Lecture 5 - Bacteriophage Vectors; Lab Practicum 2 (AMG text pp ) September 4, 2001

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1 Lecture 5 - Bacteriophage Vectors; Lab Practicum 2 (AMG text pp ) September 4, 2001 Biology of Bacteriophage Lamda Lambda 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 can integrate into the bacterial genome in a process called lysogeny. If lambda gene transcription is activated, then lytic infection occurs leading to phage release and infection 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. coli genome. Applied Molecular Genetic methods used in the lab are designed to take advantage of cell lysis. The plaque formation assay can be used to isolate pure strains of lambda phage by using a low multiplicity of infection (MOI). Bacteriophage plaques are physical areas on a petri plate where the bacteria have all lysed due to multiple rounds of infection by a single clonal phage. Each plaque contains about 1 million virus particles derived from a single 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 isolating recombinant DNA from a clonal isolate. One method to do this is to infect a liquid culture of E. coli with phage stock and then harvest the lysate 4-8 hours later. The bacterial debris (lysed cells) is removed by low speed centrifugation and then polyethylene glycol (PEG) is added to the supernatent and the phage are then pelleted by 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 all just 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 separate the recombinant phage DNA away from the phage head and tail proteins? Two important technical hurdles had to be overcome before lambda vectors could be utilized as cloning reagents for genomic and cdna libraries; 1) In vitro lambda phage packaging extracts were developed and made available commercially, 2) Genetic strategies were developed to increase the yield of recombinant phage in DNA libraries to make up for the low ratio of insert to vector DNA used in the initial ligation reactions. Page 1

2 Two such genetic strategies are shown below. The first was developed for cdna libraries and the second for genomic DNA libraries. Note that the total length of lambda DNA that can be efficiently packaged is a crucial determinant in this protein-driven process with a maximum limit of 52 kb (vector + insert). Insertional cloning into the ci gene of the lambda-gt10 cdna cloning vector (DNA inserts of ~1-5 kb) can be selected in hfl (high frequency of lysogeny) mutant strains of E. coli. In hfla strains of E. coli, expression of the lambda cii gene is elevated, resulting in transcriptional induction of the lambda ci repressor gene which promotes lysogeny. Disruption of the lambda ci coding sequence by DNA insertion into the unique EcoRI site of the lambda gt10 cdna cloning vector, blocks the lysogenic pathway leading to cell lysis and plaque formation. Replacement of lambda DNA containing the red and gam genes in the lambda EMBL3 genomic DNA cloning vector with BamHI compatible DNA inserts of ~10-20 kb, permits lytic growth of recombinant phage in E. coli strains containing the P2 bacteriophage 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 of lambda 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 Biology M13 filamentous phage have a single strand genome that exists temporarily inside infected E.coli cells as a double strand plasmid. M13 phage are budded off of an infected cell and single strand DNA can be purified for use in DNA sequencing or in vitro mutagenesis. Initially M13 phage vectors required a working knowledge of phage biology and was primarily used for creating single strand DNA molecules for DNA sequencing. Fortunately, M13-derived cloning vectors called "phagemids" have been developed 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, phagemid vectors are used to prepare single strand DNA for in vitro mutagenesis protocols using oligonucleotide primers (chapter 3). Infection of E. coli F+ cells containing phagemid DNA with replication-deficient M13 helper phage results in the packaging of single strand phagemid DNA. The orientation of the M13 replication origin (+ or -), relative to the 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 is required for M13 phage attachment, E. coli strains have been developed that include a transposon encoded antibiotic resistance gene on the F plasmid (tetr or kanr). Rolling Page 2

3 circle replication and phage packaging by helper virus proteins result in the production of 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 phage replication 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 in Biotechnology. 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 to generate anti-calreticulin antibodies in rabbits. Probably the most widely used bacterial protein expression is the pet vector system which was developed in 1984 by Bill Studier and colleagues at Brookhaven National Labs. The vector system is now sold commercially by Novagen. One of the most innovative pet vectors is petblue which utilizes a lacz gene (alpha region) on the opposite coding strand such that blue-white screening can be done but the recombinant protein does not contain any lacz fusion sequences. Research Objective A plant biologist is interested in studying the calcium binding protein calreticulin in the plant Arabidopsis thaliana to determine its possible function in floral tissues. As a first step, he wants to generate antibodies that specifically recognize Arabidopsis calreticulins in order to determine which floral cell types express these proteins using immunocytochemistry. His plan is to express an Arabidopsis calreticulin coding sequence in E. coli to allow isolation of calreticulin antigen for polyclonal antibody production in rabbits. He searched the Arabidopsis expressed sequence tag (EST) database over the internet using a known human calreticulin sequence and identified several candidate cdna clones that were available from the Arabidopsis Biological Resource Center at Ohio State University. Available Information and Reagents 1. A 1.4 kb Arabidopsis cdna has been obtained from the Resource Center and its complete DNA sequence was determined. It was found to encode a 425 amino acid protein with high amino acid homology to human calreticulin. 2. A pet(his)6 bacterial expression vector, the plyss plasmid encoding T7 lysozyme, and the BL21(DE3) E. coli lde3 lysogen strain containing the T7 RNA polymerase gene under lac control, have been obtained from a commercial source. 3. Biochemical supplies are available for purifying proteins containing a polyhistidine sequence from bacterial cell extracts using metal chelation chromatography. Page 3

4 4. Resources are available for polyclonal antibody production in rabbits using purified antigen. The plant biologist has expertise in plant immunocytochemistry and biochemistry. Basic Strategy The two essential components of this T7 bacteriophage RNA polymerase-based expression system are the pet plasmid containing the coding sequence of a DNA insert downstream of the T7 promoter (Tpr), and an E. coli strain containing a genomic copy of the T7 RNA polymerase (T7 pol) gene under the control of the lac UV5 promoter Also shown in this example is a T7 lysozyme encoding plasmid containing the chloramphenicol-resistance gene (cat), which provides a means to inhibit the small amount of T7 RNA polymerase that is expressed in the absence of IPTG. High level expression of the histidine-tagged calreticulin protein is obtained by blocking laci repressor function with the addition of IPTG to the media. This IPTG-inducible expression system is based on the finding that bacteriophage T7 RNA polymerase is highly specific, and efficient, in transcribing genes containing a T7 promoter. By transforming a recombinant calreticulin pet vector into an E. coli strain containing the T7 RNA polymerase gene under lac control, it will be possible to induce calreticulin expression by treating the cells with IPTG (see below). Since low level expression of the T7 RNA polymerase gene occurs in this system in the absence of IPTG, and appreciable levels of the plant calreticulin protein may inhibit bacterial growth, a compatible plasmid containing the T7 lysozyme gene will also be introduced into the host cells. T7 lysozyme is a bifunctional protein that not only cleaves peptidoglycan linkages in the bacterial cell wall but also stoichiometrically inhibits the function of T7 RNA polymerase. Therefore, low level expression of T7 lysozyme is able to inhibit the activity of T7 RNA polymerase molecules present in cells even before IPTG treatment. Regulated expression of genes in E. coli using the pet vector system. The two essential components of this T7 bacteriophage RNA polymerase-based expression system are the pet plasmid containing the coding sequence of a DNA insert downstream of the T7 promoter (Tpr), and an E. coli strain containing a genomic copy of the T7 RNA polymerase (T7 pol) gene under the control of the lac UV5 promoter. Also shown in this example is a T7 lysozyme encoding plasmid containing the chloramphenicol-resistance gene (cat), which provides a means to inhibit the small amount of T7 RNA polymerase that is expressed in the absence of IPTG. High level expression of the histidine-tagged calreticulin protein is obtained by blocking laci repressor function with the addition of IPTG to the media. Flow scheme for calreticulin antibody production using purified protein from bacterial extracts. The polyhistidine tag contained within the recombinant calreticulin protein can be used to purify soluble protein from cell extracts using a Ni2+ affinity column. The bound calreticulin protein is eluted from the column with imidazole which outcompetes the histidines for Ni2+ binding. Protein purification is monitored by gel electrophoresis (SDS-PAGE). In this example, calreticulin-specific polyclonal antibodies are produced in a rabbit and immunopurified with a calreticulin affinity column that is Page 4

5 made using the calreticulin antigen protein from the E. coli extracts. The immunopurified calreticulin-specific antibodies could be useful as reagents in Western blots, immunocytochemical studies and immunoprecipitation experiments. Comments The production of antigen in E. coli is the least demanding application of bacterial expression systems since denaturing conditions can be used to aid in protein solubilization. The production of polyclonal antibodies using recombinant proteins has become a standard procedure. Many researchers use commercial services that specialize in antibody production. Protein samples are sent to the service by express mail, and 7-10 weeks later, serum is sent back for analysis. Since the antigen is produced in E. coli and therefore available in large quantities, antigen-specific antibodies can be purified from the serum using affinity chromatography. Depending on the results obtained using polyclonal antibodies, the production of antigen-specific monoclonal antibodies may also be warranted. Prospective The plant biologist found that the anti-calreticulin antibodies detected high levels of the protein in developing seeds, a result which is consistent with its role as a chaperone protein in tissues with elevated rates of protein synthesis. During seed development, large amounts of storage proteins are synthesized which are later degraded to fuel seed germination. It would be interesting to determine if calreticulin was required for seed development. This could be done for example, by disrupting calreticulin function using cell-specific antisense genetics or RNA interference (RNAi) and by characterizing calreticulin in Arabidopsis mutants containing defects in seed development. Calreticulin function in the seeds could also be studied by isolating seed proteins that interact with the bacterial expressed calreticulin (His)6 protein following absorption to a Ni2+ affinity column. What are two advantages of using the T7 promoter and T7 RNA polymerase in the pet system to control expression of cloned recombinant genes. How is this better than simply using the lac promoter upstream of the calreticulin gene)? What is the purpose of running the rabbit polyclonal anti-serum over a calreticulin affinity column? How are the antibodies eluted from the column without releasing the calreticulin antigen at the same time? Describe how the bacterially-expressed His-calreticulin protein could be used to isolate calreticulin bind proteins from seed extracts? How could you identify the bound proteins beyond their molecular weight on a gel? Page 5

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