Background the grids electron microscopy grids need to have little or no charge, so that the em stain uranyl. Of the doublestranded phages, bacteriophage lambda derived vectors are the most popular tools for several reasons. Bacteriophage definition they are one of the types of virus that attacks the bacteria and infects it. Different phage also take different amounts of time to go through one growth cycle. In vivo excision of the pbluescript phagemid from the lambda zap ii vector. First you need to use a vector that can accept large fragments of dna. Can be modified for use as a cloning vector and assembled in vitro.
Restriction enzymedigested lambda dna 48,502bp may be used as a molecular weight size marker in gel analysis of nucleic acids. Our new crystalgraphics chart and diagram slides for powerpoint is a collection of over impressively designed datadriven chart and editable diagram s guaranteed to impress any audience. An understanding of the intrinsic molecular organization and of the genetic events which determine lysis or lysogeny in lambda has allowed investigators to modify it to suit the specific. Bacteriophage lambda has been in use as a cloning vector for over 25 years, and has been used extensively as an expression vector. Lambda phage can be manipulated and used as an anticancer vaccine, nanoparticle, targeting human aspartyl asparaginyl. The repressor found in the phage lambda is a notable example of the level of control. A new look at bacteriophage lambda genetic networks f les sequences cos du bacteriophage.
Jul 08, 2009 the prophages sometimes add more functions to the host cell. Learn vocabulary, terms, and more with flashcards, games, and other study tools. The number of phage that can be released from one bacterium after infection and growth by one phage is known as the burst size. Lambda phage consists of a virus particle including a head also known as a capsid, tail and tail fibers. Bacteriophage lambda phage lambda is a bacteriophage or phage, i. Ppt lambda phage powerpoint presentation free to view. As a simple example, figure 3c shows the infection time courses for dlamb. Phage sample preparation for electron microscopy objective to prepare phage sample for electron microscopy. However, the number of individual clones that can be obtained by plasmid cloning is limited by the relatively low efficiency of e. Lambda phage vectors have the advantage of high transformation. This is done most easily by phenol extraction method a. A cosmid is a type of hybrid plasmid that contains a lambda phage cos sequence. Lab 5 transposition insertion mutagenesis using minikan. The lambda phage is a typical example of bacteriophage which has a polyhedral head and tail.
In practice, the process often involves combining the dna of different organisms. A number of ingenious modifications help overcome the disadvantages associated with its mode of growth and its size. Nov 01, 2004 after excision, the iac dna was recovered by use of the qiaquick pcr purification kit qiagen, according to the manufacturers instructions. Bacteriophage lambdabased expression vectors springerlink.
The shotgun cloning method was performed to construct a genomic library of the bacteriophage lambda. The efficiency of packaging and infection, and the simplicity of plaque screening are advantages of lambda as a cloning vector. Commonly used bacteriophages are m, f1, fd and lambda. This is possible because lambda zap incorporates the signals. Samples 1 ml were collected at different time points and after. Cosmid vector the cosmid vector is a combination of the plasmid vector and the cos site which allows the target dna to be inserted into the. Lambda dna is also a commonly used substrate in restriction enzyme activity assays. If used for expressing certain gene in the dna fragment, it is called an expression vector commonly used vectors include plasmid, lambda phage, cosmid and yeast artificial chromosome yac plasmid.
Diagram of phage characteristic features of a bacteriophage they have a typical structure of outer protein capsid enclosing a genetic. Lambda phage will enter bacteria more easily than plasmids making it a useful vector that can. It is a bacterial virus, or bacteriophage, that infects the bacterial species escherichia coli. The plasmid based vectors used for cloning dna molecules generally carry up to 10 kb of inserted dna. We know when a phage has successfully reproduced when we are able. Recombinant dna and genetic techniques university of leicester. D herelle coined the term bacteriophage meaning bacterial eater to describe the agents bacteriocidal activity. General procedure for plating and titering lambda stocks. Using a host cell to carry the vector allows for easy amplification and retrieval of specific clones from the library for analysis. This is one reason why temperate phages are not used for phage therapy. The analysis of lambda transducing phage showed that approximately 40% of the wildtype genome is not required for replication by the lytic life cycle. It was used for immunoprecipitation in chip assays.
Bacteriophage lambda medical definition merriamwebster. Lambda phage immediate early gene turns on delayed early genes modifies host rna polymerase using nus factors. The hindiii digest of lambda dna ci857ind1 sam 7 yields 8 fragments suitable for use as molecular weight standards for agarose gel electrophoresis 1. Lambda phage dna, methylated from escherichia coli host. They were first described by collins and hohn in 1978. Take care not to contaminate the lambda zapcmv vector with this hightiter filamentous helper phage. As noted above, in phage lambda crosses chi stimulates homologous recombination at or near the site of the mutation. By using the hybrid primers, hybrid dna comprising a large sequence of lambda phage dna. They are often used as a cloning vector in genetic engineering. If you feel there is excess ethanol or ether in your sample, you can put the tube in the vacuum dessicator to drive off the. Stimulation is exclusively by the recbcd pathway and extends leftward from chi with respect to the direction chi is written here. Start studying lab 5 transposition insertion mutagenesis using minikan from a phage lambda vector.
Bacteriophage lambda questions and study guide quizlet. In this chapter, we will look at phage development using t4, l lambda, p1, and m as examples. Internal control dna for pcr assays introduced into lambda. The lambda phage has an icosahedral head and a long tail terminating in a single fiber. Oct 24, 20 there is an upper limit on the amount of dna that can be packed into a phage a maximum of 53 kb, therefore to allow foreign dna to be inserted into phage dna, phage cloning vectors need to have. Cloning vectors used in recombinant dna technology. Use of lambda phage dna as a hybrid internal control in a.
Agtlo and agtll are examples of this type of vector. Larger dnafragements have been cut into smaller, appearing as new bands further down. One famous example is the conversion of harmless viobrio cholerae by a phage into a highly virulent one. Molecular cloning of polyoma virus dna in escherichia coli. This virus is temperate and may reside within the genome of its host through lysogeny. The most commonly used procedures for lambda phage dna isolation include purification of intact phage particles by chromatography 34 or csci gradient centrifugation,5 and purification of dna by.
Figure s3 one hundred and twenty five simulated infection. Vectors puc18 and puc19 are small highcopy number plasmids that are widely used. Vectors for easy cloning, adaptable display and versatile applications. The isolation of phage dna from a purified phage preparation simply involves the removal of the proteins of the phage particle. The life cycle of upon infection, the phage can pursue lytic growth or lysogeny. A lambda insertion type cdna cloning vector, lambda zap, has been constructed. Lambda phage will enter bacteria more easily than plasmids making it a useful vector that can destroy or can become part of the hosts dna. The biological activity of recombinant phage and recombinant phage dna containing monomeric or dimeric polyoma dna inserts was examined in mice and cultured mouse cells. Feb 18, 2015 this feature is not available right now. The lambda zap vector has six unique cloning sites that will accommodate dna inserts from lambda zap vector. Lambda phage dna, methylated from escherichia coli host strain w3110 is suitable for use as a substrate for restriction enzymes. Bacteriophage and viral cloning vectors springerlink.
Many plasmid cloning vectors are designed and available for many prokaryotic organisms as well as for eukaryotic organisms. The approximate mass of dna in each of the bands is provided assuming a 1. Commonly used vectors include plasmid, lambda phage, cosmid and yeast artificial chromosome yac. A forwardgenetic screen and dynamic analysis of lambda phage. But the plasmid cloning vectors which carry more than 5 to 10 kb gene of interest are often unstable.
A genomic library is a set of recombinant clones that contains the entire dna of an organism. Therefore size of the gene of interest is limited in plasmid cloning vectors. The excision process eliminates the need to subclone dna inserts from the lambda phage into a plasmid by restriction digestion and ligation. Twort in great britian 1915 and felix d herelle in france1917. This repressor is important for controlling expression of fusion proteins which may be toxic to the e. Learn lambda phage with free interactive flashcards. The process depends on the ability of cut, and rejoin, dna molecules at points identified by specific sequences of nucleotide bases called. Diagram explaining the lytic and lysogenic cycle of a bacteriophage. The dna is protected by the polyhedral structure, and the tail interacts with the specific site of bacteria for attachment. All phages were produced using the fth1 vector which were grown, harvested and analyzed by dot blot and elisa using standard protocols as previously described in tarnovitsky et al. Lambda zap ii undigested vector kit 5 third, the f. Lambda phage vectors have the advantage of high transformation efficiencies due to good commercially available packaging extracts. Darrell kottons lab is published in am j respir cell mol biol. Lambda phage hostdependencies reveals an extensive.
Lecture about cloning vector which illustrates the properties of lamda. Lambda phage elements like those that comprise the plasmids. Oligonucleotides corresponding to the bt were introduced at the n terminal aspects of proteins 3, 8, 7 and 9 as follows. So, today, it is possible if we want, for example, any gene that you. Examples of these are bacteriophage and cosmid vectors and more recently yeast artificial. The plasmid based vectors used for cloning dna molecules generally carry up to 10 kb of inserted. Recombinant dna or rdna is made by combining dna from two or more sources. This article throws light upon the three types of cloning vectors used in recombinant dna technology. Temperate phages are basically bacteriophages which can choose between a lytic and lysogenic pathway of development. The limit on how much exogenous dna can be propagated in a lambda vector results from the packaging capacity of the phage particle, approximately 3550 kb of dna. Bacteriophage virus that infect bacteria is known as bacteriophage.
General procedures for phage preparation and purification materials. The central part of the lambda genome is not essential for the virus to replicate in escherichia coli, so this can be excised using an appropriate restriction enzyme, and inserts from donor dna can be spliced into the gap. The cosmid vector can carry up to 45 kb whereas plasmid and. Extensive research has been directed toward the development of multipurpose lambda vectors for cloning ever since the potential of using coliphage lambda as a cloning vector was recognized in the late 1970s. Mix phage with cells and allow a time for cell binding usually about 1530 min, centrifuge the sample at,000 x g microfuge and then titre the number of free phage remaining in the supernatant. More importantly, it is our experience that replica plaque lifts are more. The purified iac dna fragment was inserted into the ecori cloning site of lambda phage dna with use of the lambda gt11ecoriciaptreated vector kit stratagene. It was first reported by frederick twort, a british biologist and later by felix dherelle, a french microbiologist. Dna cloning with vector vectors for cloning large fragments. The restriction endonuclease, psti, was used to cleave the escherichia coli puc19 vector and the dna.
Vector is an agent that can carry a dna fragment into a host cell. If used for expressing certain gene in the dna fragment, it is called an expression vector. Construction of genomic library using lamda phage youtube. Bacteriophage lambda vectors we have talked about plasmids as vectors for cloning small pieces of dna. Enterobacteria phage lambda an overview sciencedirect. If used for reproducing the dna fragment, it is called a cloning vector. Nusa, nusg, nusb, nuse rnap now termination resistant cterm of n reacts with rnap central domain of n reacts with nusa nterm of n binds rna via boxb rna hairpin structure in at the nut site. A cloning vector is a small piece of dna, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign dna fragment can be inserted for cloningpurposes.
The large amounts of high quality dna needed for use as vectors or for markers for gel electrophoresis can be obtained in this way. But bacterial transformation with recombiant lambda phages is very ineffective. Recombinant virus lambda phage transfer plasmid long terminal repeat. Chart and diagram slides for powerpoint beautifully designed chart and diagram s for powerpoint with visually stunning graphics and animation effects. Choose from 67 different sets of lambda phage flashcards on quizlet. Overview of the lambda zap vector system the lambda zap vector1, 2 system combines the high efficiency of lambda library construction and the convenience of a plasmid system see figure 1. This presents problems when you are trying to create a genomic library of a large genome such as with plants. Recombinant preparations containing a single copy of viral dna were invariably noninfectious.
1008 726 1566 1316 1012 29 1528 1484 637 814 586 353 1476 1036 807 63 1539 82 1048 925 288 1224 296 1462 749 449 1211 75 1044 938 964 577 408 444 150 140 264 713 198 214 1353 1405 293 1287 216