BS514_2.3 Selection of Clones PDF

Selection of clones Mansi Desai Direct selection Eg.1 R6-5 a low copy number, conjugative, FII incompatibility group plasmid a molecular length of 102 kb specifies resistance against several antibiotics (chloramphenicol, fusidic acid, kanamycin,...

Selection of clones Mansi Desai Direct selection Eg.1 R6-5 a low copy number, conjugative, FII incompatibility group plasmid a molecular length of 102 kb specifies resistance against several antibiotics (chloramphenicol, fusidic acid, kanamycin, streptomycin and sulphonamide) and mercury salts EcoRI digest: 13 fragments Direct selection  Marker rescue Auxotroph A microorganism that is unable to synthesize one or more essential growth factors, and it will not grow in fermentation media lacking them. Auxotrophs are microorganisms that are unable to synthesize an essential nutrient because of a gene mutation. trpA- survive if tryptophan is added into medium trpA gene codes for tryptophan synthase Scope of marker rescue Applicable for most genes that code for biosynthetic enzymes As clones of these gene can be selected on minimal medium. Technique is not limited to E.coli or bacteria Auxotrophic strains of yeast and filamentous fungi are also available E. coli auxotroph can be used as host for selection of some genes from other organisms Limitation of marker rescue A mutant strain must be available for the gene of interest A medium on which only wild-type can survive is needed Many bacterial mutants are not auxotroph Mutant and wild-type can not be distinguished Genes from animals or plants Foreign enzyme do not function in the bacterial cell Expression vector Expression vectors are cloning vectors, specially designed for expressing genes in a cell They aid in the transcription and protein translation process of the desired DNA fragment. They contain all features of a typical cloning vector and some Additional features, promoters, enhancers, termination sequence, initiation sites, stop codon, etc., to help in protein synthesis. It is a vector widely used for protein production. They have basic features of a vector like ori (origin of replication), insertion site, a selectable marker, etc. Additionally Insertion of a strong promoter. Insertion of a strong termination codon. Considerable distance between promoter region and the cloned gene. Insertion of a transcription initiation sequence. Insertion of a translation initiation sequence. Screening based on gene expression For a screening method based on expression of the gene of interest it is necessary to construct an expression library in a vector designed to ensure that the cloned genes are expressed You will then need a way of detecting expression of your particular gene. Eg. cloning the xylE gene from Pseudomonas aeruginosa xylE gene: the enzyme, catechol 2,3-oxygenase Can be detected by a simple colorimetric test These enzymes have a key role in the degradation of aromatic molecules in the environment by soil bacteria. Catechol 2, 3-dioxygenase catalyzes the incorporation of dioxygen into catechol and the extradiol ring cleavage to form 2-hydroxymuconate semialdehyde A range of bacterial genes can be screened for in this way including those involved in the utilization of specific sugars and the genes involved in antibiotic production and resistance to antibiotics. Use of chromogenic substrate: An important aspect of the development of the technology. The most popular system uses the compound X-gal (5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside) X-gal: a colourless substrate for β-galactosidase. β-galactosidase: product of lacZ gene (part of lac operon) The enzyme is normally synthesised by E. coli cells when lactose becomes available. However, induction can also occur if a lactose analogue such as IPTG (iso-propyl- thiogalactoside) is used. The method is based on the blue pigment that forms when beta-galactosidase catalyzes hydrolysis of the synthetic substrate X-gal.  Hydrolysis of X-gal produces galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter product then undergoes spontaneous dimerization and oxidation to form a blue pigment. The expression of the lacZ (β-galactosidase) gene can be detected easily. E. coli cells that express beta-galactosidase activity thus form blue colonies when spread on agar plates that contain X-gal, while cells that do not produce active enzyme form white colonies This can be used either as a screening method for cells or plaques or as a system for the detection of tissue specific gene expression in transgenics.  Normal enzyme function: β-galactosidase cleaves the disaccharide lactose to produce galactose and glucose which then ultimately enter glycolysis. This enzyme also causes transgalactosylation reaction of lactose to allolactose which then finally cleaved to monosaccharides. The X-gal detection system can be used where a functional β- galactosidase gene is present in the host/vector system. This can occur in two ways. First approach an intact lacZ gene may be present in the vector, as is the case for the insertion vector Charon 16A. Host cells that are Lac− are used, so that the Lac+ phenotype will only arise when the vector is present.  Second approach- to employ the complementation system Deletion of a specific fragment of the lacZ ω gene Deletion: lacZΔM15 Creates a non-functional β-galactosidase enzyme. A part of the lacZ gene (encoding this section of amino acids called the α-peptide) is carried by the vector. The remaining part of the gene sequence is carried by the host cell. The region coding for the smaller, vector-encoded α- peptide is designated lacZ’. Host cells are designated lacZ−. Blue colonies or plaques will only be produced when the host and vector fragments complement each other to produce functional β-galactosidase Providing DNA encoding α-peptide to a lacZΔM15- mutant bacterial cell in trans complements the mutation allowing for a functional enzyme. This process is called α-complementation.  Insertional inactivation A foreign DNA is cloned within the coding gene responsible for a phenotype. As a result of insertion, the gene product is not available to modulate the phenotype of the host. This approach is known as insertional inactivation, and it can be used with a suitable genetic system. (i) Insertional Inactivation of antibiotic resistance gene- pBR322: two antibiotic resistance gene, Apr and Tcr. If a gene fragment will be cloned in PstI, it will disrupt the Apr gene. As a result, the clone will be ampicillin sensitive and Tcr. Where as the original plasmid will be Apr and Tcr. To select the clone, first the transformed E.coli is plated on tetracycline containing media. Subsequently, a replica plate will be made on ampicillin containing media to identify the clone growing on tetracycline media but not on ampicillin media. PstI (ii) Insertional Inactivation of Lac Z gene-  Blue-white screening provides a convenient and powerful way to distinguish bacterial colonies or phage plaques that contain a cloning vector with a DNA insert, from those containing empty vectors with no insert DNA. Alternatively, alpha complementation may be used. If DNA is inserted into the plasmid-borne gene segment, the encoded subunit is not made and β- galactosidase is not produced. When β-galactosidase is expressed, the bacteria can degrade X-gal, which turns the bacterial colony blue. If a piece of DNA is inserted into the alpha fragment gene, the bacteria cannot split X-gal and stay white. (iii) Insertional Inactivation of cI gene- During an infection cycle, virus undergoes a lytic and lysogenic stages. The lytic phase is responsible for lysis of host to release the virus particle Lysogenic stage allow the replication of virus without lysis of host. The cI gene encodes for CI protein and which is responsible for the maintenance of lysogeny. In the presence of functional cI, the plaques contains unlysed host cells and has a turbid appearance where as in the absence it will clear. This feature can be use to screen the clone to detect functional cI (absence of clone) or absence of cI (presence of insert). Extra Information Identification of clone from a gene library Genomic library??? How to prepare genomic library??? For bacteria, yeast and fungi Complete genomic library is manageable For plants and animals???? Second type of library Not specific to whole organism Specific to particular cell type  cDNA library??? How cDNA library can be prepared?? House keeping genes?? Clone identification Complementary nucleic acid strand hybridization Colony and plaque hybridization probing What is Probe? It is a small complementary sequence to the desired gene which we want to screen out. The probe will bind to desired gene and as the probe oligonucleotide are tagged or conjugated with a detectable identity such as Radioisotope or fluorescent or chemiluminescence, It’s location can be identified.  Probe labelling Labelling with radioactive marker Hazard Disposal of radioactive waste Non radioactive labelling Extra Information Extra Information  Extra Information nucleic acid hybridisation- Colony Hybridisation Probe hybridization Abundancy probing to analyze cDNA Oligonucleotide probes for genes whose translation products have been characterized Heterologous probing allows related genes to be identified Southern hybridization enabled a specific restriction fragment containing a gene to be identified Plaque hybridization PCR based screening Extra Information Immunological screening for expressed genes Identify the protein product of a cloned gene by immunological screening Required protein expression in recombinants Specific antibody is used Polyclonal Monoclonal Extra Information 2. Immunological screening method- 1.Preparation of Replica plate- As original genomic or cDNA library is precious and will be consumed in later stage, all procedure is performed with the replica plate containing clones in a indentical manner. 2. Blotting- The clone is transferred on a nitrocellulose membrane to get similar pattern of colonies on master plate. The cells on the membrane are lysed and released protein is denatured, and allowed to bind the membrane. 3. Treatment with primary antibody- membrane is incubated with antibody having immunoreactivity towards a particular protein. The primary antibody will binds to the target protein due to exclusive specificity towards antigen (Figure 12.4). The membrane is washed to remove unbound primary antibody. 4. Treatment with secondary antibody- membrane is incubated with secondary antibody recognizing primary antibody. Secondary antibody is labeled with an enzyme (Horse raddish peroxidase or alkaline phosphatase) to use to give readable signal. The secondary antibody will binds to the primary antibody and will allow to detect the location of primary antibody. The membrane is washed to remove unbound secondary antibody Extra Information Extra Information  Extra Information Immuno- screening method- Selection Direct: Screening based on gene expression catechol 2,3-oxygenase, antibiotic resistance Screening by Complementation or marker rescue: auxotrophs Eg. Blue white screening, tryptophase synthase, lysine synthesis, GFP, GUS assay Indirect: Screening using nucleic acid hybridization Screening clone bank Use of PCR screening Immunological Screening of Expression Libraries Selection vs Screening Selection: Here some sort of pressure is applied during the growth of host ,cells containing recombinant DNA. The cells with the desired characteristics are therefore selected by their ability to survive. This approach ranges in sophistication, from selection for the presence of a vector up to direct selection of cloned genes by complementation of defined mutations. Screening: A procedure by which population of variable cells is subjected to some sort of analysis that enables the desired sequences to be identified. Because only a small proportion of the large number of bacterial colony or bacteriophage plaques being screened will contain the DNA sequence of interest, screening requires method that are highly sensitive and specific. In practice, both selection and screening method may be required in any single experiment and may even be used at the same time if the procedure is designed carefully Genetic selection and screening Genetic selection and screening methods rely on the expression of certain traits. Usually these traits are encoded by the vector or perhaps by the cloned sequence (in direct selection) Eg. Use of antibiotics to select presence of vector Use of chromogenic substractes Insertional inactivation Complementation of defined mutations Clone identification Highly specific method Selecting a clone Genetic selection For presence of vector Direct selection (Complementation of a defined mutation) May incorporate Use of chromogenic substrate Insertional inactivation Screening a clone bank Nucleic acid hybridization Colony hybridization Plaque hybridization Immunological screening