Preface 'Miniature world of microbes has fascinated mankind for centuries. Man has made miracles through microbes. The small pox vaccine is one example having collective effect on life of human being on this earth. Microbes are still mysterious entities for us. Through their genome carries a few Mb genome we have not still understood the nature and properties of microbes exclusively. Microbes are assuming special importance in this era of nanobiotechnology. The diversities in the utilities of microbes interested the specialist of many branches of biological sciences. How to work with these microbes? Which are the fundamental techniques ? What sort instrumentation it involves? How to work on microbial biotechnology? These are some of the aspects addressed in this collection entitled “Molecular Biology and Biotechnology: Microbial Methods”. The text consists of 370 pages divided into 36 chapters followed by detailed glossary. Most of the required protocols have been included. This is a huge subject and has many subsidiaries like food microbiology, textile microbiology, medical microbiology, and agriculture microbiology etc. Only the common modules of microbial biotechnology with broad generalization have been considered in this text. Working with the specific microbes is a trial & error game. It require lot of commonsense and tenacity. With that one can pave the way out in the methodological contest. And there will be no end of working with microbes. Open one gate and the several gate again will be in front of you. The reward for work well done is the opportunity to do more, and in the field of observation chance favours only the prepared mind. This text is just a guide line to set the hand. In actual working you will be doing much more beyond this text and that will be going to make us wiser. 

The Laboratory Worker is Surrounded by Many Dangers such as from.......... Handling of infectious materials. Handling of broken glassware. Accidental spill of corrosive reagents. Swallowing of corrosive reagents such as concentrated Sulphuric acid, Hydrochloric acid, sodium hydroxide, trichloroacetic acid etc. Swallowing of infectious specimen. Inhalation of poisonous fumes. Infection in the conduct of necropsies. Potential hazards in the form of inflammable chemicals and gas leakages.

BC 1750 The Sumerians brew beer. 500 The Chinese use moldy soybean curds as an antibiotic to treat boils. 250 The Greeks practice crop rotation to maximize soil fertility. 100 Powdered chrysanthemum is used in China as an insecticide. AD: Before the 20th Century 1590 The microscope is invented by Janssen. 1663 Cells are first described by Robert Hooke. 1675 Leeuwenhoek discovers protozoa and bacteria. 1745 Maupertuis proposes an adaptationist account of organic design 1797 Jenner inoculates a child with a viral vaccine to protect him from smallpox. 1802 The word “biology” first appears.

Search for genetic material—nucleic acid or protein/DNA or RNA? Griffith’s Transformation Experiment Avery’s Transformation Experiment Hershey-Chase Bacteriophage Experiment Tobacco Mosaic Virus (TMV) Experiment Timeline of Events 1890 Weismann - substance in the cell nuclei controls development. 1900 Chromosomes shown to contain hereditary information, later shown to be composed of protein & nucleic acids. 1928 Griffith’s Transformation Experiment 1944 Avery’s Transformation Experiment 1953 Hershey-Chase Bacteriophage Experiment 1953 Watson & Crick propose double-helix model of DNA 1956 Gierer & Schramm/Fraenkel-Conrat & Singer Demonstrate RNA is viral genetic material

1. pH Meter pH meter/electrode systems are the most common method of measuring pH in the biology laboratory. Although more expensive and more complicated to use than chemical indicators, pH meters provide greater accuracy, sensitivity, and flexibility. When used properly, pH meters can measure the pH of a solution to the nearest 0.1 pH unit or better, and they can be used with a variety of samples. 2. UV Spectrophotometer Perhaps there is no other technique except UV-visible spectroscopy, which has been so extensively employed and has such a profound impact on progress in biochemical investigations. UV-visible spectroscopy has undoubtedly become an indispensable tool and is routinely used for identification and quantitative estimation of most of the biomolecules. Besides other applications, most of the enzyme assays and the screening of biomolecules recovered in different fractions by separation processes such as column chromatography, density gradient centrifugation etc, rely heavily on spectrophotometer or colorimetric technique.

Mole (mol) 1 mole of any substance contains 6.02 x 1023 units. The number of moles in a given weight of substance can be calculated using following equation.

DNA Migration in Agarose and Polyacrylamide Gels Recommended Gel Percentages for Separation of Linear DNA

1. Bacterial (Prokaryote) Genome The chromosome of most bacteria is a single super coiled double stranded circular DNA molecule. A complex packaging mechanism is necessary in order to ensure that the entire DNA is folded within the bacterial cell in a manner, which will not inhibit transcription nor allow entanglement of the two daughter strands to occur during replication (Highly condensed >10 mg/ml in region less than l ìm in diameter). This is achieved by two main mechanisms. First the DNA is folded into between 40-100 loops Second each of the quasicircles is it self super coiled independently of the others each loop are abolished by Ribonuclease. Deoxyribonucleic remove the super helical nature of the loops, though limited treatment with this enzyme will attack only a small number of the loops and therefore leads to removal of only some of the super coiling. The intact folded chromosome or nucleoid can be isolated in the presence of non ionic detergents and high molarities of salt in two possible forms free and membrane associated. The free form which sediments at 1600-1700s consists of about 60%DNA, 30%RNA and 10%protein the bulk of which is the enzyme RNA polymerase. The membrane bound form contains an additional 20% of membrane-attached protein. Bacterial genome size ranges from 580 to 9900kbp, & the average gene size is constant from 900 to 1100bp of which 87-94% are protein coding (Rickettisa’s 76% and Borelia Plasmid’s 41% are exceptions).

Introduction Yeast contain a hard cell wall. Spheroplasts are yeast cells where the cell wall has been enzymatically degraded. These spheroplasts are very fragile so we must be careful with them. Once we have spheroplasts it will be very easy to lyse the yeast cells and quickly release the RNA and proteins in the cell into a controlled environment where we can temporarly keep the cellular RNases from degrading the RNA. We don’t want to lyse the cells before they are in this controlled environment. Growth of Yeast Cultures Cultures of wild-type and yeast containing various mutations will be grown up the day of the experiment. Each section will be given one wild type yeast culture and a culture containing one of the mutant varieties that we have discussed in class. Yeast are grown very similarly to bacteria. Like bacteria, they have different stages of growth. Scientists divide these stages of growth into early-log phase, mid log-phase, late log-phase, and the stationary phase. These phases are based on the amount of yeast that are around in the broth and how quickly they are able to grow and divide based on nutrient use. Reading the absorbance of the sample at 600 nm is a way in which you can determine what phase of the yeast growth series you are in and how many cells you have. Haploid yeast cells contain approximatley 1.2 pg of RNA per cell Predict approximatly how much RNA you should isolate

Procedure Place a portion of your RNA sample (approximately 40 mg, hydrated) into a heavy walled Pyrex test tube. Add 1.0 ml of 1 N HCl and seal the tube. Heat the tube in a boiling water bath for 1 hour. Cool the tube, open it and place the contents into a centrifuge tube. Centrifuge the contents at 2,000 RPM in a clinical centrifuge to remove any insoluble residue. The supernatant contains your hydrolyzed RNA. Prepare Whatman filter paper No. 1 for standard one-dimensional chromatography. Using a micropipette, spot 20 µl of your hydrolyzate onto the paper, being careful to keep the spots as small as possible (repeated small drops are better than one large drop). Allow the spots to completely dry before proceeding. Place the paper chromatogram into your chromatography tank and add the solvent (acetic acid:butanol:water). Allow the system to function for an appropriate time (approximately 36 hours for a 20 cm descending strip of Whatman #1). Remove the paper and dry it in a circulating air oven at 40° C for about 2 hours. Locate the spots of nucleotides by their fluorescence under an ultra-violet light source. Expose the paper chromatogram to a UV light source and outline the spots using a light pencil. The order of migration from the point of origin is guanine (light blue fluorescence), adenine, cytilic acid and finally, uridylic acid. After cafefully marking the spots, cut them out with cutters and place the paper cut outs into separately labeled 15 ml conical centrifuge tubes. Add 5.0 ml of 0.1 N HCl to each tube and allow the tubes to sit for several hours to elute the nucleotides from the paper.

Introduction DNA denaturation, also called DNA melting, is the process by which double-stranded deoxyribonucleic acid unwinds and separates into single-stranded strands during the breaking of hydrogen bonding between the bases. Both terms are used to refer to the process as it occurs when a mixture is heated, although “denaturation” can also refer to the separation of DNA strands induced by chemicals like urea. For multiple copies of DNA molecules, the melting temperature (Tm) is defined as the temperature at which half of the DNA strands are in the double-helical state and half are in the “random-coil” states. The melting temperature depends on both the length of the molecule, and the specific nucleotide sequence composition of that molecule. Applications of DNA Denaturation The process of DNA denaturation can be used to analyze some aspects of DNA. Because cytosine / guanine base-pairing is generally stronger than adenosine / thymine base-pairing, the amount of cytosine and guanine in a genome (called the “GC content”) can be estimated by measuring the temperature at which the genomic DNA melts. Elevated temperatures are associated with high GC content. GC ratios within a genome are found to be markedly variable. These variations in GC ratio within a genome of higher organisms results in a mosaic like formation with islet regions called isochores. This results in the variations in staining intensity in the chromosomes. The isochores include in them essential protein coding genes, termed housekeeping genes and thus determination of ratio of these specific regions contributes in mapping these essential genesbacterial systematics has recommended use of GC ratios in higher level hierarchical classification. For example, the Actinobacteria are characterised as “high GC-content bacteria”. In Streptomyces coelicolor A3(2), GC content is 72%. The GC-content of Yeast (Saccharomyces cerevisiae) is 38%, and that of another common model organism Thale Cress is 36%. Because of the nature of the genetic code, it is virtually impossible for an organism to have a genome with a GC-content approaching either 0% or 100%. A species with an extremely low GC-content is Plasmodium falciparum (GC% = ~20%), and it is usually common to refer to such examples as being AT-rich instead of GC-poor.

To Estimate the DNA Using Diphenylamine Introduction DNA estimation is helpful to know about the total amount of DNA of prokaryotic as well as eukaryotic system. One method used for DNA estimation is Diphenylamine assay, is that of Burton who modified the original procedure of Dische. This procedure is based on treatment of nucleic acid with hot perchloric acid to hydrolyze DNA nucleotides. Diphenylamine reacts specifically with deoxyribose in DNA to form a blue-colored complex. Since diphenylamine does not react with ribose, one can assay for DNA in the presence of contaminating RNA. Principle The DPA method is based on the colorimetry measurement of products formed by the reaction of ù- hydroxylevulinaldehyde (from the deoxyribose released after depurination of DNA) with diphenylamine in 2.5 N perchloric acid. When DNA is treated with diphenylamine under acidic condition (PCA) a blue complex is formed with a sharp absorption maximum at 595 nm (600 nm). In acid solution, the straight chain form of the deoxypentose is converted to highly reactive ω – hydroxylevulinaldehyde that reacts with diphenylamine to give blue complex. The reaction yields two mixture of product which together have absorbance maximum at 595 nm (600 nm). Interferences include proteins, lipids, saccharides and RNA. In DNA the only deoxyribose of the purine nucleotides reacts, so that the value obtained represents half of the total deoxyribose present.

Principle In this method the estimation of pentose sugar is carried out. Acid hydrolysis of RNA releases ribose (a pentose sugar) which in the presence of strong acid undergoes dehydration to yield furfural. Orcinol, in the presence of ferric chloride as a catalyst, reacts with furfural producing a green colored compound which can be quantitatively estimated at A665 nm. DNA gives a limited positive reaction with Orcinol test. Materials and Reagents Spectrophotometer Boiling water bath 5% chloroform (HClO4) Standard RNA solution : Dissolve yeast RNA (500ìg/ml) in 5% HClO4 . make different dilutions to obtain solutions containing 100 – 500ìg RNA/ml with 5% HClO4 . Orcinol reagent : Dissolve 100mg of ferric chloride (FeCl3.6H2O) in 100 ml of concentration HCl and then add 3.5 ml of 6% solution of Orcinol prepared in alcohol.

Electrophoresis is a method of separating out large molecule ( DNA or protein). An electric current is passed through a medium containing the molecules, and each molecule travels at a different rate depending on its electrical charge, size and shape. Separation by electrophoresis is based on these differences. In electrophoresis, agarose and acrylamide gels are used for electrophoresis of nucleic acids and proteins. What is Gel Electrophoresis? A gel is a solid form. The term electrophoresis describes the migration of charged particle under the influence of an electric field. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means “to carry across.” Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. A molecule’s properties determine how rapidly an electric field can move the molecule through a gelatinous medium.

Introduction Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the net charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique. SDS-PAGE is the most widely used method for qualitatively analyzing any protein mixtures. The method is based on the separation of proteins according to their size and then locating by binding them to a dye. It is particularly useful for monitoring protein purity and determines their relative mass. The SDS-PAGE enables the students to understand the theory behind the techniques in checking the purity of the protein and identifying the protein in a test sample. Separation of Proteins Proteins are amphoteric compounds; their net charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The net charge carried by a protein is in addition independent of its size - i.e.: the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules.

History of PCR A 1971 Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro. However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis. At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90°C (>195°F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures. So the early procedures for DNA replication were very inefficient, time consuming, and required large amounts of DNA polymerase and continual handling throughout the process. The discovery in 1976 of Taq polymerase (a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot (50 to 80 °C (120 to 175 °F) environments) paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation, thus obviating the need to add new DNA polymerase after each cycle. This allowed an automated thermocycler-based process for DNA amplification. Developed the PCR in 1984 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993 Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.

Introduction The primary tools used by the molecular biologists in manipulating DNA are restriction enzymes and other DNA/RNA modifying enzymes. Over the years, the number and uses of these enzymes have increased as new molecules have been discovered. Restriction endonucleases are bacterial enzymes that cleave double-stranded DNA. They are present in bacteria presumably to destroy DNA from foreign sources. By cleaving the foreign DNA at specific sites. The host bacterial DNA os protected from cleavage because specific recognition sites are modified, usually by methylation form methylase at one of the bases in the site, making the site no longer a substrate for RE cleavage. Host bacteria used to propagate cloned DNA in the laboratory arte usually mutant in the host restriction genes, thus their intracellular enzyme activities will not destroy the foreign recombinant sequences. The endonuclaase with its accompanying methylase is called a restriction modification (R-M) system. At least four different kinds of R-M systems exist characterized on the basis of the subunit composition, cofactor requirements and type of DNA cleavage. Most characterized enzymes belong to type II class, together with type IIS class. They comprise the commercially available restriction enzymes used for DNA analysis and manipulation. Type I and Type III enzymes are relatively uncommon and a few additional enzymes fit none of the classes. Among more than 3000 different enzymes isolated from bacterial strains many share common specificities. Restriction enzymes that recognize identical sequences have been called as isoschizomers.

DNA can be easily isolated and purified after size selection on an agarose gel. The fragment of interest is simply cut out of the gel with a razor blade and purified by a number of different methods.The easiest is to use a method that involves first dissolving the agarose slice in a solution at 50°C, then binding the DNA from the melted agarose to a silica-gel membrane. Principle In this method following gel electrophoresis using low gelling/ melting temperature agarose, the band of interest is removed from the gel. The agarose slice is then melted and subjected to phenol extraction.

Introduction This method can be scaled to yield amounts of DNA ranging from less than ten to more than hundreds of micrograms of DNA. However, shearing forces are generated at every step, with the result that the DNA molecules in the final preparation rarely exceed 100-150 kb in length. DNA of this size is adequate for Southern analysis on standard agarose gels, as a template in Polymerase chain reactions (PCRs), and the construction of genomic DNA libraries in bacteriophage Lambda vectors. Principle The method described in protocol involves digesting eukaryotic cells or tissues with proteinase K in the presence of EDTA (to sequester divalent cations and thereby inhibit DNases) and solubilizing membranes and denaturing proteins with a detergent such as SDS. The nucleic acids are then purified by phase extractions (isopropanol). Contaminating RNA is eliminated by digestion with an RNase, and low molecular-weight substances are removed by dialysis (or centrifugation).

By Alkaline Lysis Method Introduction DNA of prokaryotic cells is relatively simple in comparison to that of eukaryotic cells. In addition to chromosomal DNA, a bacterium may also carry an additional piece of DNA called a plasmid. Bacterial plasmids are double stranded closed circular DNA molecules that range in size from 1 kb to more than 200kb. Plasmids are useful to bacterial cells since they carry genes for antibody resistance, RM system etc that allow bacteria to survive in non-ideal conditions. Plasmids constructed in the laboratory are used as cloning vehicles. These synthetic plasmids contain only essential features such as origin of replication, antibiotic resistance, multiple cloning sites etc. various copy number of plasmid For e.g. pBR322 20 copy/cell – low copy number, pUC 18, pUC 18- 800 copy/cell – high copy number. Alkaline lysis, in combination with the detergent sds, has been used for more than 20 years to isolate plasmid DNA from E.coli (Birnboim and Doly 1979). This experiment describes how to extract plasmid from bacteria – which involves growth of bacterial culture, harvesting and lysis of bacteria and isolation of plasmid DNA from the lysate using alkaline lysis method. The DNA is analysed on agarose gel.

RAPD stands for Random Amplification of Polymorphic DNA. It is a type of PCR reaction, but the segments of DNA that are amplified are random. The scientist performing RAPD creates several arbitrary, short primers (8-12 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify. By resolving the resulting patterns, a semi-unique profile can be gleaned from a RAPD reaction. No knowledge of the DNA sequence for the targeted gene is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where. This makes the method popular for comparing the DNA of biological systems that have not had the attention of the scientific community, or in a system in which relatively few DNA sequences are compared (it is not suitable for forming a DNA databank). Because it relies on a large, intact DNA template sequence, it has some limitations in the use of degraded DNA samples. Its resolving power is much lower than targeted, species specific DNA comparison methods, such as short tandem repeats. In recent years, RAPD is used to characterize, and trace, the phylogeny of diverse plant and animal species.

Introduction The AFLP amplified fragment polymorphism also known as selective restriction fragment amplification (SRFA) technique is used to visualize hundreds of amplified DNA restriction fragments simultaneously. The AFLP band patterns, or fingerprints, can be used for many purposes, such as monitoring the identity of an isolate or the degree of similarity among isolates. Polymorphisms in band patterns map to specific loci, allowing the individuals to be genotyped or differentiated based on the alleles they carry. produces highly complex DNA profiles by arbitrary amplification of restriction fragments ligated to double-stranded adaptors with hemi-specific primers harboring adaptor-complementary 5' termini.The technique has been widely used in the construction of genetic maps containing high densities of DNA marker loci. The AFLP protocol amplifies restriction fragments obtained by endonuclease digestion of target DNA using “universal” AFLP primers complementary to the restriction site and adapter sequence. However, not all restriction fragments are amplified because AFLP primers also contain selective nucleotides at the 3' termini that extend into the amplified restriction fragments. These arbitrary terminal sequences result in the amplification of only a small subset of possible restriction fragments. The number of amplified fragments can therefore be “tailored” by extending the number of arbitrary nucleotides added to the primer termini. Alternatively, the use of endonuclease combinations that vary in their restriction frequency can also be used to tune the number of amplicons. Generally, the abundant restriction fragments produced from complex genomes require of AFLP primers with longer selective regions. Conversely, analysis of small genomes require of only few arbitrary nucleotides added at the primer 3' termini. The resulting AFLP fingerprints are usually a rich source of DNA polymorphisms that can be used in mapping and general fingerprinting endeavors.

Introduction Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome. The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing. This diagram shows the basic steps involved in a Southern blot.

Introduction Ligation of DNA fragment is one prime important in genetic engineering because without ligation we could not transfer the interest gene.DNA cloning requires the DNA sequence of interest to be inserted in a vector DNA molecule. For this, both the vector as well as insert DNA is prepare by digestion with compatible restriction enzymes, so that the ends produced during digestion is complementary in both. When setting up ligation, it is important to consider the permutations that can occur and bias the relative concentration of DNA accordingly. Usually a 5- to 10-fold excess of insert over the vector DNA is the norm. This ensures that enough ligated product will be produced in the right orientation.

To Prepare the Competent Cell and Carry Out Transformation Introduction Transformation results from the uptake of purified DNA by bacterial cells and is the most frequently used procedure for introducing recombinant DNA molecules into bacteria. It is now widely used to transfer small plasmids from one bacterial strain to another. DNA is extracted from donor cell and used to treat the recipient cells that have been rendered more susceptible to DNA uptake. These competent cells allow DNA to enter through pores or channels in the cell membrane and in the case of plasmids, permit subsequent plasmid replication.

Introduction The most common method of bacterial transformation utilizes high levels of calcium chloride to facilitate the entry of plasmid DNA vectors into E.coli in conjunction with structural alteration of the bacterial cell wall. In general, there are three basic steps in the introduction of plasmid DNA unto cells. Preparation of “competent” cells to enable cells to accept DNA. Transformation of these competent cells, and Selection of transformants. Most E.coli strains exhibit transformation efficiency (measured as transformants per microgram plasmid DNA) in the range of 105 to 108. The factors influencing this efficiency depends upon the condition that render cells competent.

Introduction Genetic variability is essential for the evolutionary success of all organisms. In diploid eukaryotic the processes of crossing over exchange of genetic material between homologous chromosomes and meiosis contributes to his variability. In haploid, asexually reproducing prokaryotic organisms, genetic recombination may occur by conjugation transduction, and transformation. In this experiment only the process of conjugation is considered. Conjugation is a matting process during which a one direction transfer of genetic material occurs at physical contact between two sexually differentiated cell types. This differentiation or existence of different mating strains in some bacteria, is determined by the presence of a fertility factor of F factor, within the cell. Cells that lack the F factor are recipients (females) of the genetic material during conjugation and are designated as F- . cells possessing the F factor have ability to act as genetic donors (males)during the mating. If this F factor is extra chromosomal (a Plasmid or episome), the cells are designated as F+; most commonly only the F factor is transferred during conjugation. If this factor becomes incorporated into the bacterial chromosome there is a transfer of chromosomal genes, although generally not involving the entire chromosome or the F factor. The resulting cells are designated as Hfr, for high frequency recombinants.

Introduction Mutation refers to any heritable change in the base sequence of a DNA molecule. A mutagen is a physical of chemical agent that causes mutations to occur (or increase the frequency of their occurrence). Mutagenesis is the process of producing a mutation. If the mutation occurs in nature without the addition of a mutagen, it is referred as a spontaneous mutation, and the resulting mutants are spontaneous mutants. If it caused by a mutagen, the process is induced mutagenesis. Ultraviolet light is a fairly potent mutagen. The main DNA lesions are two chemically different types of covalent dimers: cyclobutane-pyrimidine dimers and pyrimidine dimers. The pyrimidine dimers only account for 20% of the total dimers but are correspondingly more mutagenic. In E. coli the number of mutants induced by ultraviolet (UV) light can be reduced by exposure to visible light (photoreactivation), which implicates cyclobutane pyrimidine dimers since dimers can not be photoreactivated. Aromatic amino acid of proteins, and purine as well as and pyrimidine bases of nucleic acid absorb ultra violet radiation. Pyridine bases, particularly thymine of DNA is the main target compound involved in bactericidal action of ultra violet radiation. The energy absorbed by thymine causes it to under go a photochemical reaction with adjacent ( adjoining) thymine molecule in same DNA strand. DNA replication is blocked because the formation of thymine: thymine (T:T) dimmers. As the dosage increase, cytosine-thymine (C:T) to (C:C) cytosine-cytosine dimmers can be found to add the lethality of ultra violate radiation.

Introduction The isolation of mutants is often critical to the study of cellular process, especially when such studies are performed at the molecular level. The phenotype of a particular mutant is attributable to the loss of a particular cellular function; the loss of that function is actually attributable to a mutation in a gene necessary for that function. In this experiment we will isolate auxotrophic mutant which can grow only if provided with specific amino acid. For example arg- mutant have lost the catalytic function of an enzyme involved in arginine biosynthesis due to mutation in the structural gene that encodes the enzyme. Mutagenic Agent Mutation is an alteration in the coding sequence of a gene. Mutation occur spontaneously at a frequency of about 10-8 per bacterial cell per generation. The rate of mutation can be increased with mutagenic agents. Mutants are strains with any structural alteration of their DNA that gives rise to a mutant phenotype. Mutagens are chemical agents (e.g. nitorsoguanidine, 5 bromouracil), physical (e.g.UV) and biological agents (e.g. trnasposons) that cause mutation to occur.

To isolate a streptomycin resistant mutant of E.coli by Direct selection using Gradient Plate Method. Introduction Zylbalskii devised this technique. A mutation is any change in the nucleotide sequence of DNA. These changes are heritable. Although most mutations are harmful to the organism, a small percentage is beneficial under certain environmental conditions. Mutation can occur during chromosome duplication and they may be spontaneous of they may be induced by mutagenic agent. In this experiment, we select for a mutation causing streptomycin resistance in E. coli. Resistant mutants will be detected directly using thy gradient plate method. Principle Microbiologist developed a highly efficient method for isolation of antibiotic resistant mutant. The gradient is said as gradient of antibiotic formed on an agar plate. A gene imparting resistant toward an antibiotic is present in plasmid or chromosome. The occurrence of mutant with different level of streptomycin resistant involves mutation of gene with unequal potential. This means that under certain cells degree of mutation is more while few shows less mutation. Some cells show intermediate resistant while others show completes resistant. The resistant in cell against streptomycin of this type, while in case of penicillin resistant increases step arise & so resistant mutant of high concentration of penicillin cannot be isolated/ obtained. While it is possible with streptomycin obtaining high resistance to penicillin stepwise & so this stepwise increase in resistance involves mutation of gene with equal potential. This theory of mutation through multi step procedure. The gene direct the organism to produce an enzyme, which inactivates an antibiotic & thus resistance to antibiotic, is observed in case of cells. Resistance of antibiotic is a plasmid-mediated resistance.

To isolate a streptomycin resistant mutant of E.coli by Indirect selection using Replica Plate Method. Introduction J. Lederberg & his wife E. M. Lederberg devised this technique in 1952. U.V. light coming from the sun emits strong radiation to extent the antibacterial effect in the microbial population. However it has a very short penetration power, UV light is used as a mutagenic agent & was used by Beedle & Tatum to obtain biochemical mutation. The mutation rate is only once in 10-9 cells per population. Then hence using mutagens like UV light by 10 to 1000 folds will increase this mutation rate. In isolation of the mutant using UV light the dose of UV light is measured by its units/nm2 organisms. The time selected for the mutation is calculated on the basis of 0.1 to 1% survival & a suitable biochemical character is used to evaluate the mutation.

Limitations of Bacteria Normally genetically engineered bacteria are very useful for the large scale production of valuable eukaryotic proteins. Bacteria grow rapidly, They grow on relatively simple media, they are easy to handle, they are easily transformed, they can grow on wide range of substrates, they multiply, fast, they contain simple and well characterized genome, they can be easily modified but even though, they have their own problems… Bacteria do not have intrudes and so they do not have any system or machinery to remove introns. Many eukaryotic proteins undergo different types of modifications after they are produced. These modifications are known as “post translation al modifications”. Each bacterial cell usually contains many copies of the plasmid but there is no mechanism to ensure the equal distribution of plasmids in daughter cells.

Introduction Western blot analysis can detect one protein in a mixture of any number of proteins while giving you information about the size of the protein. It does not matter whether the protein has been synthesized in vivo or in vitro. This method is, however, dependent on the use of a high-quality antibody directed against a desired protein. So you must be able to produce at least a small portion of the protein from a cloned DNA fragment. You will use this antibody as a probe to detect the protein of interest. Western blotting tells you how much protein has accumulated in cells. If you are interested in the rate of synthesis of a protein, Radio-Immune Precipitation (RIP) may be the best assay for you. Also, if a protein is degraded quickly, Western blotting won’t detect it well; you’ll need to use (RIP). See the section on RIP for more information, as well as a helpful comparative chart that illustrates the differences between these two techniques.

Introduction The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence. The term ‘northern blot’ actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane; however the entire process is commonly referred to as northern blotting. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot, named for biologist Edwin Southern. The major difference is that RNA, rather than DNA, is analyzed in the northern blot.

Sanger Method for DNA Sequencing One necessity first know a little about the structure of DNA: A segment of DNA, which is ordinarily double stranded, has a specific orientation, as it has a 5' (read as “5 prime”) and a 3' (“3 prime”) end. This can be simply thought of as a front and tail end to the DNA segment. When DNA is synthesized in the lab, the two strands are separated and new bases are added to the 3' end-thus DNA is assembled from the 5' to 3' end. DNA cannot be synthesized from scratch. A short piece of DNA, called a primer, is required for the reaction to begin. Primers are designed such that they are able to bind to the target DNA, the binding of which is the initiator for DNA synthesis.

Metagenomics is a relatively new field that combines molecular biology and genetics in an attempt to identify, and characterize the genetic material from environmental samples and apply that knowledge. The genetic diversity is assessed by isolation of DNA followed by direct cloning of functional genes from the environmental sample. But more importantly, it is extremely beneficial in the study of synthetically made genomes. Metagenomics is described as “the comprehensive study of nucleotide sequence, structure, regulation, and function”. Scientists can study the smallest component of an environmental system by extracting DNA from organisms in the system and inserting it into a synthetic model organism. The model organism then expresses this DNA where it can be studied using standard laboratory techniques. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent thevast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that avoided the bias imposed by culturing and led to the discovery of vast new lineages of microbial life. Metagenomics is employed as a means of systematically investigating, classifying, and manipulating the entire genetic material isolated from environmental samples. This is a multi-step process that relies on the efficiency of four general steps as shown in the diagram. The procedure consists of: the isolation of genetic material, the manipulation of the genetic material, library construction, and the analysis of genetic material in the metagenomic library. Metagenomics provides a second tier of technical innovation that facilitates study of the physiology and ecology of environmental microorganisms. Novel genes and gene products discovered through metagenomics include the first bacteriorhodopsin of bacterial origin; novel small molecules with antimicrobial activity; and new members of families of known proteins, such as an Na+(Li+)/H+ ant porter, RecA, DNA polymerase, and antibiotic resistance determinants. Reassemblyof multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and micro heterogeneity, and lateral gene transfer among members of an uncultured community. The application of metagenomic sequence information will facilitate the design of better culturing strategies to link genomic analysis with pure culture studies.

NTSYSpc 2.0 software is used for various types of analysis including calculation of similarity index values and construction of dendrogram. Construction of dendrogram using gel data in the form of bands. This software accepts zero-one data.

Glossary Abiotic factors : Non living; moisture, soil, nutrients, fire, wind, temperature, climate Absorption : Movement of ions and water into as organism as a result of metabolic processes, frequently against an electrochemical potential gradient (active) or as a result of diffusion along an activity gradient (passive). Absorption field : A system of properly sized and constructed narrow trenches partially filled with a bed of washed gravel or crushed stone into which perforated or open joint pipe is placed. The discharge from the septic tank is distributed through these pipes into trenches and surrounding soil.While seepage pits normally require less land area to install, they should be used only where absorption fields are not suitable and wellwater supplies are not endangered. Acetogenic bacterium : Prokaryotic organism that uses carbonate as a terminal electron acceptor and produces acetic acid as a waste product. Acetyleneblock assay : Estimates denitrification by determining release of nitrous oxide (N2O) from acetylenetreated soil.