Cloning Vectors

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

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)

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

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)

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

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

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…

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)

Vectors for Bacterial Cells

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

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

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)

Purification of his-tagged protein on an affinity column

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

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

Structure of Plasmid pBR322 Antibiotic resistance Restriction enzyme cut site

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

Cloning Genes with pBR322

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.

The ampicillin resistance gene is a selectable marker.

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.

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

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

Bacteriophages

Bacteriophage λ Life Cycle.

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

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)

Genetic map of bacteriophage λ and λgt11 vector

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

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)

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

M13 Vector

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

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)

Cosmid replicates like a plasmid and is packaged like phage λDNA

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.

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

Yeast Cloning Vectors

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)

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)

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.

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

Yeast expression vector

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

Vectors for Mammalian Cells

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.

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.

Cloning strategy using SV40

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

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.

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

Retrovirus life cycle

Structure organization of Retrovirus vector

Production of retrovirus safe vector

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