Cloning vectors

Details of different cloning vectors.

1. CLONING VECTORS Dr. Mayank Chaudhary Assistant Professor Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana-Ambala, Haryana, INDIA

2. PLASMIDS These are extra-chromosomal, double-stranded, circular DNA molecules capable of autonomous replication within bacterial cells. Size of plasmids range from 1kb-250kb. Episomes /Integrative Plasmids: P lasmids that replicate by integration into bacterial chromosomal DNA. Copy number: Number of molecules of individual plasmid that is found in single bacterial cell. On the basis of copy no.: Stringent: Have low copy no. per cell (1 or 2) Relaxed: Present in multiple copies per cell.

3. Plasmid Classification: Fertility or F Plasmids: Conjugative plasmid carrying transfer gene ( tra ) and having the ability to form conjugation bridge (F pilus ) with F - bacterium. Eg . F plasmid of E.coli. Resistance or R Plasmids: Carry genes which give resistance to bacteria from one or more antibacterial agents. Eg . RP4 plasmid found in Pseudomonas. Col Plasmids: Have genes that code for colicins (proteins that kill other bacteria). Eg . ColE1 of E.coli . Degradative Plasmids: Allows host bacterium to metabolize unwanted molecules such as toluene and salicylic acid. Eg . TOL of Pseudomonas putida . Virulence Plasmids: Confers pathogenicity to host bacteria. Eg . Ti plasmid of Agrobacterium tumefaciens . Cryptic Plasmids: Do not have any effect on the phenotype of the cell carrying them.

4. pBR322 Plasmids can have insert size of ≤15kb . Nomenclature: P: indicates that it is a plasmid BR: identifies laboratory where it was constructed (stands for Bolivar and Rodriguez who investigated it) 322: distinguishes it from other vectors developed in the same laboratory. Useful Properties: Suitable size ( 4363 bp ‹ 10kb)- To avoid problems such as DNA breakage during purification. 2 marker genes (ampicillin and tetracycline resistant genes) High copy number

5. pUC Nomenclature: p: indicates plasmid UC: University of California Useful Properties: Small size (2686 bp) High copy numer Contain lacZ sequence (easy identification through blue/white screening) Ampicillin resistant marker gene

6. Bacteriophages Viruses that infect bacteria. Simple structure: Nucleic acid surrounded by protein coat (capsid). Phage infection cycle: lytic or lysogenic

7. Typical insert size for phage is 5-20kb . Features of lambda DNA molecule: Size of lambda DNA molecules is 49kb. Gene clustering (genes performing related functions are grouped that allows switching on/off of genes as a group rather than individually. DNA conformation: Linear DNA has two free ends with 12nt single-stranded stretch on either side. These single strands are complementary and can base pair to form circular, double-stranded molecule. Complementary single strands are called as sticky ends/cohesive ends. Also known as Cos sites. Function of Cos sites: 1) Allows circularization of linear DNA molecule injected into bacterial cell. 2) Acts as recognition sites for RE to separate out large number of DNA molecules formed through rolling circle mechanism.

8. Types of vectors developed using Lambda genome: Insertion Vectors Replacement Vectors Insertion vectors: Foreign DNA is inserted into lambda genome without significant change of wild-type genome resulting int small insert size ( upto 10kb).

9. Replacement vectors: Stuffer fragment is replaced by foreign DNA resulting in insert size of 10-23kb.

10. Cosmid Hybrid plasmid with bacterial ori sequence and cos sequences derived from lambda phage. Within a cell, Cosmid replicates as a plasmid (Recombinant DNA is therefore obtained from colonies rather than plaques). Contains gene for selection. Insert size: 35-50 kb. Widely used for synthesis of genomic libraries. Example of Cosmids: pJB8 Limitations of Cosmid vector: Slower replication Higher frequency of recombination Unstability inside host ( E.coli )

11. Phagemid Cloning vectors that are hybrid of filamentous phage M13 and plasmids. Components of Phagemid vector: Origin of replication of plasmid Intergenic region (IG) containing packaging signal for phage particle Gene encoding phage coat protein Selection marker Restriction enzyme recognition sites Commonly used for phage display technology (proteins are expressed as fusions to phage coat proteins and displayed on the viral surface) Example: pEMBL Advantages of P hagemids over Phage vectors: Higher carrying capacity, higher transformation efficiency and more stability.

12. Artificial chromosomes These are DNA molecules assembled in-vitro that can function as natural chromosomes. Types: BACs: Bacterial a rtificial chromosomes YACs: Yeast artificial chromosomes MACs: Mammalian artificial chromosomes PACs: P1-derived artificial chromosomes

13. BACs Insert size: 100-300 kb Components of BACs: oriS , repE -F: for plasmid replication and regulation of copy number parA and parB : To maintain low copy number and to avoid two F plasmids in single cell Antibiotic resistance marker gene lacZ gene for blue/white selection T7 and Sp6 promoters for transcription of inserted genes Example: pBeloBAC11

14. YACs Insert size: 100-500 kb YAC has sequences to exist as circular plasmid inside bacteria and as linear nuclear chromosome in yeast. Components of YAC vectors: E.coli origin of replication Yeast origin of replication Elements of eukaryotic yeast chromosome (centromere and telomere) Selection markers for both the host Can be used to express eukaryotic proteins that require post-translational modifications.

15. MACs Insert size: 100 kb-1 Mb MACs also depend upon presence of centromeric , telomeric sequences and origin of DNA replication. Two procedures for generation of MACs: Telomere directed fragmentation of natural chromosomes. ( eg . HAC from chromosome 21) De novo assembly of cloned centromeric , telomeric and replication origins in-vitro . MACs have application in gene therapy and eukaryotic protein expression.

16. REFERENCES T.A. Brown. Gene Cloning and DNA Analysis-An Introduction. Seventh Edition. NPTEL notes (Module 1).