Cloning Vectors

1. Cloning Vectors

2. DNA Technology 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. 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 structuralelements. • 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. 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 Advantages of Using pBR322

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 λand 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 100-200 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. λgenome λ Replacement vector λ insertion vector R R R 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 ~60-100 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 • 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