Pseudomonas Aeruginosa Gene Expression

Pseudomonas Aeruginosa Gene Expression Pseudomonas aeruginosa is the gram negative rod shaped bacteria is found in all natural and man made environments. the organism is so stubburn so that it can grow in any environment and also at high temperatures such as 420C. The unique ability of the organism is forming biofilm. The biofilms are formed by the organism with the association with a surface. The surface may be anything such as cloth, paper, glass etc and any artificial surface. It is the opportunistic pathogen which behaves neutral to the normal tissues but it infects once the defences of them are compromised. The development of the biofilm starts with the attachment of the freely moving planktonic cell to a surface. Generally the twitching motility of the P.aeruginosa helps to use its unipolar flagellum to adhere a surface. The bacterium develops colonies known as microcolonies and adhere to the surface strongly which are known as pili structures (Baron S et al.,1996). During the biofilm development several factors pla ys a key role mainly the exopolymeric matrix. The EPS matrix is not visualised directly, but it can be done by using some fluorescent dyes. The functioning of the EPS matrix in holding cells of Pseudomonas aeruginosa together and forming the mature biofilms. The biofilm develops with cell division and recruitment. The cells interacts via quorum sensing and these interactions are studied under scanning electron microscopy. The biofilm formation takes place in stages and many changes are observed in its size and shape during the development. The EPS matrix is made up of polysaccharides, proteins and nucleic acids. The exopolysaccharide matrix is the important component of the biofilm. The exopolysaccharide genes such as pilB, pslA, lecA, ureB and alginate genes are responsible for the formation of pseudomonas biofilms. The locus of the psl polysacchide genes encode the cells to adhere to a surface and thus maintaining the biofilm structure. In this research project we try to quantify the gene expression of the above genes pilB, pslA, lecA, ureB using rpoD as the house keeping gene. The biofilms are grow invitro on microcarriers. The microcarriers made up of dextrons in the range of 60-90 µm in size are used as a surface for Pseudomonas aeruginosa to form biofilms. The microcarriers are spherical in size where the bacteria can easily adhere on surface. In this present work we try to grow planktonic cultures and biofilms cultures on the microcarriers in a fermentor separately. The genes responsible for the biofilm formation are quantified by comparing the planktonic and biofilm samples. The planktonic cells grow very rapidly whereas the bacterial adhering takes some time to form biofilm in other case. The samples are collected at regular intervals and mRNA is extracted. The gene expression is quantified using real time PCR. The relative quantification method of qPCR or real time PCR method does not require any standard curve for the analysis where the samples are normalised by using an internal control. The threshold CT values obtained from the curve are used to calculate relative quantification by comparative à ¢Ã‹â€ Ã¢â‚¬  Ãƒ ¢Ã‹â€ Ã¢â‚¬  CT method. The results from the planktonic and biofilm gene expression results are compared. On the other hand we also assay the nutrients such as glucose, ammonia, phosphorus and protease during the fermentation. The concentration levels of the nutrients are also compared for both biofilm and planktonic fermentation. Review of literature: Pseudomonas aeruginosa is a member of gamma proteobacteria class of bacteria. The bacteria is gram negative, rod shaped and aerobic belongs to the bacterial family of pseudomonadanceae. The recent developments in the taxonomy based on the 16s ribosomal RNA or conserved macromolecules, the family members are divided into eight groups. Pseudomonas aeruginosa is a species present in this group sharing the with other twelve members. Pseudomonas aeruginosa is a free-living bacterium, commonly found in soil, water, skin flora and in all man made environments. The organism regularly occurs on the surfaces of the plants and animals. Pseudomonas aeruginosa is an occasional pathogen to plants. It is also an increasingly emerging pathogen of clinical relevance. The organism not thrives in the normal atmosphere but also in the places with little oxygen and thus forms the colonies in natural and artificial environments. Pseudomonas aeruginosa is an opportunistic pathogen for humans, which means that it exploits some break in the host defences to initiate an infection. The bacteria never infects the uncompromised tissues, but it can infect any if the tissue defences are comprised. It can cause infections such as urinary tract, respiratory system infection, soft tissue infection dermatitis, gastro intestinal infections. It causes a variety of systemic infections in patients particularly with severe burns and immune suppressed in cases of cancer, AIDS, cystic fibrosis in lungs. Characteristics Pseudomonas aeruginosa is rod shaped gram negative bacteria. The size of it ranges between 0.5 to 0.8  µm by 1.5 to 3.0  µm. almost all the strains of Pseudomonas are motile with a unipolar flagellum. It is ubiquitous in soil ,water and on the surfaces in contact with water or soil. It has respiratory metabolism and can grow in the absence of oxygen if NO3 is available as respiratory acceptor for electron. Generally the bacterium is found in nature in the form of biofilm attached to a surface or substrate, or in the planktonic form as a unicellular organism swimming actively with its unipolar flagellum. It is one of the most vigorous and fast swimming organism found in pond waters. It can grow at temperatures at 42OC or high, but the optimum temperature is 30 to 37OC. it is resistible to a wide variety of physical conditions such as temperature and can even resist the salts, dyes, antiseptics and most commonly used antibiotics. Pseudomonas aeruginosa produces different types of colonies from the source they have isolated. They appear like small and rough colonies. Moreover the samples collected from the clinical sources look like a fried egg like structure which is smooth, large with elevated appearance. The other type of colonies obtained from the respiratory and urinary tract secretions looks like mucoid.this mucoid appearance is attributes for the production of alginate slime. These smooth, mucous colonies presumably plays an important part in the colonization and virulence. Pseudomonas aeruginosa produces two types of soluble pigments. Pyocyanin which is a blue-green and a yellow-green and fluorescent pigment called as pyoverdin. Pyocyanin is produced abundantly in the media with low levels of iron and also functions for the iron metabolism of the organism. Many strains of Pseudomonas aeruginosa produces pyocyanin but not all the strains does it. Although the organism is classified as the aerobic, sometimes the organism is considered as facultative anaerobe where it can adapt and proliferate the conditions with less or total O2 depletion. The organism can grow in the absence of oxygen if NO3 is available as respiratory acceptor for electron. It can ferment arginine by substrate level phosphorylation where the layers of alginate present surrounding the mucoid cells of the bacteria controls the diffusion of oxygen(Collins FM, 1955), this is witnessed in the cystic fibrosis of the lung infection. Pseudomonas aeruginosa which is found in the biofilm form is responsible for a variety of activities involved which are natural and artificial. Resistance to antibiotics Pseudomonas aeruginosa is opportunistic pathogen, has a notorious resistance to antibiotics. The organism is very resistance towards many antibiotics due to the potential permeability barrier provided by the gram negative membrane. Pseudomonas aeruginosa in the biofilm form makes the cells impervious to the concentrations of the therapeutic antibiotic. The organism gets associated with others like actinomycytes, moulds and bacillus because of the natural environment they live. Moreover the organism have antibiotic resistance plasmids R factors and RTFs, the genes are able transferred by conjugation and transduction following the mechanism horizontal gene transfer(HGT) (Todar.K,2008). Biofilms Biofilms are defined as a matrix enclosed bacterial populations which are adherent to each surfaces or interfaces. Microbial aggregates and floccules and also adherent populations within the pore spaces of the media are spoken in the definition (Costerton J.W, et al 1995).bacterial biofilms came into existence and became significant from their first recognition in their ubiquity. The past researches show that profoundly the growth phase of the biofilms is distinct from the planktonic. In response to the surfaces proximity, bacterial cells alter their phenotypes (Costerton J.W, et al, 1995). The early stages of biofilm formation the bacteria is in stable juxta position with those of same and other species. The micro environmental conditions, cellular juxtapositions and the extra cellular polysaccharide matrix constitute to the development of the bacterial biofilm. Different bacteria responds to the specific environmental conditions with certain growth patterns, and a structurally comp lex and mature biofilm develops(Hamilton W.A et al, 1987). Physiological cooperation is the major criteria in shaping structure and juxta positions establishment to make biofilms adherent for the surfaces. It has been proposed that large suites of genes are expressed differentially regulated. Formation The formation of the biofilm initially starts with the attachment of the freely floating bacterial cells to a surface. The bacterial colonies adhere to the surface with weak vander wall forces. These colonies can lead themselves to more adhesion called as pili structures (Baron S et al.,1996). The organism adheres more to the available surface by binary fission, exopolymeric matrix is produced to develop mature biofilms. The matrix produced holds the biofilm together. Biofilm develops with cell division and recruitment, the cell in the biofilm communicate via quorum sensing. We can observe changes in shape and size in the final stages of biofilm formation. Distribution and ubiquity of biofilms The general phenotypic strategy of organism is to change in the mode of growth that to adopt to the altering growth conditions. The cells of Pseudomonas aeruginosa which is a ubiquitous bacterial species respond to all favourable nutrient conditions. The organism adheres to the available surface by binary fission, exopolymeric matrix is produced to develop mature biofilms. The rod shaped bacteria grows in matrix which is enclosed in a sessile growth mode. The cells are protected from the biological, antibacterial, chemical agents and adverse environmental conditions. The periodically shed planktonic cells from the biofilm provide and sacrifice the protection to the new fresh habitats can colonize with the biofilm. The adherent biofilm populations has a significant metabolic activities( Fletcher.M, 1986). The extent of biofilm formation is predicted in the particular system is based on the following principles: Metabolically active bacteria show tendency towards the surfaces for adhesion in all natural environments. In all environmental conditions the extent of biofilm formation is controlled by the available nutrient for cell respiration and exopolysaccharide production. The organic nutrients sometimes tend to associate with the surfaces, in such cases the local biofilm is difficult to trigger. This is more in extremely oligotrophical environments, but bacteria normally does not adhere to surfaces in nutrient less environments. Pseudomonas aeruginosa Biofilms The communities of microbial bacteria(Pseudomonas aeruginosa) forms a definite structures with the association with surfaces are abundantly available environment. These structures are often called as biofilms which are less susceptible to antimicrobial agents. These biofilms causes chronic infections and very difficult to remove. The extracellular polymeric matrix(EPS) are known to holds the biofilm structure and it performs as a matrix. The EPS matrix holds the biofilm cells together and protects them from shear forces in fludic environments. The extra cellular matrix for Pseudomonas aeruginosa forms the biofilm development in different stages. The matrix holds the cells together on the surface and forms a critical structural design resists to many environmental changes. Pseudomonas aeruginosa biofilm develops in a five stage multicellular cycles, the process is initiated by the attachment of planktonic (free) cells to a surface. This is followed by the formation of microcolonies. T he microcolonies are seeded and gets dispersed where the cells form the microcolonies comes out to occupy a new surface( Ma. L et al, 2009;Stoodley.P et al,2002). The EPS of Pseudomonas aeruginosa composed of polysaccharides, proteins and nucleic acids. It is the important component of the biofilms. The exo polysaccharide genes lec A , Pil B, Psl A, alginate are responsible for the formation of Pseudomonas biofilms. The locus of the Psl exopolysaccharide encodes bacterial cells to surface and thus maintaining the biofilm structure. The matrix which is anchored by the Psl is not visualised directly during the developmental stages of Pseudomonas aeruginosa biofilm. In addition to the present scenario of exopolysaccharides, extracellular DNA also plays an important part in . P.aeruginosa biofilm matrix (Allensen-Holm M et al, 2006). The extracellular DNA is derived from the chromosomes and it functions for signalling between the cells interconnecting the biofilm matrix. In the biofilm matrix, extracellular cellular DNA promotes cation gradients, release of genomic DNA and induces antibiotic resistance ( Mulcahy H, et al. 2008). Real time PCR Polmerase chain reaction or simply PCR is introduced by Kary mulis in 1980, which really a revolution in the molecular biology. PCR is the standard procedure to amplify small quantities of DNA molecule into large quantities. The method depends on thermal cycling which involves the continuous heating and cooling of reaction for DNA melting and enzymatic replication of DNA. PCR applications employ a general heat stable DNA polymerase such as Taq polymerase enzyme isolated from the bacterium Thermus aquaticus. PCR method is involves three major steps.1) Denaturation 2) Annealing 3) Elongation. In the first step DNA template is denaturated to single strand at high temperature of 940C. In the second step, single stranded DNA template annealed with primers, the temperature is 650C. At 720 C the DNA starts elongation which is third step. DNA polymerase synthesizes new complementary strand to the template DNA strand. Real time polymerase chain reaction or qualitative polymerase chain reaction is a laboratory technique which not only amplifies but also quantifies the target DNA molecule. it helps the user for detection and quantification(absolute and relative) of many specific genes in a present in a given DNA sample. The general principle of polymerase chain reaction (PCR) is also used in the RT PCR. Unlike PCR, RT PCR is a new technology product is detected at the end of the reaction. There are two common methods for the product detection in RT PCR 1) Non -specific fluorescent dyes which interacts with the double stranded DNA molecule 2) Sequence specific DNA probes consists of oligonucleotides that are fluorescently labelled with reporter. The product is detected after the hybridization of the probe and complementary strand. The RT PCR is used to amplify the reversly transcribed cDNA products from the mRNA, which is becoming the effective and powerful tool in the field of molecular biology to quantify and study the gene expression. The RT PCR method is very easy to apply provides the reliable, accuracy and rapid gene expression quantification. The accurate nucleic acid quantification requires mathematical application for data analysis(Michael W.P., 2001). The real time PCR provides the very accurate and reproducible quantification of gene copies (Heid C.A., 1996). Unlike other PCR methods, RT PCR does not require post process sample handling, prevents product contamination, provides faster and accurate assay results. Real time PCR is very accurate and less labour usage than the present gene quantification methods. Nucleic acid sequence quantification has an important role in the biological research. Measuring the gene expression have been extensively used in the biological responses to various external s timuli( Tan et al, 1994; Huang et al,1995). Gene and genome quatification also used for HIV demonstrates the change in the expression level of virus in the disease period( Conner et al. 1993); (piatak et al, 1993). There are two types of quantification methods used in real time PCR: Absolute quantification Relative quantification Absolute quantification determines the input copy number generally by relating the PCR signal to a standard curve. In this method equal input amounts of sample, each sample under analysis amplifies with identical efficiency up to the point of qualitative analysis( Heid et al,1996). We quantitate the unknowns based on a known standard quantity. The unknown samples are compared to the known by creating a standard curve. The input copy number of the PCR signal is related to standard curve in absolute quantification method. Sometimes it is not necessary to know the copy number but it is important to calculate the relative change gene expression (Livak.K.J et al, 2001). The second method is relative quantification, we analyse the changes in gene expression in a given sample relative to another reference sample(untreated control sample). Relative standard again consists of two types of methods they are relative standard curve and comparative Ct method. In the relative quantification the PCR signal is related to the targeted transcript of the samples in the group. The new applications of comparative Ct method is the 2-à ¢Ã‹â€ Ã¢â‚¬  Ãƒ ¢Ã‹â€ Ã¢â‚¬  Ct method which is efficiently useful for the analysis of qPCR data. The 2-à ¢Ã‹â€ Ã¢â‚¬  Ãƒ ¢Ã‹â€ Ã¢â‚¬  Ct method(Livak.K.J et al , 2001): Derivation of 2-à ¢Ã‹â€ Ã¢â‚¬  Ãƒ ¢Ã‹â€ Ã¢â‚¬  Ct method: The equation for the amplification of PCR can be written as: Xn=X0 (1+Ex)n(1) Where Xn is the number of target molecules at cycle n of the reaction. X0 is the initial number opf target molecules. Ex is the efficiency of the target amplicon. n is the number of cycles CT is the threshold cycle, indicates the fractional cycle at which the amount of amplified target reaches a fixed threshold. XT =X0 (1+Ex)CT.X=Kx(2) Where XT is the threshold number of target molecules. CTX is the threshold cycle for target molecule and Kx is constant. A similar reaction for the internal control gene is RT =R0 (1+ER)CTR=KR-(3) Where RT is the threshold number of reference molecules R0 is the initial number of reference molecules ER is the efficiency of the reference molecule. CT.R is the threshold cycle for reference amplicon. KR is a constant. Dividing XT and RT, we get , (XT/ RT )= (X0 (1+Ex)CT.X) / (R0 (1+ER)CT.R )= (Kx / KR ) = K-(4) For real time amplification using Taqman probes the exact values of the XT and RT depend upon the number of factors including the reporter dye and fluorescence properties of the probes, efficiency and purity of the probe, florescence threshold setting. Thus k cannot be equal to one. Assume the efficiencies of target and the reference as equal. ER = Ex=E (X0 / R0) (1+ E) CT.X- CT.R =K(5) XN (1+E)-à ¢Ã‹â€ Ã¢â‚¬  CT =K-(6) Where XN is equal to the normalised amount of target gene (X0 / R0) and à ¢Ã‹â€ Ã¢â‚¬  CT is the difference of the threshold cycles of target and reference (CT.X- CT.R ). The expression can be written as XN=K(1+E) -à ¢Ã‹â€ Ã¢â‚¬  CT -(7) If we divide the whole equation by XN for any sample q by XN as the calibrator(cb) XN.q /XN.cb ={[ K(1+E) -à ¢Ã‹â€ Ã¢â‚¬  CTq ]/[ K(1+E) -à ¢Ã‹â€ Ã¢â‚¬  CTcb ]} =(1+E) -à ¢Ã‹â€ Ã¢â‚¬  Ãƒ ¢Ã‹â€ Ã¢â‚¬  CT Here -à ¢Ã‹â€ Ã¢â‚¬  Ãƒ ¢Ã‹â€ Ã¢â‚¬  CT = -(à ¢Ã‹â€ Ã¢â‚¬  CTq -à ¢Ã‹â€ Ã¢â‚¬  CT.cb) For amplicons which are designed less than 150 bp, primer and magnesium concentration are optimised, the efficiency is nearly equal to 1. Therefore the amount of target gene, normalized to an endogenous reference which is relative to a calibrator, taken as Amount of target =2-à ¢Ã‹â€ Ã¢â‚¬  Ãƒ ¢Ã‹â€ Ã¢â‚¬  CT . When the threshold is set for n standard deviations in the plot , above the mean of the base line emission from cycles 0 to n. The standard deviations is calculated from the data attained from base line of the amplification plot. When the fluorescence exceeds the threshold limit, CT values are determined at that point. The CT values are ten times more than the standard deviation of base line. The point at which amplification plot crosses the threshold can be defined as CT (Heid et al, 1996). Materials and methods Identifying pseudomonas aeruginosa Gram straining: Prepare a small amount of smear on a clear glass slide. Heat fix the smear to slide. Add a drop of crystal violet on to smear allow for 60 seconds. Rinse the slide with water. Add a drop of grams iodine to the smear allow for 60 seconds. Rinse the slide with water. Add 95% absolute ethanol to destain the crystal violet. Rinse again with water. Add a drop of saffronin over the smear, allow for 60 seconds. Rinse with water. Blot dry the slide, do not rub. Observe the smear under microscope. 10. If the colour of the bacteria appears pink it is gram negative, it is gram positive if it appears purple. Rod shaped pink coloured bacteria is clearly observed for gram negative pseudomonas aeruginosa. Shake flask method Prepare media of nutrient broth about 13 grams per litre of distilled water. Mix well and pour 50 ml of media into a 250ml conical flask. Plug the conical flask and wrap the mouth with aluminium foil. The less volumes of media in the flask increases surface area and oxygen transfer rate(OTR). The flasks are autoclaved at 1210 C and 1.5 lbs pressure for 1 hour 15 minutes. Planktonic-contains 50 ml of nutrient broth Biofilm-contains 25ml of PBS with microcarriers plus 25 ml of nutrient broth. Inoculate a loopful of pseudomonas aeruginosa into 50 ml nutrient broth aseptically. Allow the bacteria to grow overnight. Take the optical density at 550nm using spectrophotometer.The amount of the inoculums to be added to the shake flasks for analysis is calculated using the formula below: Amount of sample to add into shake flask = [working concentration X volume(ml)] / stock(OD) obtained After inoculation the flasks are kept in the orbital shaker at 300C. The samples of 1 ml is collected for RNA extraction at particular time intervals at 0,1,2, 4,24 and 48 hours for planktonic. In respect the biofilm samples are collected at time 0,22,24,26,44,46 and 48 hours as their growth is slower than the freely suspended planktonic cells. Then 2 ml of RNA later solution is added to the collected samples for RNA stabilization. The optical density is regularly monitored to check the growth of pseudomonas aeruginosa . Fermentation The fermentation process we perform here is batch fermentation. The bioreactor used for the study is a small scale fermentor from APPLICON BIOTECHNOLOGIES. The process will take place in a 3L fermentor(consists the working volume of 2.5L) for about 24 hours. During the run the criteria of temperature is kept at 300C . The pH is run free, stirrer speed is set at 600rpm for planktonic culture. The fermentor is run free for 24 hours overnight to check the production of foam. The cold finger helps to control the temperature by feeding water from/to the reactor. The o ring is checked for any leakage. The sample port is a tube for sampling using back pressure and forces the liquid into a glass recipient. The inlets for acid, alkali and antifoam are linked to durham bottles, going through the pumps. The durham bottles are blank, because above parameters are not controlled in this process. Air is pumped through a filter. The condenser is connected to water that comes out of the fermentor. Wh en the water is warmer than outside, condensed water reaches the condenser. The gas goes from an outlet connected to the condenser. The dissolved oxygen probe is connected to dO2 cable from the machine. It measures the amount of dO2 in the media. This probe needs to be kept in the buffer, when not in use. The probe should be plugged for 10 to 12 hours before the inoculation inorder to polarize. The stirrer is placed on the top of fermentor. The vessel is heated with the help of heating pad and temperature is detected using temperature probe. The air inlet is set up through the pumps and its volume is controlled with a rotometer in front of the machine. In this case 2.5L of air/min is supplied to the fermentor. For the gas analysis of oxygen and carbon dioxide the gas is passed through a drying column and reaches the monitor which is already set up for calibration. we run two runs of fermentor for both planktonic and biofilm cultures. The parameters such as temperature, aeration for planktonic and biofilm cultures are almost same. The only parameter varies for both is stirring is maintained at 600rpm for planktoni c and 200rpm for biofilm fermentation. Procedure: The fermentor is sterilised and all the valves are sealed with foil before going into the autoclave. The fermentor is run for about 24 hours before adding the inoculum to check any formation of foam. About 20 ml of an overnight culture is added to the fermentor. Sampling :About 7ml of sample is collected for every 20min for planktonic and every 2 hours for biofilm until the exponential phase is reached. The sample collected is preserved in the freezer for different analysis. The sample is assayed for gene expression by adding 2 ml of RNA later solution for RNA stability. The samples are assayed for ammonia, phosphorus, glucose and protease. The fermentation conditions are listed below. RNA EXTRACTION (protocol provided by applied biosystems) Materials required: 2-mercapto ethanol,100% ethanol, 10% SDS(in RNase free water), 0.5  µl/sample. Lysozyme solution: 100  µl/ sample 10mM Tris HCl (PH 8.0)mM EDTA 10mg/ml lysozyme (in RNase free water) Lysis buffer: Before beginning the lysis and homogenization steps, prepare a fresh amount of lysis buffer containing 1% 2-mercapto ethanol for each purification procedure. Add 10 µl 2-mercaptoethanol for each 1ml lysis buffer. Use 350  µl of freshly prepared lysis buffer for 1109 bacterial cells. Lysis and homogenization: Harvest 1109 bacterial cells and transfer them to an appropriately sized microcentrifuge tubes. Centrifuge at 500xg for 5 minutes at 40C to pellet cells. Discard the supernatant. Add 100  µl of prepared lysozyme solution to the cell pellet and resuspend by vortexing. Add 0.5  µl 10% SDS solution vortex to mix well. Incubate the cells in the tube for 5 minutes at room temperature. Add 350  µl lysis buffer prepared with 2-mercaptoethanol. Vortex to mix well. Transfer the lysate to a homogenizer inserted in an RNase free tube and centrifuge at 12,000xg for 2 minutes at room temperature. Remove the homogenizer when done. Purifying the RNA from bacterial cells: These steps are followed to bind wash and elute the RNA from the P.aeruginosa cells sample. Add 250  µl 100% ethanol to each volume of bacterial cell homogenate. Mix thoroughly by vortexing to dispersing any visible precipitate that may form after adding ethanol. Transfer the sample(including any remaining precipitate) to a spin cartridge( with a collection tube. Centrifuge both the spin cartridge and collection tube at 12,000xg for 15 seconds at room temperature. Discard the flow through and re-insert the spin cartridge in the same collection tube. Add 700  µl of wash buffer I to the spin cartridge. Centrifuge at 12,000xg for 15 seconds at room temperature. Discard the flow through and the collection tube. Place the spin cartridge into a new collection tube. Add 500  µl wash buffer II with ethanol to the spin cartridge. Centrifuge at 12,000Xg for 15 seconds at room temperature. Discard the flow through and re insert the spin cartridge into the same collection tube. The steps are repeated once again. Centrifuge the spin cartridge and collection tube at 12,000Xg for 1 minute at room temperature to dry the membrane with attached RNA. Discard the collection tube and insert the spin cartridge into a recovery tube. Add 50  µl of RNase free water to the centre of spin cartridge. Incubate at room temperature for 1 minute. Centrifuge the spin cartridge and recovery tube for 2 minutes at 12,000Xg at room temperature. Store the purified RNA or proceeded for DNase treatment after RNA treatment. (store purified RNA at -800C for long term). Agarose gel electrophoresis: The RNA samples after extraction are checked for the RNA signal. To visualise the RNA 1% agarose gel electrophoresis. The materials required for agarose gel electrophoresis is TAE buffer and agarose. TAE buffer(50x Tris acetate) 242 g of Tris base 57.1 ml glaciel acetic acid 100 ml of EDTA 0.5M PH .8.0 700ml of dH2O . About 20 ml of above solution is mixed with 980ml of distilled water to make it 1X. 1 gram of agarose is added to 100 ml of TAE buffer. The solution is mixed well. The solution is heated until the agarose is completely dissolved and solution becomes colourless. About 2.5  µl of ethidium bromide is added to the solution to visualise the RNA bands. Special care is taken while adding the ethidium bromide because it is bio-hazardous. Then about 5  µl RNA sample is mixed with 1  µl of DNA loading dye. The RNA samples are RUN with the potential difference of 80V with DNA Hyper ladder II as a standard. DNase treatment (protocol and kit provided by applied biosystem) The DNase I treatment is performed to remove any traces of genomic DNA from the eluted RNA. Protocol: Add 10x DNase buffer and DNase I to the RNA sample in the following composition: Amount component 1/9 th volume 10x DNase buffer 4  µl DNase I The components are mixed with gentle pippeting. Then the mixture is incubated at 370C for 90 minutes. DNase inactivation reagent is added to the solution and mixed well, leave at room temperature for 2 minutes. The samples are centrifuged at maximum speed for 1 minute to pellet the DNase inactivation reagent, after that the RNA solution is transferred to a new RNase free tube. The samples are run on 1% agarose gel electrophoresis to check the DNA contamination. We fou

Foreign Investment in Malaysia and Its Impact on Economic Growth Essay

Foreign direct investment (FDI) means an international capital flows in which a firm in one country creates or expands a subsidiary in another (Krugman & Obstfeld, 2006). Directly, it means the subsidiary not only has the financial obligation towards its parent company, it extends to the same organizational structure and value. Theoretically, companies involve in FDI due to cost saving on the location, usage of abundance resources, technology transfer, vertical integration (coordinating supply and demand to an agreed price) and currency exchange that will reduce cost and increase value to shareholders. FDI in a host country is expecting to boost the manufacturing and services industry and consequently boost up the economy. FDI impact on economy and social The area has been widely studied by economist and among others, in East Asia, FDI is used as channel of increasing capital stock and it has positive effect on the economic growth in Vietnam (Thu Thi, Paitoon, & Bangorn, 2010) and more growth in Vietnam if the invest is done in education, training, financial market development (Anwar & Lan Phi, 2010). FDI increase wages of skilled and unskilled labour (Oladi, Gilbert, & Beladi, 2011) and it could increase the household spending in the host country. However, the distance of investors from origin country to destination or host country plays an important role in promoting FDI in the latter. This is a sample of macroeconomic gravity impact whereby the investors easily commute from their home country and understanding of the custom and language could reduce the barrier in communication. Foreign investment could contribute in ethical and structural norm in an organization rather than the western cultural transfers. Local cultural norm shall be adhered to during the negotiation process in order to have a win-win situation between investors and local entrepreneur. It is also discussed that political stress may impacted the inflow of FDI by tightening the rules and regulation which in turn will make the investment environment in destination country is less attractive compare to global environment. FDI are positive correlated with network (Shaner & Maznevski, 2011) and regional integration (Nathapornpan Piyaareekul & Peridy, 2009); host countries’ levels of financial market and institutional development, better governance and appropriate macroeconomic policies (Polpat, Bangorn, & Paitoon, 2011; Vadlamannati, Tamazian, & Irala, 2009); productive improvement and learning experience from previous FDI (Takechi, 2011). Therefore, a good support from the government is vital in promoting the FDI in host country. Not only FDI expect good support from the government, study shows that FDI creates instability and worsen crisis (Kazi, 2011). The way to control FDIs in one country are defined the terms and sectors which they are allowed to invest; do a thorough risk assessment on the portfolio; and resolve global dispute in an organization such as World Trade Organization (Cohen, 2009). FDI and determinants are co-integrated. Among determinants FDI factors in Malaysia are openness of a company, interest rates, inflation rate, China joining WTO1 and level of corruption.(Ting-Yong & Tuck-Cheong, 2010). Comparing to ASEAN as a whole, FDI is looked as more market-seeking rather than profit-seeking due to growing internal markets (Siew-Yong, Chen-Chen, & Hui-Boon, 2010). Contrary, Prema-chandra and Swarnim (2011) found that FDI in Malaysia has eroded compare to outflow to another countries. World Trade Organization Facts on FDI in Malaysia (2002-2011) Annual percentage growth rate of Gross Domestic Product (GDP) at market prices based on constant local currency. Aggregates are based on constant 2000 U.S. dollars. GDP is the sum of gross value added by all resident producers in the economy plus any product taxes and minus any subsidies not included in the value of the products. It is calculated without making deductions for depreciation of fabricated assets or for depletion and degradation of natural resources2. Data from World Bank (Chart 1 and Chart 2) revealed that FDI into Malaysia has a significant increment over past decade. However, there was a drop of FDI net inflows in 2009, due to world economic recession in 2008. The uptrend is picking up to a highest point at approximately USD12 billion from the last decade. Comparing to our neighboring country, Thailand, whom has a higher GDP, it has the same effect except the decline trend after 2010. It might be influenced by political crisis in Thailand since 2008 that effected international companies’ decision to extend their business in Thailand. From Chart 3, we gathered that the gross capital formation for Malaysia approximately between 20% to 25% of our GDP, with the lowest point at 17.84% in 2009 after 2008 recession. Foreign investment inflows are following the same trend and it clearly shows that FDI dropped synchronize with capital formation following the recession.

Appropriation of Brand Extension - Free Essay Example

Sample details Pages: 27 Words: 8073 Downloads: 3 Date added: 2017/06/26 Category Statistics Essay Did you like this example? LITERATURE REVIEW This chapter intends to set the theoretical frame of the thesis by introducing the main areas needed to create the basis of our analysis, shaping the ways towards our own main purpose. Thus, it begins with the roots of brand extension and starts of with the concepts of brand, brand identity and brand hierarchy and then leads into brand extension and explains it as a means of growth for a brand. Narrowing down the scope, it goes into the typology of brand extension and identifies the successful and unsuccessful factors of brand extension. Don’t waste time! Our writers will create an original "Appropriation of Brand Extension" essay for you Create order Finally it identifies certain rules for the success of brand extension and looks at different models used for the effectiveness of brand extension. 2.1 BRANDING: In todays world of increased competition and consumer awareness, the marketing of new products has become ever more complex. We have moved into a time where consumers are literate enough to choose their own products on the basis of their judgment and where competition among products and services gives them an opportunity to select the best product that would suit their need. Branding has become one of the most important issues in the launch of a new product. Having functional and emotional attributes attached to it, branding has gained popularity as consumer relates more and more to it. Historically examples of branding can be found as early as 9000 years ago when owners or manufacturers used to give distinctive and distinguishing symbol or name to their property or product. However it was the 19thcentury that arguably saw the dawn of the modern branding era and it was the industrial revolution that caused its birth. It was the industrial revolution that created the mass production that meant an ever- increasing proportion of people worked for a manufacturer and not themselves. They no longer needed to mark the products that they produced as their own; rather what they produced was collectively produced for one company. Before we proceed further, lets look more deeply into branding and then link that to the concept of brand extension. 2.1.1 DEFINITION AND EXPLANATION Different scholars have defined the word brand differently as different meaning or contexts have been attached to them. Balmer and Greyser (2003) have given the most explanatory definition of branding explaining both the traditional and their own perspective about branding. They have stepped forward from the traditional definitions of branding and have defined branding on the corporate level having corporate implications. According to them three type of definitions have been identified. The first two are traditional whereas the third one is the advanced version of branding which incorporates their point of view about branding. They are: Erstwhile. In its simplest sense a brand denotes a name, logotype, or trademark and was originally used to signify ownership as with branding of live stock. These are, increasingly, seen to be points of entry to the essence of a brand rather than the essence of branding per se. (Definition similar to the one given in Oxford Concise Dictionary) Established. This refers to the added values that a brand brings to a product. Products may or may not have brand values. Product brand values are superimposed by the organization by its marketing and communication experts and advisers. They are made memorable. In the main, such values are fashioned in the mind; not on the production floor. They are, essentially, synthetic. Whereas products are made in a factory, brand values exist in the mind. Brands can be timeless in a way that products may not be. However Balmer and Greyser(2003) have identified a new understanding about brands. They call this aspect of branding as emergent Emergent. While the category most certainly is established, the fundamental differences between this category and the other two are only beginning to be appreciated. This category refers to brands at the corporate level. Corporate brand values are not contrived; they need to be bona fide. The role of personnel and of culture in establishing and maintaining and understanding corporate brand values is of essence. In the words of Sir Michael Perry, a former Chairman of Unilever, brand is much more than a symbol to differentiate goods and services: In the modern world, brands are a key part of how individuals define themselves and their relationships with one another. More and more we are simply consumers We are what we wear, what we eat, what we drive. This description of brand explains that brand is much more than the physical and functional value that it holds. Its a bundle of attributes both functional and emotional. Thus brands not only meet our physical needs but also address our emotional needs. A blind test was conducted on Pepsi and Coca Cola. It was found that Pepsi was preferred over Coke in regards to its taste. Yet the sales of Coke are much higher than Pepsi that shows that despite being functionally better, people are emotionally attached to coke. Stephen King was Director of planning at one of the largest advertising agencies, J Walter Thompson, when he described brand as: People choose their brand as they choose their friends. You choose your friends not usually because of specific skills or physical attributes (though of course these come into it) but simply because you like them as people. It is the total person you choose, not a compendium of virtues and vices. 2.2 BRAND IDENTITY Brand identity refers to the public image of a product, line or service in the eyes of a consumer. McClendon (2003) considers that brand identity is something that exists in the minds and hearts of the consumers when they hear the name of the brand. He further adds that it is the identity of the brand that provides the real strength to the business. It is the visual link between the company and the consumer. Brand identity includes brand names, logos, positioning, brand associations and brand personality. Upshaw (1995) has identified brand identity as a brands DNA configuration. He supposes that the particular set of brand elements is blended in a unique way to establish how the brand will be perceived in the market place. According to Kapferer (2001), it is critical for each business to understand that the attributes of a brand represent the indispensable elements. Not all brand managers are aware of this. Yet in order to find out which of the extended brand elements is needed to m ediate with the market, pre testing is done and this is considered to be the best method to avoid trails and errors. In his book, Aaker (2000) argues that a brand is more than a product. Creating an extension can benefit the parent brand by helping it break out of the box. According to him, there are several reasons for building a rich extended brand identity, reasons that are going to be illustrated in the following figure and explained underneath it. A richer brand identity is a more accurate reflection of the brand. Just as a person cannot be described in one or two words, neither can a brand. Three word taglines or an identity limited to attributes will simply not be accurate (Aaker, 2000, p. 54). Aaker (2000) considers that the identity of a brand represents what the brand stands for. Taking into consideration that the brand identity is inspirational, it must comprise and reflect the values and cultures of the entire organization. Moreover, customer concern should dominate the strategy of the business. And lastly, Aaker emphasizes in his picture that the extended identity provides a home for constructs that help the brand move beyond attributes. In particular, brand personality and symbols normally fail to make the cut when a terse brand position is developed, yet both are often extremely helpful strategically as well as tactically (Aaker, 2000, p.54). Balmer (2003) has emphasized on the concept of corporate identity and in his historiography model, we are currently in stage 4 in which the emphasis would be on organizational identity, corporate identity, corporate communication, corporate reputation and finally but most importantly corporate branding. 2.3 BRAND HIEARCHY TREE Brand structure can be illustrated logically by using the brand family tree together with all the related sub-brand branches. The figure below can be viewed as an organizational chart. The horizontal and vertical dimensions are grouped after numerous factors such as segment, product, quality and design (Aaker, 2000). The horizontal dimension shows the scope of the brand in terms of the sub brands that lie under the brand umbrella in the box visualized Colgate as a parent brand. The vertical dimension represents the brands and sub-brands that exist for an individual product- market entry (Aaker, 2000). The visualized overview of the whole brand guides the brand managers to keep an eye on its entire brand and to analyze if there are too many or too few. The question is how these brands can be reinforced, what message they deliver to the consumer and what improvements to the particular message can be done (Aaker, 2000). Keeping an eye on this hierarchy is quite important as it enables a company to identify the fit for new extension and also helps to maintain a clear vision of each product keeping in view the rest of the brands in the hierarchy. Thus its easy to maintain fit and leverage in brand extension with the help of this brand hierarchy. Every company would like to see its brand growing and prospering. Brands grow through two principle means. The first mean is called organic growth whereas the second one is called growth through extension. 2.4 ORGANIC GROWTH FOR BRANDS In this case making a brand or product frequently available or adding incentives to the brand makes it more popular. Sales of any one brand increase because what they have to offer becomes attractive to somebody, somewhere. Brands can be made more attractive by improving either the functional or emotional attributes of the brand. Thus in functional attributes we can improve any of the four Ps whereas in emotional one can improve the personality or image of the brand. A good example would be of Coca Cola and their distribution. Not only have they made it available from Atlanta to Zanzibar, from Moscow to Melbourne but also you can buy it from supermarkets, newsagents, cinema, restaurants, street corners, cafÃÆ' ©, football stadium, pop concert and even at car parks where you have vending machine1. Whilst there are numerous marketing tools to achieve organic growth, this type of growth stems from three things: getting that brand used by more people, getting it used by the same people more often or getting people to use more of it on any of the occasions they use it in the first place. 2.5 GROWTH THROUGH EXTENSION The second and relatively newer way of growing brand is through extension, which is the core focus of this study. Before going into detail about how brands grow through extension, I will firstly define extension and try to differentiate the various types of extensions. Due to the relative immaturity of the concept, there is no standard definition of brand extension and various marketing scholars have given different definition to the same terminology. From the readings that I have conducted of books and research papers, its obvious that around a decade back scholars used to give a more generalized definition of brand extension. The generalization of the definition can be observed from the fact that brand extension was used for extension into both related and non-related products. The following definition will clarify my point of view. In a typical brand extension situation, an established brand name is applied to a new product in a category either related or unrelated, in order to capitalize on the equity of the core brand name (DeGraba and Sullivan, 1995; Pitta and Katsanis, 1995). Also certain research papers indicated that brand extension being generalized was then differentiated into two types as indicated by this piece of research work. Brand extensions come in two primary forms: horizontal and vertical. In a horizontal brand extension situation, an existing brand name is applied to a new product introduction in either a related product class, or in a product category completely new to the firm (Sheinin and Schmitt, 1994). A vertical brand extension, on the other hand, involves introducing a brand extension in the same product category as the core brand, but at a different price point and quality level (Keller and Aaker, 1992; Sullivan, 1990). In a vertical brand extension situation, a second brand name or descriptor is usually introduced alongside the core brand name, in order to demonstrate the link between the brand extension and the core brand name (e.g. Marriott Hotels, Courtyard Inn by Marriott). Most recently the word line extension has been given to extension done in the same product category whereas brand extension would refer to extension in unrelated products and in this study I will undertake this understanding of extension. Taylor (2003) has referred to them as direct and indirect stretch. Jobber (2003) has given the term brand extension to line extension whereas brand extension has been referred to as brand stretch. The current emphasis on the subject has been due to its enormous success. Consumers being the end users have become friendlier to the concept and are now accepting it as illustrated on the next page. Its obvious from this graph that consumers are becoming much friendlier to the concept then they were a decade ago and this shows the popularity of the concept and the frequency at which it has been used in the past decade. Lets get an insight into the various types of extension. 2.5.1 LINE EXTENSION OR DIRECT STRETCH Line extension is defined as being a variant of the same basic product. It might be a new flavor or a new size. Basically its a slight variation to the original product. Examples would be of Colgate. We used to have Colgate regular but now we have Colgate total, Colgate Max fresh Gel, my first Colgate for kids, Platinum, Deep clean etc. The basic purpose of this strategy is to encourage more people to use a brand. It can also be considered as a first step towards brand extension. But the only bad thing about too many variations in the products or having too many line extension is that it may confuse the user in regards to which product should he/she use. Also it may cause a cannibalization affect within the product range. 2.5.2 BRAND EXTENSION OR INDIRECT STRETCH Brand extension on the other hand would refer to extending your product range into a product category that wouldnt be commonly associated with it. A simple definition described below will illustrate my point of view. Brand extension is using the leverage of a well known brand name in one category to launch a new product in a different category. (Brandextension.org) Giles Lury in his book about Brand Watching has defined brand extension as: Brand extension is the use (and occasional misuse) of an existing brand name and equity to launch a product or service into a category or market not normally associated with that brand. (Lury, 1998) Thus in contrast to earlier scholars, who had generalized the concept of brand extension, new researchers have distinguished the concept well from line extension. 2.6 RATIONALE BEHIND USING BRAND EXTENSION Brand extension has gained a lot of popularity and is considered to be the key tool for launching new innovations. A survey was conducted by Brand gym in 2003 in which marketing directors were asked about brand extension. The following graph illustrates the response. The results indicated that 83% of the marketing directors thought that brand extension would be the main way of launching new innovation in the next two to three years. Yet research has also shown that only 50 percent of brand extension survives after the first three years. Firstly brand extension differs from line extension because where line extension offers customers more varieties or styles of the original brand in its original market, a brand extension takes an existing brand to pasture new ones. Taking Mars as an example we see that the original chocolate bar has been line extended into different styles including Mars Kingsize, Mars miniature and for a limited period Dark chocolate Mars. However when Mars launched the Mars ice cream, it entered a new market for the brand and as such had extended the brand franchise. Mars also extended into flavored milk drinks market with Mars in a bottle. The rationale behind brand extensions popularity is that its difficult and expensive to launch a completely new brand. The most often quoted statistic being that nine out of every ten new brands fail. New brands are therefore seen as a high, though sometimes high return strategy. On the other hand, brand extension is a cheaper and more reliable method of building on what already exists. Not surprisingly companies who have already invested a lot of money in creating a brand are keen to maximize its full potential. Finally it can be concluded that companies would like to leverage and thus give initial success to the new brand by exploiting the equity that has been established by the parent brand. 2.7 KELLER AND AAKERS WORK ON BRAND EXTENSION Keller and Aaker (1998) extending on their typology of product range extension and corporate brand extension have examined the impact of corporate marketing on a companys brand extension. In their research paper they have described how consumers evaluate brand extension in general and then concentrating on corporate brand extension, they have studied the impact of corporate marketing on consumer evaluation of corporate brand extension in the presence and absence of supporting product advertising. The initial research work describing product brand extension is as follows: Research on consumer responses to extensions of product brands, suggest that two key factors influence consumer evaluation. the types of association that make up the parent brand image the relationship between the parent brand and the extension product These factors affect the consumer belief about whether the new product fits as a member of the product line. In sum, the record therefore suggests that a variety of different associations for the parent brand can be transferred to an extension, assuming a basis of fit exists. Now an extension that they made in regards to brand extension was that they applied this concept to corporate brand extension. But before going further its important to know Aakers three dimensions of corporate credibility. They are: 1. Corporate expertise is the extent to which a company is thought able to competently make and sell its products and services. 2. Corporate trustworthiness is the extent to which a company is thought to be honest, dependable, and sensitive to consumer needs. 3. Corporate likability is the extent to which a company is thought likable, prestigious and interesting. This results gathered from this study have strategic implication both to the benefits/risks associated with brand extension and also to the effectiveness of brand extension. Thus a summary of the results are as follows. Firstly by showing that corporate marketing related to product innovation enhances perceptions of corporate credibility and extension fit, and thus much favorable extension evaluations, this study showed benefits for brands with reputation of high quality products. Secondly this study provided a more detailed account of particular dimensions of corporate credibility, namely corporate expertise, trust worthiness and likeability. Thus this study concluded that corporate expertise appeared to play a more influential role in evaluation of corporate brand extension than either corporate trustworthiness or likeability. Thirdly this study suggested the merits of leveraging a strong brand to introduce a new product. One advantage of using a brand extension strategy to name a new product is that a less concerted advertising effort may be necessary. To the extent that brands extensions are able to leverage existing parent brand associations in consumer memory, a company should find it easier to achieve brand image with an extension branding strategy instead of giving a new product a new name. The fact that corporate marketing activity impacted consumer evaluations of a corporate brand extension in the absence of any product specific advertising is further an empirical support for the benefit of adopting a brand extension strategy. Fourthly this study suggested that corporate marketing activity significantly influenced extension evaluations even when the extension was advertised on the basis of another image dimension point. Thus corporate image associations are more likely to transfer to an extension on the basis of the branding strategy. Lastly this study also suggested that where a company is in a situation of having a trade off between various strategies like reinforcing a strong association, strengthening a weak association or creating a new association, then it wholly depends on the situation of each of the elements to decide which strategy to choose. For example: In some cases, existing associations may be so strong that they may be better off emphasizing other information to fortify a weak or supply a missing association. 2.8 TYPES OF BRAND EXTENSION Limited work has been done on the typology of brand extension. From various research papers, books and websites that I have consulted regarding brand extension, very few have distributed brand extension into different types. (Brandextension.org) have generated the following typology of brand extension taking functional and emotional attributes of the brand into consideration. 1. Similar product in a different form from the original parent product. This is where a company changes the form of the product from the original parent product. An example is (frozen) Snickers Ice Cream Bars. The original Snickers bar is a shelf stable candy. The brand extension is a similar product, but in a different form. Jell-O Portable Pudding and Pudding Cups is Jell-O pudding in a different form and section of the store. 2. Distinctive flavor/ingredient/component in the new item. When a brand owns a flavor, ingredient or component, there may be other categories where consumers want that property. E.g. Peanut butter is a characteristic ingredient in Reeses Peanut Butter Cups candy. Chocolate is a characteristic ingredient of Hershey. Brand Extension Research identified Reeses Peanut Butter as a logical extension that capitalizes on this association. 3. Benefit/attribute/feature owned. Many brands own a benefit, attribute or feature that can be extended. E.g. Brand Extension Research showed Armor All brand was defined by automotive surface protection which can go beyond vinyl dressing. Paint needs protecting also. Arm Hammer owns a benefit of deodorizing. Their baking soda product has claimed that it removes odors from refrigerators, etc. As a result, they extended the brand into other products such as Arm Hammer underarm deodorant and cat litter deodorizer. 4. Expertise. Over time, certain brands may gain a reputation for having an expertise in a given area. Leverage can be achieved when extending into areas where this special expertise is deemed important. E.g. Hondas expertise in reliable engines led to lawn mowers, gas powered generators and a variety of other gasoline engine powered devices. What brand comes to mind when we think of baby products? Gerber. As a result of this acceptance of their expertise, they successfully launched Gerber Baby Powder, Gerber Baby Bottles, etc. Sara Lee is known for baked desserts, so why not other baked goods like bread. 5. Companion products. Some brand extensions are a natural companion to the products the company already makes. E.g. Contadina was a tomato paste and sauce brand. In brand extension research, consumers thought Contadina pasta was a logical companion product that would have the leverage of the Italian heritage of the parent. Aunt Jemima (the pancake mix brand) launched pancake syrup, as a companion to compete with Log Cabin syrup. 6. Vertical extensions. Some brand extensions are vertical extensions of what they currently offer. A brand can use their ingredient/component heritage to launch products in a more (or sometimes less) finished form. E.g. NestlÃÆ' ©s Toll House chocolate refrigerated cookies is an example. Most Toll House chocolate chips are used in cookies, so why not make a brand of Toll House chocolate chip cookies. Mrs. Fields Cookies were ready-to-eat. They offered frozen cookie dough, moving backwards as a vertical extension. Rice Krispies has always been used in kids treats. Kellogg offered Rice Krispies Treats ready-to-eat. 7. Same customer base. Many brand extensions represent a marketers effort to sell something else to its customer base. This works particularly well when that customer base is large and to some extent captive. E.g. VISA launched travelers checks directed to its credit card customers. 8. Designer image/status. Certain brands convey status and hence create an image for the user. E.g. Designer clothing labels have been extended to furniture, jewellery, perfume, cosmetics and a host of other items. Some brands promote a lifestyle and can extend to items that people wear, as a badge of identifying themselves with that lifestyle. The above-mentioned typology is quite useful as it indicate the key areas where extension is done along with the methodology used to extend the product line. Yet it must be said that not all research work would agree with this typology as it is felt that certain types confuses line and brand extension or in ways generalizes it more to extension rather than brand extension. For example: Adding attribute to the products in the same product line would be line extension and not brand extension. Still it is a good base for my research work and also for further research into the typology of brand extension. Aaker (1998) has described two types brand extension differentiating the concept on a corporate level. The first type described by him is product brand extension. A company makes a product brand extension when it uses an existing brand name distinct from its corporate name to introduce a new product outside its current product offering. With product brand extension consumers are often completely unaware of the company involved. The second type described by him is corporate brand extension. A corporate brand extension is one which relies on the corporate name to launch a new product . A corporate brand extension clearly identifies an organization with a product, and so evokes different reactions from consumers than a product brand extension. A corporate brand may create associations in consumers minds that reflect the values, program, and activities of the firm. 2.9 SEQUENTIAL STRETCHING AND UMBRELLA BRANDING Extension to parent brand is usually a sequential process in which brands are initially line extended and then brand extended. This sequential stretching of brands leads to the formation of a whole family of brands thus giving rise to the concept of Umbrella branding. As the name indicates, umbrella branding refers to extension of a parent brand into a variety of products such that a whole range of products would come under the same brand. Taylor (2003) has divided the sequential extension into three main steps namely core brand extension, direct stretch and indirect stretch. I will illustrate the concept using Dove as an example. Brand extension was a key driver of Doves explosive growth during the 1990s. Coupled with geographic expansion, it helped grow sales fivefold, to almost $1 billion. The brand continues to grow at 20 percent per year and is well on its way to hitting the $ 2 billion mark in the next few years. Let go through the sequential process and apply it to Dove. The first and most crucial step to be noted is that Dove didnt extend its product line until it had achieved the following two things. à ¢Ã¢â€š ¬Ã‚ ¢ A strong bar business had been built à ¢Ã¢â€š ¬Ã‚ ¢ The brand had satisfactory scores on attributes rating for mildness and moisturizing. An important thing to be noted is that extension took place only after Dove had secured its soap bar business and had improved it. Thus once there was strength in the brand, it extending it to other products. Stretching went through the following stages. Stage One: Core Range extensions: Dove remained a product brand with a single format at this stage. It extended (line extension) its product range by adding new versions such as sensitive skin that now accounts for up to a third of sales. Further growth of the bar through product and pack innovation, remains a key source of profitable growth. Diagrammatic illustration of this step would indicate the extension into the two types. Stage Two: Direct stretch: In this stage extension is done into markets that are quite relevant to the product line. In the case of dove, it extended its product range into bath and shower products. Yet till now dove is focused on personal washing. The key reasons of dove extension at this stage were strong product delivery and innovative packing that differentiated them from other products in the range. The following diagram illustrates their stretch in to shower and bath products. Stage three: Indirect stretch: Capitalizing on their skin care outlook, Dove decided to be ambitious and to move beyond the washing and bathing market. Although they started off selectively, they introduced products like deodorants and hair gels etc. that were once again a big success. This process of broadening a product range is referred to as Umbrella Branding as illustrated by the diagram given below. The dove success has been due to consistent marketing and a consistent communication campaign. Consistency has been a key part of building brand identity and has been an additional glue to tie together the extension. 2.10 BENEFITS AND DRAWBACKS ASSOCIATED WITH BRAND EXTENSION Brand extension being the most popular mean of brand growth has some surprising statistics. Success rate of brand extension is hard to find, especially as what constitutes a success varies enormously. Yet a survey conducted by OCC using a simple and effective definition of success (still being on shelf after six years after launch) found out that 50 percent of all brand extension fails. This figure is certainly an eye opener for most companies as half of the product fails using brand extension. Taylor (2003) has associated this huge failure figure due to Brand ego tripping and also gives effective steps to avoid it. But before we go into the detail of this concept, lets look into the benefits and drawbacks of brand extension. 2.10.1 BENEFITS ASSOCIATED WITH BRAND EXTENSION The remarkable popularity of the concept over the last decade is a confirmation of the fact that there are marked benefits that can be associated with brand extension. Taylor (2003) has described the consumer benefits of brand extension in which he has identified consumer knowledge, consumer trust and lower cost as the major benefits of brand extension. Tauber (1988) has differentiated the benefits on the basis of efficiency and effectiveness emphasizing more on the cost benefits. An existing strong brand promotes a new product or service as there is less need to create awareness and imagery. Thus in a way awareness is already present and the only thing left is communicating the message of a new product to the end consumer. Consumers trust strong brands to deliver against a particular promise. A survey by Brandgym (2003) indicates that 58% of UK consumers are more likely to try a new product from a brand they know versus only 3% for a new brand. Thus an extension uses the reputation of an established brand to create a compelling value proposition in a new segment or market. Taylor (2003) has referred to this as consumer knowledge and trust. The cost benefit attached to brand extension is certainly one of the key reasons of the concepts popularity. Tauber (1988) has described awareness as an efficiency benefit in which cost advantage is achieved. This leads to the second most important benefit of brand extension that is lower cost. Tauber (1981) has pointed towards lower cost to achieve awareness and trial target level as the cost benefit of brand extension. Brandgym (2003) have shown that cost per unit of trial is 36 percent lower with brand extension and that repurchase is also higher. Brand extension also opens the door to the possibility of other extensions thus broadening the scope of the parent brand. Every product has a definite life cycle and once in maturity has to be rejuvenated other wise it will end up in the decline stage of its product life cycle. Brand extension gives a new life to the brand name and survives the brand name over a longer period of time. Brands are the barrier to entry into new categories; they are also the means to entry.(www.brandextension.org) An example can be taken of typewriters. With computers coming into the market, the demand for typewriters declined dramatically. Now if the typewriter companies would have extended their brand to electronic typewriters or computers at that time, then their brand name would have still existed with even better success chances in comparison to a new computer company although the parent product would no longer exist. Another major benefit of brand extension is that which it brings to the rest of the brands in the product range. A successful brand extension creates synergies among the rest of the brands in the umbrella and in particular the parent brand. It reinforces the consumers perceptions of the parent brand name that in return affects the sales of the parent brand and rejuvenates it. In a study MartÃÆ' ­nez (2004) has shown that the perceived quality of the brand and consumers attitudes towards the extension positively influence both the general brand image (GBI) and the product brand image (PBI) after the extension. While familiarity with the products of the brand only affect the GBI, the perceived degree of fit affects the PBI. Thus this study indicates that positive attitudes are established due to successful brand extension. 2.10.2 RISKS ASSOCIATED WITH BRAND EXTENSION Brand extension has a number of risks that can be associated with it. Taylor (2003) in his risk analysis model has described three things that can seriously damage the health of the core brand. The first one is cannibalization. As the name suggest, this is the risk of an existing eating up other family members in the hierarchy tree. The biggest risk occurs with range extensions that are brand clones lacking differentiation versus the existing brand. Thus with cannibalization not only is the volume of the core brand decreased, but also the profit margins of the extended product is also decreased due to extra cost owing to goodies yet failing to be priced up. Tauber (1981) in his risk benefit table has described cannibalization as a risk to existing lines. Chung and Anne (1996) have concluded that the effect of a step down brand extension in quality would affect the parent brand in three principle ways. 1 Cannibalization of the core brands sales, 2 Tarnishing the prestige of the core brand name, and 3 Negative feedback effects among the core brands consumer franchise The second risk described by Taylor (2003) is stealing thunder. According to him, when extension would not improve the profitability of the business, then it would be better to improve the core brand rather than investing on new brands. The third major risk described by Taylor (2003) is new toy syndrome. When efforts are concentrated on new extended products, it leaves the core branded unattended to and the competition seeking this weakness would strike on the core brand and thus the extended product would fail automatically with the fall of the core brand. Aaker (1990) has also expressed this view when he wrote that extension (successful or unsuccessful) may potentially dilute the equity built up by the parent brand. Chen (2000) has also shown that unsuccessful brand extension may dilute the equity of the brand irrespective of the level of equity held by the parent brand. Tauber (1981) has also mentioned that new product might create confusion or negative connotations in the minds of consumers and thus weaken the core values of the brand. Much has been written as scare tactics about the negatives and risks of brand extension. In reality, if a brand extension is so off target or lacks fit and or leverage, it likely will fail and will damage the brand image and reputation of the parent brand. Most of these misfires though die in limited test market. In a study Chen (2004) studied the impact of a parent brand on the trial of the extension and the reciprocal effect of a successful trial of new brand extensions positioned horizontally and vertically on the parent brand. Results showed positive influence of the parent brand on the trial of the extension. Yet there can be real damage to the parent brand, however, when too many unrelated brand extensions are launched. Names like Betty Crocker, General Electric and Kraft have been extended profusely. While they have not lost their awareness as household words, the strong associations they once had to specific products and related qualities (e.g. cake mix, light bulbs and chee se) may be diluted. This is especially dangerous when a brand is used synonymously with a specific product. Brands that are not legally generic but are used that way such as Kleenex, Scotch (Tape), and Band-Aid should not be extended broadly or they risk losing this valuable quality. It is crucial that one weigh these advantages and disadvantages of brand extension before launching a new product. Lets look into the concept of brand ego-tripping and then try to make brand extension an effective tool. 2.10.2.1 Brand ego-tripping and its detection: Taylor (2003) has described brand ego tripping as the biggest risk for brand extension. Brand ego-tripping refers to a companys inability to properly assess the challenge of creating a truly compelling and credible extension. Thus a company loses sight of what made them famous in the first place, what helped them deliver differentiation, relevance and value. They end up focusing internally on the needs of the business and its management rather than externally on the needs of the consumer. Thus Taylor (2003) taking the example of Virgin has explained and given five points where a company may lead to brand ego tripping and has recommended ways on which if emphasis is given would lead to the success of the company. Neglecting the core: Taylor (2003) recognized that Virgin was neglecting the core brands of travel and entertainment and if it would have focused on this sector then it might have made a greater success. Forgetting what made you famous: Taylor (2003) recognized that part of virgins failure in cola, vodka and jeans products could be because they couldnt differentiate effectively as they did in the airline and entertainment business. Failing to understand consumers and markets: Taylor (2003) also thought that the lack of real success in jeans, cola and vodka also reflected that virgins could understand these markets effectively. Consumers mostly want inspirational, fashionable badge values. Their differentiation on price wasnt quite successful. Scatter gun stretching: If virgin would have concentrated on a few products, then they would have achieved greater success as they would have emphasized adequately of each of them. Neglecting execution: Finally the last reason for brand ego tripping is neglecting execution. Virgin Energy was not able to meet the demands of the consumer and could not offer the service as promised. Its quite important to recognize this concept and identify the five reasons for brand ego tripping because it can damage the brand identity of the parent brand and can create negative connotation about the brand. Thus once brand perception is developed negatively then its hard to rectify the image of the brand and convey a positive image about the brand. 2.11 EFFECTIVENESS OF BRAND EXTENSION Now we come to the most important part of the literature review and that is seeking ways to making brand extension an effective tool for the launch of new products. In order to study the effectiveness of brand extension different principles and models will be presented that would discuss the effectiveness of brand extension. A few principles have been indicated below which can be viewed as a gauge to measure the success of brand extension. The first and foremost principle is that brand extension should have a balance between fit and leverage. In order to illustrate this concept further, following are the definition and explanations of fit and leverage. Fit: What categories will consumers accept from a brand? Thus it defines the ability of a brand to stretch or to identify the boundaries of the brand. According to Tauber (1988) perceived fit is achieved when the consumer accepts the new product as logical and would expect it from the brand. Aaker and Keller (1990) suggested that perceptual fit is whether a consumer perceives the new item to be consistent with the parent brand. For example, Consumers accepted the concept of Duracell flashlights but when asked about Duracell cameras, they replied no. Duracell didnt have that expertise in the mind of the consumer. Whether they did or not didnt matter. Perceptions dictated that Duracell (www.brandextension.org) Leverage refers to the distinctive properties, which a brand owns that provide a competitive advantage to the brand extension in its new category. Leverage is the impact of these distinctive properties that lead customers in the new category to perceive the brand extension as superior to existing competitive products on an important dimension. There is often an inverse relationship between fit and leverage (www.brandextension.org). Martinez and Pina (2003) have concluded in a study that if the consumer does not get the connection between the main brand and the extension the link is too distant, tenuous or seems downright daft then the extended brand is likely to fail. In a study Zimmer and Bhat (2004) investigated the impact of extension quality and fit on the parent brand. The results showed that the impact of good fit on the brand attitude of the parent brand was quite positive whereas the impact of bad fit on the brand attitude of the parent brand was relatively less. Although this study would decrease the importance of fit yet I would still recommend brand extension to posses fit and leverage as little negative affect from one product would lead into another extension and would gradually become significant enough to create an impact on the parent brand. Another important aspect of brand extension which is crucial to the concept is that the product should be well distinguished from the parent brand and other brands in the range other wise it would lead to cannibalization of the brand. Crest spent decades launching new toothpaste twists such as tartar control, gum protection and whitening. In the USA, share halved from 50 per cent with one product to 25 per cent with 50 products (Lury 1998). Speed (1998) has indicated in his study that management decisions about branding strategy are driven by consideration of risk and return. According to him managers seek to minimize their exposure to cannibalization risk and capture possible benefits from transfer and reciprocity. Taylor (2003) has proposed a brand extension work out in which he has presented six practical steps that would help in applying the principles of brand added value to boost the chances of success. The following figure would illustrate things further. Step One: Strengthen the core step emphasizes the importance of a strong core brand and product for successful stretching. Thus in this first step it is essential that the core brand is strong and healthy and one that can add real value to new extensions. Taylor emphasizes on the fact that if the core brand is under performing, it may decrease the appeal of the new product or services. Step two: With a healthy core product, attention can be turned to the future. Developing a clear and ambitious brand vision is a great stimulus for successful extension. By developing a broader definition of the market, opportunities for brand extension and potential threats from competitors can be highlighted. It also gives the whole team a sense of direction, assisting in ensuring that extensions not only build sales, but also play a role in building one big brand idea. Step three: With a clear and inspiring vision in place, the next step is generating ideas. This should start with a search for opportunities to extend the core range, using a number of different insight springboards. Importantly, consumers are only one source of ideas. Many successful ideas come from borrowing ideas from competitors and companies in other categories, or looking within the company itself. Step four: Having generated extension idea, teams need to employ a process of focus on those with the best potential to build the business and the desired brand vision. Clarity is also needed on the companys competences to ensure that the product promise can be fully delivered. Without such a disciplined approach, companies risk launching too many small extensions that add limited value for consumers and so build little extra business. Step five: Even when one has focused his/her efforts and investments on extensions with the best potential, poor execution can still let a company incur losses. Failing to deliver on the promises made in the concept is rated by marketing directors as one of the main causes of extension failure. In contrast, excellence of execution has several positive effects. First and foremost, it helps boost repurchase and so increase the probability of profitable growth. It also helps generate positive word of mouth and free publicity, by far the best forms of extension promotion. Finally product quality can allow a product to push the brand extension boundaries: people will tend to buy a fantastic product even if the fit with the brand is not obvious. Step six: As a brand extends it becomes a bigger challenge to manage and risks running off in too many directions. A multitude of messages confuses the consumer and dilutes the image of the parent brand. It also leads to a loss of clarity on priorities within the company. Brand architecture (Brand Hierarchy tree) helps to structure all the brand extended product range so that brand values are developed consistently within the umbrella of brands. This sequential, logical, simple and practical model given by Taylor (2003) will help all companies in identifying the pitfalls in brand extension and would help them in having a successful brand extension as it not only emphasizes on the sequence of steps leading from strengthening the core to brand architecture but also within each step identifies the possible mistakes that companies have been making over the years. This model also seeks to address the issue of brand ego-tripping. The table described on the next page will summarize the activity of this model and will link it to the problems created by brand ego-tripping. Solutions to each problem are also provided. 2.12 CONCLUSION FROM LITRETURE REVIEW From the literature review its obvious that brand extension should occur only if the core brand is strong enough to be translated on to another brand. Thus there is no use of extending a brand that isnt strong because then it would risk having negative effects on the parent brand. Also its concluded that brand extension should be done only if it has fit and leverage. The significance from a consumer perspective is that if it doesnt have fit and leverage then the consumers would be able to associate the image of the parent brand to the new one that would cause it to fail. Weighing the benefits against the drawbacks determines the strength of any brand extension. Although it is recognized that brand extension may serve different functions yet its imperative that a clear vision is kept about the product category in which extension is expected. The matrix given by Taylor (2003) summarizes the use of brand extension for building the business of any organization. On one axis he has business build and on the other axis he has taken brand vision build as the key elements that a brand extension would achieve. Thus he described four kinds of extension According to Taylor (2003) hero extension are those that intend to build business and the brand vision of the parent brand. The bulk of the marketing activities should be concentrated on this extension. On the other extreme is drain extension, which doesnt intend to build business and brand vision. This is the type of extension that business should be aware of and companies that arent aware of the concept usually end up in this box. The other two extensions are used for tactic and are situational in nature. Thus taking the above matrix into consideration, I would conclude this literature review by emphasizing on the fact that the aim of extension should be to become a hero extension. Yet ambitions have to be controlled and the benefits and drawbacks of the extension should be taken into consideration. If done properly brand extension can turn out to be an excellent mean of brand growth.

The First World War And The Reconstruction Of Britain

This essay will explain why Brutalism became one of the 20th Century’s most controversial architectural movements and help explain what influenced the development of the style. Firstly by explaining and describing the characteristics of brutalist structures and discussing the most monumental brutalist buildings during the 1950s and 1960s. By exploring historical and contextual factors, this essay will discuss what technological advancements occurred and contributed towards the birth of Brutalism. Within this essay there will be discussion about the way the Second World War and the reconstruction of Britain changed the direction of British architecture and which architects were most influential in this redevelopment process. Furthermore, it will explore the Modernist movement by evaluating key historical information which contributed to the progression of a new, mid-century style. 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