YAC)
Artificially produced mini chromosome, consist of:
Centromere: important in cell division
Telomeres: Mark the end of chhromosome.
Origin of replication,
Marker genes
Able to accommodate very large inserts (~1,000 – 2,000 kb)

42. The 2μ Circle Developed based on plasmid 6318 bp in size
Present in the nucleus of most Saccharomyces strains at ~ copies
Contain the origin of replication from the 2μ circle or autonomously replicating sequence (ARS) from the yeast chromosomal DNA
"integrative" vectors: the vector DNA integrates into the yeast chromosome (without 2μ circle or ARS)

43. The 2μ Circle………cont Selectable marker LEU2 URA3
Gene codes for β-isopropylmalate dehydrogenase, an enzyme involved in the synthesis of leucine
Only transformant with LEU2 grow in the medium lack of leucine
URA3
Mutant yeast strains (used as host) lacks the gene cannot synthesize uridine monophosphate.
Only transformants harboring the vector (with URA3) can grow in the medium.

44. The 2μ Circle………cont
A suitable promoter is needed for gene expression.
Two types of promoters are used:
For constitutive expression (i.e. The gene is expressed continuously during the culture of the yeast cells)
Low growth
Unfavorable selection of transfectant
Gene expression is low
For regulated expression (i.e. The gene is expressed in response to an external signal.)
"shuttle vector" : contain the plasmid ori – can work for either yeast and bacteria

45. Yeast expression vector

46. Bacterial Artificial Chromosome (BAC)
Based on the naturally-occuring F plasmid in E. coli.
F plasmid is relatively large.
Have larger capacity to accepting inserted DNA.
Able to clone up to 300kb DNA fragments

47. Vectors for Mammalian Cells

48. Methods for transferring DNA into mammalian cells:
Mediated by virus infection
simian virus 40 (SV40)
Bovine papilloma virus (BPV)
Epstein-Barr virus (EBV)
retrovirus.
Baculovirus
Transfection with mammalian expression vectors.

49. SV40 Viral Vectors Genome size of ~5 kb.
It consists of 2 promoters that regulate
Early genes (encoding large T and small t antigens)
Late genes (encoding viral capsid proteins VP1, VP2, and VP3).
Contains a replication origin that supports autonomous replication in the presence of the large T antigen.
Disadvantages of the use of SV40 viral vectors:
Limited to applications using only monkey cells;
The expression is unstable due to cell lysis
DNA rearrangement occurs during replication.

50. Cloning strategy using SV40

51. Direct DNA Transfer
For transient expression of transfected DNA in mammalian cells
Methodes
Coprecipitation with calcium phosphate
Electroporation
Other methods

52. Structural features of mammalian expression vectors
A replication origin for efficient amplification in mammalian cells (e.g. SV40 ori for COS cells)
An eukaryotic promoter for transcriptional regulation of the foreign gene targeted for expression (Viral gene promoters are usually used.)
A selectable marker and/or reporter gene (including an appropriate promoter) for the selection of the transfected host
An enhancer sequence to increase transcription from the eukaryotic promoter
A multiple cloning site
Transcription termination sequence and poly(A) sequence
A bacterial replication origin
Marker gene (e.g. antibiotic resistance) for selecting transformants in bacterial cells.

54. Retrovirus RNA Genome
long terminal repeats (LTR) carrying the transcriptional initiation and termination
Three coding regions for viral proteins
gag for viral core proteins
pol for the enzyme reverse transcriptase
env for the envelop
a psi (Ψ) region carrying signals for directing the assembly of RNA

55. Retrovirus life cycle

56. Structure organization of Retrovirus vector

57. Production of retrovirus safe vector

58. Further Reading
Lodge J, Lund P & Minchin S, 2007, Gene Cloning: Principle and Aplications, Taylor & Francis Group, NY
Wong DWS, 2006, The ABCs of Gene Cloning 2nd Ed, Springer, NY