Fundamental Biotechnology

1. Fundamental Biotechnology Lecture# 6 Haji Akbar M.Phil

2. Introduction Bacteriophage • Virus infecting Bacteria. (eater of bacteria) • Short Phage • Groups on structure base • i. tailless • ii. Head with tail • iii. filamentous

3. Continue!! • Genome: (50% of total mass) • DNA (ssDNA or dsDNA) or • RNA (ssRNA or ds RNA) • in tailless and tailed DNA is encapsulated in capsid.

4. Historical: • Frederick Twort (1915) and Felix d'Herelle (1917) were the first to recognize viruses which infect bacteria, which d'Herelle called bacteriophages (eaters of bacteria).

5. Diversity • There are at least 12 distinct groups of bacteriophages, which are very diverse structurally and genetically; the best known ones are the common phages of E.coli

6. Virulent vs. Temperate Phages • Virulentphages: do not integrate their genetic material into the host cell chromosome and usually kill the host cells (lytic infection) (e.g. T-phages of E.coli). • Temperate phages: may integrate into the host DNA, causing LYSOGENY. (best example λ phage)

7. Bacteriophage T4? • DNA is packaged in the head of T4 phage. • Genome of 173 kb of linear ds DNA. • During the early stages of a infection cycle T4 nucleases encoded by so called early genes • degrade the chromosomal DNA of E. coli in order to obtain large quantities of nucleotide precursors for its own DNA synthesis.

8. λ page • Genome 48.5 kb (46 genes) • Entire Genome has been sequenced and regulatory site are known. • At the ends short (12bp) ss- complementary region “cohesive or sticky” ends--- circulation after infection. • Region is known as cos site.

9. Phage infection begins!! • Adsorption • Lytic or lysogenic cycle depends on # of factors: • Nutrional & metabolic state of host cell • Multiplicity of infection (m.o.i- the ratio of page to bacteria during adsorption) • In lysogenic phage genome integrates into host chromroms =prophage • Packaged into mature phage

10. Bacteriophage infection can be easily monitored by plaque formation. (in the lytic cycle, anyway) Plaque forming Unit (p.f.u): is a measure of the number of particles capable of forming plaques per unit volume (1,000 PFU/µl indicates that there are 1,000 infectious virus particles in 1 µl of solution)

11. Page M13 • Filamentous • ss-circular DNA (size 6407 bp) • DNA enter in to cell converted to double stranded molecule known as replicative form or RF. • Replicates until there are about 100 copies in the cell.

12. Vectors based on Bacteriophage λ • The λ genome is 48.5 kb, in which 15 kbor so is ‘optional’ • it contains genes that are only needed for integration into the E. coli chromosome (controlling lysogenic properties) • These segments can therefore be deleted without impairing the ability of the phage to infect bacteria and direct synthesis of new λ particles by the lytic cycle.

13. Types of vector • Insertion vectors: (single recognition sit for one or more restriction enzyme) e.g. λgt10, Charon 16A. (both having EcoRI) • Replacement vectors:/ Substitution vector: Having two restriction sits (RS) which flank a region known as stuffer fragment (hatched) e.g. EMBL4 and Chapron 40

15. λgt10: 7.5 kb (insert size), EcoRI lies in cI gene which is basis of selection/ screening. • Charon 16A: 9kb, EcoRI lies in β-galactosidase gene (lac Z). • EMBL4: 13.2 kb (stuffer fragment) and can clone 9 & 22 kb. • Chapron 40: (polystuffer) similar size fragment as EMBL4

16. Vectors based on M13 • Two aspect valuble for G.E. • 1. RF similar to plasmid • 2. ssDNA useful for sequencing by Dideoxy method. 507 bp intergenic region is the only part available for manipulation. • Vectors constrict by introducing polylinker /lacZ α-peptide sequence into this region. • Mark reduction in cloning efficiency when DNA fragment longer than 1.5 kb inserted.

17. Other vectors

18. Cosmid • Hybrid of plasmid/Phage vectors • contain E coli ori that allow it to maintained as a plasmid in the cell and carries a λ cos site. • a plasmid that carries a λ cos site. • cos sites, act as substrates for in vitro packaging • because the cos site is the only sequence that a DNA molecule needs in order to be recognized as a ‘λ genome’ by the proteins that package DNA into λ phage particles.

19. A cosmid can be 4-8kbin size, so up to 45-47 kbof new DNA can be inserted. • inside the cell the cosmid cannot direct synthesis of new phage particles and instead replicates as a plasmid. • Recombinant DNA is therefore obtained from colonies rather than plaques.

20. e.g: • pLFR-5 has two cos sites from page λ, separated by ScaI RE sit, a multiple cloning site with six unique restriction site origin of replication and Tetr gene

21. Cosmid Cloning System • cos sites inserted into a small plasmid Target DNA ligated between two cosmid DNA molecules Recombinant DNA packaged and E. coli Infected as before Can clone DNAs up to 45 kb

22. Cosmid vectors • Advantages: • Useful for cloning very large DNA fragments (32 - 47 kbp) • Inherent size selection for large inserts • Handle like plasmids • Disadvantages: • Not easy to handle very large plasmids (~ 50 kbp)

23. Phagemid vectors • plasmids which contain a small segment of the genome of a filamentous phage, such as M13, fd or f1. • contain all the cis-acting elements required for DNA replication and assembly into phage particles. • They permit successful cloning of inserts several kilobases long.

24. Continue!! • Following transformation of a suitable E. coli strain with a recombinant phagemid, • the bacterial cells are superinfected with a filamentous helper phage, such as f1, which is required to provide the coat protein. • Phage particles secreted from the superinfected cells will be a mixture of helper phage and recombinant phagemids.

25. The mixed single-stranded DNA population can be used directly for DNA sequencing. • because the primer for initiating DNA strand synthesis is designed to bind specifically to a sequence of the phagemidvector adjacent to the cloning site. • Commonly used phagemid vectors include the pEMBL series of plasmids and the pBluescript family.

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