Lecture 4: Bacteriophages and its Vectors PDF

BACTERIOPHAGES TYPES: λ phage M13 phage PHAGE INFECTION CYCLE VIRULENT PHAGES Infect bacteria Specific phage enzymes coded by the phage chromosome cause phage DNA replication Capsid proteins synthesized and phage particles reassembled and re...

BACTERIOPHAGES TYPES: λ phage M13 phage PHAGE INFECTION CYCLE VIRULENT PHAGES Infect bacteria Specific phage enzymes coded by the phage chromosome cause phage DNA replication Capsid proteins synthesized and phage particles reassembled and released from the cell LYTIC CYCLE PHAGE DNA IS NEVER MAINTAINED IN A STABLE CONDITION IN THE HOST  INTEGRATIVE PLASMID TEMPERATE PHAGES hage DNA (λ phage) in a similar fashion gets integrated into the bacterial genom PROPHAGE LYSOGEN  Prophages remain quiescent, and the lysogen is indistinguishable from an uninfected cell  Prophage is eventually released from the host genome by reverting to lytic mode  LYSOGENIC INFECTION CYCLE OF BACTERIOPHAGE λ http://www.wwnorton.com/college/biology/microbiology2/ch/10/etopics.aspx New phage particles synthesized λ DNA excises from the host chromosome Can possibly maintained for 1000s OGENIC GENE TRANSCRIPTIONS 1. PR and PL get activated upon circularization leading to synthesis of immediate early transcripts (N and cro) that terminate at tL and tR 2. N protein allows tR and tL terminators being overridden, so subsequent transcription gives rise to delayed early transcripts – cII and cIII. 3. cIII protein protects cII against degradation by host proteases. 4. cII activates promoter PRE (promoter for repressor establishment) that stimulates production of cI, which is a repressor molecule inactivating P R and PL thus switching off the lambda genome. (confers immunity to superinfection) 5. cII also activates promoter PINT that, in conjunction with host proteins, mediates site-specific recombination across att and a similar sequence in the bacterial TIC GENE TRANSCRIPTIONS 1. PR and PL get activated upon circularization leading to synthesis of immediate early transcripts 2. N protein allows subsequent transcription gives rise to cII and cIII. 3. If conditions don’t favor stabilization of cII, PRE is not activated, cI not synthesized, and rest of phage genome continues with transcription (further ensured by Cro that inactivates PRM. 4. O and P cause DNA replication. Q causes activation of P R’ and expression of late genes (encoding head and tail proteins). Int and Xis cause phage excision.  ISOLATION OF PHAGE DN Deproteinizati on to remove capsid The extracellular λ titre is extremely low. To increase the yield, cultures need to be “induced” such that lytic processes predominate. Most strains of λ carry a temperature-sensitive mutation (ts) in the cI gene. cI+ cells in lysogeny cIts cells in lysogeny at 30 C cIts cells enter lytic cycle at 42 C  Phage infection is visualized as plaques on an agar medium Each plaque is derived from a single infected cell and therefore contains identical phage particles Virus titers are measured in terms of “plaque forming units/mL” 17 plaques = 17 / (0.1 * 10-6) = 1.7 * 108 PFU/mL PLAQUE ASSAY: Dulbecco, R., & Vogt, M. (1953). Cold Spring Harbor Symp. Quant. Biol., 18, 273- 279 tp://www.virology.ws/2009/07/06/detecting-viruses-the-plaque-assay/ 17 plaques  GENETIC MAP OF BACTERIOPHAGE λ Essential features of the λ genome (useful for cloning purposes) include:  clustering of functionally-related genes (46 genes encoded that can be turned “on” or “off” as a group rather than individually).  Linear double-stranded DNA with two free ends (but contain a 12-nt stretch of ssDNA on either ends that are complementary to each other). se of the cos sites: w circularization of the linear DNA, which is necessary for insertion into the host g ing circle” mechanism of replication If the overall length of the genome is not altered greatly, then addition of “new” genes in the internal regions (excluding cos sites and its immediate neighbours) will not affect the phage life cycle. ning strategies using λ phage Potential drawbacks: 1. Only upto 3 kb DNA can be inserted 2. The entire genome contains more than one recognition sequence for every known RE 15 kb Region can be replaced with “new” genes without affecting the ability of the phage to infect E.coli cells oval of this region causes the phage to be only in “lytic” mode ural selection to get rid of all (or all except one) RE recognition sequenc  INSERTION VECTORS Remove the non- essential region and religate the DNA Can carry up to 8 kb new Can carry up to 10 kb new DNA in the DNA in the cI region polylinker containing lacZ’ gene (single RE (single RE sites) sites)  REPLACEMENT VECTOR Contains two recognition sites for RE for inserting new D Digest with other REs to prevent its own re- insertion Can carry larger fragments of DNA Utilize different REs for cloning Recombinants λ phage cannot infect E.coli with integrated DNA from P2 usually selected phages (sensitive to P2 prophage inhibition, spi+). Insertion on the basis of of new DNA causes change from Spi+ to Spi- enabling size  M13 DNA does not integrate into the bacterial genome M13 phage M13 DNA is circular and entirely single stranded New phage particles are continuously assembled and released without lysing the host cells (infected bacterium survive but grow at a slower rate) Infects only E.coli that have F-pili (conjugation- proficient cells) Upto 1000 phage particles may be released into the medium per cell per generation  Can be isolated similar to a plasmid Replicates until about 100 copies occur per cell Replication of RF becomes asymmetric with accumulation of viral-encoded ssDNA-binding protein that binds to the phage DNA and prevents synthesis of complementary strand. he M13 Genome M13 genome is only 6407 nt in length (circular and entirely ssDNA) Capsid consists of only 3 proteins (3 genes) unlike λ with 15 proteins Simpler infection cycle – no genes for insertion into host genome Small size ideal for use as a cloning vector. Most useful for obtaining single stranded versions of cloned genes for sequencing in vitro (oligonucleotide-directed) mutagenesis. nstruction of M13 based vectors M13 genome has ten genes essential for the replication of the phage Intergenic sequence where new DNA can be inserted is only 507nt long and contains the ori Blue plaques on X-gal agar No unique RE site present  Introducing a unique EcoRI site in the lacZ (Gronenborn and Messing, 1978) using in vitro mutagenesis Addition of a polylinker with multiple RE sites and sticky EcoRI site Four possible cloning sites have now been generated Maximum limit for cloning