Cloning Vehicles and Transformation:

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1 Cloning Vehicles and Transformation: Once recombinant DNA molecules have been constructed in vitro the desired sequence can be isolated In some experiments, hundreds of thousands of different DNA fragments may be produced and isolation of particular sequence would seem to be an almost impossible task It is bit like looking for the proverbial needle in a haystack with added complication that the needle is made of the some material as the haystack Fortunately the methods available provide a relatively simple way to isolate specific gene sequences Three things have to be done to isolate a gene from a collection of recombinant DNA sequences: The individual recombinant molecules have to be physically separated from each other The recombinant sequences have to be amplified to provide enough material for further analysis and The specific fragment of interest has to be selected by some sort of sequence dependant method

2 In this chapter, we will consider the first two requirements which in essence represent the systems and techniques involved in gene cloning This is essential part of most genetic manipulation programs Even if the desired result is transgenic organism, the gene to be used must first be isolated and characterized and therefore cloning systems are required The biology of gene cloning is concerned with the selection and use of: a suitable carrier molecule or vector a living system or host in which vector can be propagated and methods for getting DNA into cells Vectors: Plasmid Vectors; Plasmids are widely used as cloning vehicles Plasmids are replicons which are stably inherited in any extra chromosomal state Most plasmids exist as double-stranded circular DNA molecules

3 Therefore, desirable properties of plasmid cloning vehicles are: o o o Low molecular weight Ability to confer readily selected phenotypic traits on host cells Single sites for a large number of restriction endonucleases preferably in genes with a readily scorable phenotype For genetic engineering, naturally occurring plasmids have been extensively modified to produce vectors that have the desired characteristics psc101: It was the first cloning vector used as a cloning vector in genetic cloning experiments Low copy number plasmid from Salmonella Developed by Herbert Boyer and Stanley Norman Cohen a DNA plasmid in 1973 They demonstrated that a gene from a frog could be transferred into bacterial cells and Then expressed by the bacterial cells

4 Fig down loaded from /basic_cloning_vectors/psc101/ pbr322: an important plasmid in the history of gene manipulation It has all features of a good vector such as: o o o o Low molecule weight i.e. 4363bp Antibiotic resistance genes i.e. amplicine and tetracycline An origin of replication and Several single-cut restriction endonucleases recognition sites Fig 5.1 Nicholl 3 rd Ed puc Family: More exotic, derived from pbr 322 Fig 5.2 Nicholl 3 rd Ed Although plasmid vectors have many useful properties and are essential for gene manipulation, they do have a number of disadvantages One of the major drawbacks is the size of DNA fragment that can be inserted into plasmids is 5kb before cloning efficiency or insert stability are effected Genomic library

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6 Mexican creators: Bolivar and Rodriguez

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8 Copy number of plasmid vector Plasmids can also be categorized on the basis of their being maintained as multiple copies per cell (relaxed plasmids) or as a limited number of copies per cell (stringent plasmids) Generally conjugative plasmids are of relatively high molecular weight and present as 1-3 copies per chromosome whereas non-conjugative plasmids are of low molecular weight and present as multiple copies per chromosome Table down loaded Bacteriophage Vectors: They are more specialized than plasmid In the 1940s Max Delbruck and the phage group laid the foundation of Modern Molecular Biology by studying bacteriophages These are literally eaters of bacteria and are viruses that are dependent on bacteria for their propagation so called bacteriophages or phages Structurally phages fall into three main groups: o o o Tailless Head with tail an Filamentous

9 DNA construct Origin of replication Copy number Classification Plasmids puc vectors pmb1* High copy pbluescript vectors ColE High copy pgem vectors pmb1* High copy ptz vectors pmb1* >1000 High copy pbr322 and derivatives pmb1* Low copy pacyc and derivatives p15a Low copy psc101 and derivatives psc101 ~5 Very low copy Cosmids pmb1 SuperCos Low copy pwe15 ColE Low copy 20bacterial%20cultures/

10 Fig 5.3 Nicholl 3 rd Ed The genetic material may be single or double stranded DNA or RNA. Double-stranded DNA (ds DNA) being found most often In typical ds DNA phages, the genome makes up about 50% of the mass of the phage particle. Thus phages represent relatively simple systems when compared to bacteria and for this reason they have been extensively used as models for the study of gene expression Phages may be classified as either virulent or temparate depending on their life cycles Fig 4.11 Primrose 6 th Ed The genome of phage is 48.5 kb in length and encodes some 46 genes The entire genome has been sequenced (this was the first major sequencing project to be completed and represents one of the milestones of molecular genetics) and all the regulatory sites are known At the ends of the linear genome there are short (126p) singlestranded regions that is complementary These act as cohesive or sticky ends which enable circularization of the genome following infection The region of the genome that is generated by the association of the cohesive ends is know as the cos site

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13 Fig 5.4 Nicholl 3 rd Ed The genome circularizes and the phage initiates either the lytic or lysogenic cycle depending on a number of factors that include the nutritional and metabolic state of the host cell and the multiplicity of infection (m.o.i) the ratio of phage to bacteria during absorption To determine the number of bacteriophage present in a suspension, serial dilutions of the phage stock are mixed with an excess of indictor bacteria (m.o.i. is very low) and placed onto agar On incubation, the bacteria will grow to form what is termed a bacterial lawn Phage that grown in this lawn will cause lysis of the cells that the phage infects and as this growth spreads a cleared area or plaque will develop Fig 5.6 Nicholl 3 rd Ed Plaques can then be counted to determine the number of plaque forming units (p.f.u.) in the stock suspension Two main classes of vectors obtained by manipulating nonessential parts of genome: Insertion vectors and Replacement vectors

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16 Fig 5.7 Nicholl 3 rd Ed Further innovations have been made by adding different characteristics on basis of selection and/or screening can be done Fig 5.8 Nicholl 3 rd Ed Fig 5.9 Nicholl 3 rd Ed The filamentous phage M13 differs from λ both structurally and in its life cycle Fig 5.3 Nicholl 3 rd Ed The M13 genome is a single stranded circular DNA molecule 6407bp in length The phage will infect only E.coli that has F-pili (threadlike protein appendages found on conjugation-proficient cells When DNA enters the cell, it is converted into double stranded molecule known as the replicative form or RF, which replicates until there are about 100 copies in the cell At this point DNA replication becomes asymmetric and single stranded copies of the genome are produced and extruded from the cell as M13 particles The bacterium is not lysed and remain viable during this process, though growth and division are slower than in non-infected cells

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18 ci λ repressor- plaque formation and morphology

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21 Two aspects of M13 infection are of value to the genetic engineer i. The RF is essentially similar to a plasmid and can be isolated and manipulated using the same technique ii. A single stranded DNA produced during infection is useful in techniques such as DNAsequencing by dideoxy method Unlike phage λ, M13 does not have any non-essential genes Only part available for manipulation is a 507bp intergenic region which has been used to construct vectors Fig 5.10 Nicholl 3 rd Ed Cosmid Vectors: Today the Molecular Biologist has available an enormous range of vectors and these are notable for three reasons: Many of them combine elements from both plasmid and phages and are known as phasmids or if they contain an M13 ori region then called phagemids Many different features that facilitates cloning and expression can be found combined in a single vector Purified vector DNA plus associated reagents can be purchased from molecular biology suppliers There are two general uses of cloning vectors: cloning large pieces of DNAand manipulating genes

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23 BACs-Bacterial Artificial Chromosome: - A multi purpose vector that has been developed for mapping and analysis of complex eukaryotic genomes is based on the F factor of bacteria and is called a bacterial artificial chromosome - What are F factors? - Independently replicating plasmids that are involved in the transfer of genetic information during bacterial conjugation - Because F factors can carry fragments of bacterial chromosome up to one Mb in length, they have been engineered to act as vectors for eukaryotic DNA Fig Klug Vectors for Cloning Large Fragments of DNA: Cosmid Vectors / Hybrid Vectors: Fig Klug Table 5.1 Primrose 6 th Ed Method for Getting DNA into Cells: Fig 1 Chapter 6 Walker 3 rd Ed

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27 Calcium Phosphate Co-precipitation; It possibly concentrates the DNA on the cell membrane to further facilitate uptake by phagocytosis and also protects the DNA from hydrolytic nucleases The transfect ion efficiency or level of gene expression depends upon both the efficiency of uptake by the cells and the transcriptional capability of each DNA template introduces into the successfully transected cells The efficiency of DNA uptake is critical upon the ph of the calcium phosphate DNA co-precipitate formation and the amount of DNA in that complex Efficiency can be enhanced by application of glycerol shock DEAE-Dextran; Work as calcium phosphate co-precipitation Efficiency can be enhanced by application of glycerol shock Polybrene-DMSO Treatment; It is a synthetic cation which can be used to facilitate DNA transfection by serving as an electrostatic bridge between the negatively charged DNA and positively charged components on the host cell membrane

28 Various methods to let DNA go into cells: - Chemical - Physical - Viral

29 As such, this technique exploits the same strategy as described above for calcium phosphate co-precipitation and DEAE-dextran Thus, similar to DEAE-dextran, the uptake of the adsorbed DNA on the cell surface is enhanced by treatment with DMSO which, in the manner of the glycerol shock, serve to permeabilize the DNA coated cells Lipofection; Synthetic cation lipids can be used to generate liposomes in the presence of DNA Liposomes entrap 100% of DNApresent These vesicles may be fused to cell membranes and thus deliver the DNA directly to the cell interior The rationale behind this procedure is that a single DNA plasmid is surrounded by sufficient cationic lipid to completely neutralize the negative charge of DNA and provide a net positive charge which facilitates as association with the negatively charged surface of the cell Depending upon the cell line, lipofection is reported to be fold more effective than either the calcium phosphate or DEAEdextran mediated transfection techniques

30 Electroporation; This method requires a specialist equipment Based upon the induction and stabilization of permeation sites within cell membrane via an interaction of lipid dipoles in an electric field It does not require carrier DNA and is very quick compared the above techniques Fig 2 Chapter 6 Walker 3 rd Ed Protoplast Fusion; This is a general method for introducing macromolecules into cells and is therefore not restricted to DNA Fig 14.4 Primrose 5 th Ed Fig 3 Chapter 6 Walker 3 rd Ed Scrapefection; This technique was initially describe as a general method for introducing functional macromolecule into adherent cell lines This protocol is very simple and involved incubating a washed monolayer of cells with a buffered DNA solution prior to scraping with a rubber policeman

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34 The scraped cells are then distributed to fresh culture plates and analyzed for transient expression of the transfected DNA 1 to 5 days later Microinjection; As mentioned DNAdirectly introduced in to nucleus with a glass micro-pipette with the aid of micromanipulator Fig 5.13 Nicholl 2 nd Ed Fig 11.1 Primrose 2 nd Ed /Fig 16.5 Primrose 5 th Ed Fig 13.9 Nicholl 3 rd Ed Fig 16.4 Brum Fig 16.5 Brum Table 17.1 Glick 3 rd Ed Fig 17.9 Glick 3 rd Ed Table 17.2 Glick 3 rd Ed Table 17.3 Glick 3 rd Ed

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37 Large in size copied of GH gene/mouse metalothionine promoter were injected -Subsequently fuse to form the diploid zygote nucleus of fertilized egg - Embryo culture in vitro until they have undergone a number of division / Surrogate mother This was the ground work for development of genetically engineered livestock carrying genes that improve their food value. For example Pig - leaner meat as increased amount of GH stimulates the conversion of nutrients into proteins rather than fat

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44 Host Cells: The type of host cell used for a particular application will depend mainly on the purpose of the cloning experiment If the aim is: To isolate a gene for structural analysis, the requirements may call for a simple system that is easy to use To express the genetic information in a higher eukaryotic such as a plant, a more specific system will be required These two aims are not necessarily mutually exclusive Often a simple primary host is used to isolate a sequence that is then introduced into a more complex system for expression Table 5.1 Nicholl 3 rd Ed Prokaryotic Hosts; An ideal host cell should be: o easy to handle and propagate o available as a wide variety of genetically defined strains and o should accept a range of vectors

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46 The bacterium E. coli fulfills these requirements and is used in many cloning protocols: Studies in great detail and many different strain were isolated by microbial genetics as they investigated the genetic mechanism of this prokaryotic organism Such studies have provided the essential information on which genetic engineering is based E. coli is a Gram-negative bacterium with a single chromosome packed into compact structure know as the nucleoide Genome size is 4.5x10 6 base pairs and complete sequence is known The process of gene expression (transcription and translation) are coupled with newly synthesized m RNA being immediately available for translation No post transcriptional modification of the primary transcript as is commonly found in eukaryotic cells E. coli can therefore be considered as one of the simplest host cells Much of gene cloning that is carried out routinely in laboratories involves the use of E. coli hosts, with many genetically different strains available for specific applications

47 In addition to E. coli other bacteria may be used as hosts for gene cloning experiments with examples including species of Bacillus, Pseudomonas and streptomyces There are however certain drawbacks with most of these: - Fewer suitable vectors available - Getting recombinant DNA into the cell can cause problems, direct introduction of ligated DNA into the cell - Often sensible to perform the initial cloning in E. coli, isolate the required sequence and then introduce the purified DNA into the target host Eukaryotic Hosts; One disadvantage of using an organic such as E. coli as a host for cloning i.e. that it is a prokaryotic and therefore lacks the membrane bound nucleus and other organelles, found in eukaryotic cells Eukaryotic cells range from microbes such as yeast algae to cells from multi-cellular organisms such as human beings Microbial cells have many of the characteristics of bacteria with regard to ease of growth and availability of mutants

48 Higher eukaryotes present a different set of problems to the genetic engineer, many of which require specialized solutions Often the aim of gene manipulation experiments in a higher plant or animal is to alter the genetic make up of the organism by creating a transgenic The yeast saccharomyces cervisiae is one of the most commonly used eukaryotic microbes in recombinant DNA technology For centuries it has been used for production of bread and beer and has been studied extensively The organism is amenable to classical genetic analysis and range of mutant cell types is available Genome is 1.35x10 7 base pairs Complete genome sequence is known now Concept Map 5 Nicholl 3 rd Ed x x x x

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