Bioc 471a/571a - Applied Molecular GeneticsFall 2001 - Dr. Roger MiesfeldPage 1Lecture 5 - Bacteriophage Vectors; Lab Practicum 2 (AMG text pp. 42-55)September 4, 2001Biology of Bacteriophage LamdaLambda phage is a bacterial virus that infects E. coli, and depending on early events(and genetics), can either multiply within cells leading to cell lysis, or the viral DNA canintegrate into the bacterial genome in a process called lysogeny. If lambda genetranscription is activated, then lytic infection occurs leading to phage release andinfection of nearby E. coli cells. Alternatively, if lambda gene transcription is repressed,then the infection is lysogenic resulting in lambda DNA integration into the E. coligenome. Applied Molecular Genetic methods used in the lab are designed to takeadvantage of cell lysis.The plaque formation assay can be used to isolate pure strains of lambda phage byusing a low multiplicity of infection (MOI). Bacteriophage plaques are physical areas ona petri plate where the bacteria have all lysed due to multiple rounds of infection by asingle clonal phage. Each plaque contains about 1 million virus particles derived from asingle infection event - the viral particles in a single plaque are therefore clonally related(same DNA sequence).Sometimes it is desireable to purify large amounts of phage for the purpose of isolatingrecombinant DNA from a clonal isolate. One method to do this is to infect a liquidculture of E. coli with phage stock and then harvest the lysate 4-8 hours later. Thebacterial debris (lysed cells) is removed by low speed centrifugation and thenpolyethylene glycol (PEG) is added to the supernatent and the phage are then pelletedby high speed ultracentrifugation.What does it mean to "titer a phage stock" and how is it done?Why are the host bacteria grown in media containing the sugar maltose prior to infection?Why does it matter how much phage stock you add to the bacterial liquid culture, doesn't it alljust mix around and lead to 100% infection eventually?What is the biochemical explanation for adding PEG to the phage suspension?Once you had obtained a highly concentrated stock of purified phage, how would you separatethe recombinant phage DNA away from the phage head and tail proteins?Two important technical hurdles had to be overcome before lambda vectors could beutilized as cloning reagents for genomic and cDNA libraries; 1) In vitro lambda phagepackaging extracts were developed and made available commercially, 2) Geneticstrategies were developed to increase the yield of recombinant phage in DNA librariesto make up for the low ratio of insert to vector DNA used in the initial ligationreactions.Bioc 471a/571a - Applied Molecular GeneticsFall 2001 - Dr. Roger MiesfeldPage 2Two such genetic strategies are shown below. The first was developed for cDNAlibraries and the second for genomic DNA libraries. Note that the total length of lambdaDNA that can be efficiently packaged is a crucial determinant in this protein-drivenprocess with a maximum limit of 52 kb (vector + insert).Insertional cloning into the cI gene of the lambda-gt10 cDNA cloning vector (DNAinserts of ~1-5 kb) can be selected in hfl (high frequency of lysogeny) mutant strains ofE. coli. In hflA strains of E. coli, expression of the lambda cII gene is elevated, resultingin transcriptional induction of the lambda cI repressor gene which promotes lysogeny.Disruption of the lambda cI coding sequence by DNA insertion into the unique EcoRIsite of the lambda gt10 cDNA cloning vector, blocks the lysogenic pathway leading tocell lysis and plaque formation.Replacement of lambda DNA containing the red and gam genes in the lambda EMBL3genomic DNA cloning vector with BamHI compatible DNA inserts of ~10-20 kb,permits lytic growth of recombinant phage in E. coli strains containing the P2bacteriophage lysogen.Does lambda DNA packaging require any enzymes or ATP hydrolysis?Why is it important to use low ratios of insert DNA to vector DNA in the construction oflambda DNA libraries, wouldn't it just be easier to increase the ratio to decrease the number of"empty vectors" in the library stock?M13 Bacteriophage BiologyM13 filamentous phage have a single strand genome that exists temporarily insideinfected E.coli cells as a double strand plasmid. M13 phage are budded off of aninfected cell and single strand DNA can be purified for use in DNA sequencing or invitro mutagenesis. Initially M13 phage vectors required a working knowledge of phagebiology and was primarily used for creating single strand DNA molecules for DNAsequencing. Fortunately, M13-derived cloning vectors called "phagemids" have beendeveloped which take advantage of M13 replication to produce single strand molecules,but can be propagated as conventional ColE1-based replicating double strand plasmids.Bluescript KS+ is an example of a phagemid cloning vector. Often times, phagemidvectors are used to prepare single strand DNA for in vitro mutagenesis protocols usingoligonucleotide primers (chapter 3). Infection of E. coli F+ cells containing phagemidDNA with replication-deficient M13 helper phage results in the packaging of singlestrand phagemid DNA. The orientation of the M13 replication origin (+ or -), relative tothe insert DNA, in the phagemid vector, determines which strand of the insert DNA(coding or non-coding) will be contained in the packaged phage.Since the bacterial sex pilus proteins are encoded on the F’ plasmid, and the pilus isrequired for M13 phage attachment, E. coli strains have been developed that include atransposon encoded antibiotic resistance gene on the F’ plasmid (tetr or kanr). RollingBioc 471a/571a - Applied Molecular GeneticsFall 2001 - Dr. Roger MiesfeldPage 3circle replication and phage packaging by helper virus proteins result in the productionof recombinant M13 phage.Why is a helper phage required to produce single strand DNA from an M13 phagemid?What does the orientation-dependence of the M13 ori tell you about filamentous phagereplication and how is this different than lambda phage replication?Lab Practicum 2 - Protein expression in E. coli using the pET system.High level expression of recombinant proteins in bacteria is a common process inBiotechnology. The production of human insulin in bacteria is one such example.Another example is the production of plant calreticulin protein in bacteria as a means togenerate anti-calreticulin antibodies in rabbits.Probably the most widely used bacterial protein