Cloning vectors

Vectors, Plasmid, Different types of vectors, Cloning, Types of plasmids, pBR322, pRSET A, B, and C, Cosmids, BACs, YACs

1. CLONING VECTORS Dr. M. THIPPESWAMY

2. Recombinant DNA Technology

3. Vector is an autonomously replicating (inside a host cell) DNA molecule designed from a plasmid or phage DNA to carry a foreign DNA inside the host cell. Transformation vectors are of two types : • Cloning vector is used increasing the number of copies of a cloned DNA fragment. Eg ., pBR322, pUC18/19 • Expression vector is used for expression of foreign gene into a protein. Eg ., pET28, pRSET -A, B & C • If a vector is designed to perform equally in two different hosts, it is called a shuttle vector. Eg ., pYES2, Ti-Plasmid VECTORS

4. Properties of an ideal vector: A good vector should have the following characteristics: • Autonomously replicating i.e. should have ori (origin of replication) region. • Contain at least one selectable marker e. g. gene for antibiotic resistance. • May contain a scorable marker ( β- galactosidase , green fluorescent protein etc.) • Presence of unique restriction enzyme site. • Have multiple cloning sites. • Preferably small in size and easy to handle. • Relaxed control of replication to obtain multiple copies. • Presence of appropriate regulatory elements for expression of foreign gene. • High copy number The selection of a suitable vector system depends mainly on the size limit of insert DNA and the type of host intended for cloning or expression of foreign DNA.

5. Types of vectors • Plasmid vectors • Bacteriophage vectors • Cosmids • BACs & YACs • Mini chromosomes

6. • These Plasmids are extrachromosomal circular DNA molecule that autonomously replicates inside the bacterial cell. • Cloning limit: 100 to 10,000 base pairs or 0.1-10 kilobases (kb) . • In their simplest form, plasmids contains a bacterial origin of replication, an antibiotic resistance gene, and at least one unique restriction enzyme recognition site which helps in cloning. PLASMIDS

7. Based on the origin or source of plasmids, they have been divided into two major classes: such as natural and artificial. i ) Natural plasmids: They occur naturally in prokaryotes or eukaryotes. Example : ColE1. ii) Artificial plasmids: They are constructed in-vitro by re-combining selected segments of two or more other plasmids (natural or artificial). Example: pBR322. PLASMIDS

8. Characteristics of natural plasmids

9. Artificial plasmids vectors are classified into two broad types based on their use: 1. Cloning vector 2. Expression vector Artificial plasmids A cloning vector is defined as a vector used for replication of a cloned DNA fragment in a host cell. These vectors are frequently engineered to contain “ ori ” – origin of replication sites particular to the host organism. Examples of commonly used cloning vectors are: pUC18, pUC19, pBluescript vectors etc.

10. Basic components of a plasmid

11. Antibiotic Ampicillin Mode of action Inhibits the cell wall synthesis by dirupting peptidoglycon cross linking Resistance gene B- galactamase ( amp ) gene product is secreted and hydrolyse the ampicillin Application Amp gene is included on plasmid vector as positive selection markers Antibiotic Tetracycline Mode of action Inhibits the binding of aminoacyl tRNA to the 30S ribosomal subunit Resistance gene tet gene product is membrane bound and prevents tetracycline accumulation by an efflux mechanism Application tet gene product is a positive selection marker on some plasmids Antibiotics and antibiotic resistance genes used in gene cloning

12. Antibiotic Kenamycin Mode of action Inactivates translation by interfering with ribosome function Resistance gene Neomycin or amino glycoside phospho transferase ( neo ) gene product anactivates kanamycin by phosphorylation Application Neo gene is a positive selection marker used in plasmids Antibiotic Bleomycin Mode of action Inhibits DNA and RNA synthesis by binding to DNA Resistance gene The bla gene product binds to bleomycin and prevents it from binding to DNA Application Bla gene is a positive selection marker used in plasmids

13. Antibiotic Hygromycin B Mode of action Inhibits the translation in prokaryotes and eularyotes by interferring with ribosome translation Resistance gene Hygromycin -B- phosphotransferase ( hyg ) gene product inactivates hygromycin B by phosphorylation Application hyg gene is used as a positive selection marker in eukaryotic cells that are sensitive to hygromycin B Antibiotic Chloromphenicol Mode of action Binds to the 50S ribosomal subunit and inhibits translation Resistance gene Chloramphenicol acetyl transferase (CM) gene product metabolises chloramphenicol in the presence of acetyl CoA Application CM gene is used as a selectable marker, and as transcriptional reporter gene of promoter activity in eukaryotic cells

14. Advantages of Plasmids in Molecular Biology • Easy to work with - Plasmids are a convenient size (generally 1,000-20,000 base pairs). • Self-replicating - Endless number of copies of the plasmid was obtained by growing the plasmid in bacteria. • Stable - Plasmids are stable long-term either as purified DNA or within bacteria (as glycerol stocks). • Functional in many species and can useful for a diverse set of applications - Plasmids can drive gene expression in a wide variety of organisms, including plants, worms, mice and even cultured human cells.

15. Major Limitation of Cloning in Plasmids  Upper limit for clone DNA size is 12 kb  Requires the preparation of “competent” host cells  Inefficient for generating genomic libraries as overlapping regions needed to place in proper sequence  Preference for smaller clones to be transformed  If it is an expression vector there are often limitations regarding eukaryotic protein expression.

16. Examples of Cloning Vector:

17. pBR322 • pBR322 is a widely-used E. coli cloning vector. It was the first created plasmid in 1977. The p stands for " plasmid" and BR for "Bolivar" and "Rodriguez", researchers who constructed it. • pBR322 is 4361 base pairs in length.  “rep” replicon from plasmid pMB1 which is responsible for replication of the plasmid.  “ rop ” gene encoding Rop protein. Rop proteins are associated with stability of RNAI-RNAII complex and also decrease copy number.  “ tet ” gene encoding tetracycline resistance.  “ bla ” gene encoding β lactamase which provide ampicillin resistance.

18. pUC plasmids: • pUC plasmids are small, high copy number plasmids of size 2686bp. • This series of cloning vectors were developed by Messing and co-workers in the University of California. The p in its name stands for plasmid and UC represents the University of California. pUC vectors consists of following elements:  pMB1 “rep” replicon region derived from plasmid pBR322 with single point mutation (to increase copy number).  “ bla ” gene encoding β lactamase which provide ampicillin resistance which is derived from pBR322. This site is different from pBR322 by two point mutations.  E.coli lac operon system. • “ rop ” gene is removed from this vector which leads to an increase in copy number.

19. A vector used for expression of a cloned DNA fragment in a host cell is called as an expression vector. These vectors are frequently engineered to contain regulatory sequences that act as promoter and/or enhancer regions and lead to efficient transcription. Commonly used expression vector series are: pET vectors, pBAD vectors, pRSET -A, B & C vectors etc. Expression Vector For an expression vector following features are essential: • Promoter: Promoter sequence recognizes the RNA polymerase which is required for initiation of transcription of gene of interest. • Terminator: It is a DNA element present at the end of a gene where transcription of gene ends. • Ribosome binding site: It is a short nucleotide sequence recognized by the ribosome as the point at which it should attach to the messenger molecule. The initiation codon of the gene is always a few nucleotides downstream of this.

20. pET28 vector: • pET vector system is a cloning and expression vector system for recombinant protein production in E.coli . This product is registered under trademark of Novagen Inc. • The original pET vector system was constructed by Studier and colleagues. That plasmid is developed at Novagen with enhanced characteristics. • Target genes are cloned under strong T7 bacteriophage promoter. • The expression of the target protein is inducible by providing T7 RNA polymerase in the host cell as an inducing signal. • Ampicillin and kannamycin resistance genes are available in pET vectors as selection marker. • pET28 and pET32 are the most commonly used pET vectors.

21. The pRSET vectors are pUC -derived expression vectors designed for high-level protein expression and purification from cloned genes in E. coli. High levels of expression of DNA sequences cloned into the pRSET vectors are made possible by the presence of the T7 promoter. T7 RNA polymerase specifically recognizes this promoter. For expression of the gene of interest, it is necessary to deliver T7 RNA polymerase to the cells by either inducing expression of the polymerase using the gratuitous inducer isopropyl β-D- thiogalactoside (IPTG), or infecting the cell with phage expressing the polymerase. pRSET A B C – Expression vector

22. Vector for cloning Saccharomyces genes. The presence of a yeast origin of DNA replication (ARS) and a yeast centromere (CEN) allows, stable replication and segregation in yeast. Also included is a yeast selectable marker such as URA3, which allows a ura3 mutant to grow on medium lacking uracil . Typical protocol for constructing a yeast genomic library. Components of a typical plasmid shuttle

23. Phage Vectors: To insert DNA fragments of more than 15 kb, normally plasmids are not the suitable vehicles, as large inserts may trigger plasmid rearrangement or affect plasmid replication. This leads to development of a new class of vectors based on bacteriophages . Amongst various bacteriophages available such as λ, T4, T5, and T7 phages; the λ phage gained favourable attention due to its unique life cycle.

24. λ phage vector Bacteriophage λ contains ~49kb of DNA and has a very efficient mechanism for delivering its genome into a bacterium. Two key features contribute to its utility as a vector to clone larger DNA fragments: One-third λ genome is nonessential and could be replaced with foreign DNA. Approximately 24.6kb of λ genome can be deleted, hence maximum insert size could be upto 26 kb. Applications: • DNA sequencing • Mutagenesis study • probe generation • Phage display

25. • Cosmids are medium sized cloning vectors. • The cloning capacity of these vectors is 35-45 kbp . • Cosmid vector are developed by combining the features of plasmid vector and bacteriophage vector. • The first cosmid vector was described by Collins in 1978. COSMID VECTOR In Brief • A cosmid is a plasmid that contain phage sequence that allows the vector to be packaged and transmitted to bacteria like phage vector. Or • A cosmid is a type of hybrid plasmid that contains a Lambda phage cos sequence. Cosmids (cos sites + plasmid = cosmids ) DNA sequences are originally from the lambda phage.

26. THANK YOU