Chemistry

Introduction

A cell’s plasma membrane is known to be selectively permeable. This implies that the membrane is selective on what substances can pass in and out of the cell. There are two methods of transport that occur through the plasma membrane. One method of transport is called the active process which uses ATP energy to transport substances through the membrane. The other method is called a passive process which does not require the use of ATP energy. During passive processes, molecules are transported through the membrane by differences in concentration or pressure between the inside and outside of the cell.

Two important types of passive processes are diffusion and filtration. Every cell in the human body uses diffusion as an important transport process through its selectively permeable membrane. During diffusion, molecules that are small enough to pass through a membrane’s pores or molecules that can dissolve in the lipid section of a membrane move from an area of higher concentration to an area of lower concentration. The kinetic energy that all molecules possess is the motivating force in diffusion. Facilitated diffusion occurs when molecules are too large to pass through a membrane or are lipid insoluble.

In this process, carrier protein molecules located in the membrane combine with solutes and transport them down the concentration gradient. Filtration is another type of passive process and, unlike diffusion; this is not a selective process. The pressure gradient on each side of the membrane as well as the membrane pore size depends on the number of solutes and fluids in the filtrate. During filtration, water and solute molecules pass through a membrane from an area of higher hydrostatic pressure to an area of lower hydrostatic pressure.

This means that water and solutes would pass through a selectively permeable membrane along the pressure gradient. To gain a better understanding of a cell’s selectively permeable membrane and the passive processes of simple diffusion facilitated diffusion, and filtration, three experiments were conducted.

Materials and Methods

Activity 1: Simulating Dialysis (Simple Diffusion)

Materials:

• two glass beakers
• four dialysis membranes: 20 (MWCO), 50 (MWCO), 100 (MWCO), and 200 (MWCO)
• membrane holder
• membrane barrier
• four solutes: NaCl, Urea, Albumin, and Glucose solution dispenser
• deionized water
• timer
• beaker flush

This experiment was conducted first by placing the 20 (MWCO) dialysis membrane into the membrane holder.

The membrane holder joined the two glass beakers; one on the left side and one on the right side. Then, 9. 00 mM of NaCl concentration was dispensed into the left beaker. Deionized water was dispensed in the right beaker. When the timer was started, the barrier that surrounded the membrane holder was lowered to allow the contents of each beaker to come in contact with the membrane.

After the 60 minutes of compressed time elapsed, results were read and recorded. Finally, each beaker was then flushed for preparation of the next experiment run. These exact steps were followed using each dialysis membrane size (20, 50, 100, and 200) as well as with each solute (NaCl, Urea, Albumin, and Glucose). There were a total of sixteen runs in this experiment.

Activity 2: Simulating Facilitated Diffusion

Materials:

• two glass beakers
• membrane builder
• membrane holder
• glucose concentration
• solution dispenser
• deionized water
• timer beaker flush

In this experiment, the first step was to adjust the glucose carrier to 500 in order to correctly build the membrane. Next, a membrane was built in the membrane builder by inserting 500 glucose carrier proteins into it. Then, the newly built membrane was placed into the membrane holder that joined the two glass beakers. The two glass beakers were joined on the left and right sides of the membrane holder. After that, 2. 00 mM of glucose concentration was dispensed into the left beaker. The right beaker was filled with deionized water.

The barrier around the membrane holder dropped when the timer was started. After 60 minutes of compressed time elapsed, the results were read and recorded. Finally, both glass beakers were flushed to prepare for the next experimental runs. The above-mentioned steps were repeated by increasing the glucose concentration to 8. 00. Both the 2. 00 mM and the 8. 00 mM glucose concentration solution was tested using membranes built with 500, 700, and 900 glucose carrier proteins. There were a total of six experimental runs.

Activity 4: Simulating Filtration

Materials:

• two glass beakers membrane holder
• 4 dialysis membranes: 20 (MWCO), 50 (MWCO), 100 (MWCO), and 200 (MWCO)
• 4 solutions: Na+Cl₂, Urea, glucose, and powdered charcoal
• solution dispenser
• pressure unit
• timer
• filtration rate indicator
• membrane residue analysis analyzer
• beaker flush

In the final experiment, the two glass beakers were placed one on top of the other with the membrane holder between them. The pressure unit that rested on the top beaker was used for forcing the solution from the top beaker through the selected membrane and into the bottom beaker.

The bottom beaker contained nothing; however, the filtration rate indicator was attached to it from one side. The experiment began by placing the 20 (MWCO) dialysis membrane into the membrane holder. Then, 5. 00 mg/ml of each of the following solutions: Na+Cl₂, Urea, glucose, and powdered charcoal was dispensed into the top beaker. The pressure unit was adjusted to 50 mmHg of pressure. The timer was set to 60 minutes of compressed time and when the timer started, the membrane holder retracted. The solution then flowed through the membrane and into the beaker underneath.

When the timer stopped, the membrane was then placed in the membrane residue analysis analyzer. The results were read and recorded and the beakers were flushed for the next experimental runs. All the above steps were repeated using the 50 (MWCO), 100 (MWCO), and 200 (MWCO) membranes.

Results

Table 1: Activity 1: Simulating Dialysis (Simple Diffusion)

Key: Solutes that we’re able to diffuse into the right beaker are indicated by a “+”. Solutes that were not able to diffuse into the right beaker are indicated by a “-“.

Graph 1: Activity 2: Simulating Facilitated Diffusion Glucose Transport Rate (mM/min)

Table 2 and 3: Activity 4: Simulating Filtration

Table #2: Solute Residue Presence in the Membrane Key: If solute residue was present on the membrane, it is indicated by a “+”. If solute residue was not present on the membrane, it is indicated by a “–“.

Table 3: Filtration Rate and Amount of Solute Detected in Filtrate

Discussion

The first lab experiment, Simulating Dialysis (Simple Diffusion), demonstrated how only certain molecules pass through a selectively permeable membrane down its concentration gradient. The four membranes utilized in this experiment consisted of each one being different in pore size (MWCO). The smallest pore-sized membrane was 20 (MWCO), and the largest was 200 (MWCO). The solutes that were tested in this experiment were NaCl, Urea, Albumin, and Glucose.

The first solute tested, NaCl, showed that with a 20 (MWCO) membrane, no diffusion occurred into the right beaker. (Table 1) The NaCl molecules were evidently too large to pass through the 20 (MWCO) membrane because its pores were too small. Membranes 50, 100, and 200 (MWCO) did allow the NaCl to pass through. (Table 1) One of the reasons this occurred is because the pores in the above-mentioned membranes were large enough to permit the passage of the NaCl molecules. The other reason diffusion occurred is because the NaCl molecules moved down its concentration gradient and into the beaker filled with deionized water. For all three membranes, equilibrium was reached in ten minutes at an average diffusion rate of 0. 0150 mM/min.

As for the solute Urea, the experiment conducted showed that no diffusion occurred with all four membranes. (Table 1) Urea should have passed through membranes 100 (MWCO) and 200 (MWCO) for the reasons that its molecules are small enough and Urea is also soluble. This experiment showed that none of the Albumin molecules diffused through any of the four membranes tested. (Table 1) This is because the Albumin molecules were too large to pass through the pores of all four membranes. The final solute tested in this experiment, Glucose, showed that the molecules only diffused through the 200 (MWCO) membrane. (Table 1) Equilibrium was reached in thirty-seven minutes at an average diffusion rate of 0. 0040 mM/min.

The Glucose molecules were too large to diffuse through the 20 (MWCO), 50 (MWCO), and 100 (MWCO) membranes. The second experiment, Simulating Facilitated Diffusion, explained how carrier protein molecules in the membrane effectively transported molecules that are too large or are insoluble to diffuse through the membrane. The carrier proteins in this experiment were glucose carriers and the solution was a 2. 00 (mM) and an 8. 00 (mM) glucose concentration. The 2. 00 (mM) glucose concentration was tested first with the 500 glucose carrier protein-membrane then the 700 and 900 glucose carrier protein membranes. The glucose transport rate for the membrane with 500 glucose carrier proteins was 0. 0008 (mM/min). Graph 1) The membrane with 700 glucose carrier proteins showed a rate of 0. 0010 (mM/min) and the 900 glucose carrier proteins membrane had a rate of 0. 0012 (mM/min). (Graph 1) The 8. 00 (mM) glucose concentration also showed an increase in glucose transport rate with membranes that contained more glucose carrier proteins. The membrane with 500 glucose carrier proteins showed a rate of 0. 0023 (mM/min). (Graph 1) Membranes that had 700 and 900 glucose carrier proteins showed a rate of 0. 0031 and 0. 0038 (mM/min). (Graph 1) These results show that with an increase in the amount of glucose carrier proteins in the membranes, transport of the glucose molecules in the concentration is more effective.

A higher concentration of glucose (8. 00 mM) also increases the rate of glucose transport in a membrane with the same amount of glucose carrier proteins as a lower glucose concentration (2. 00). In the final experiment, Simulating Filtration, four different solutes were forced through four membranes that contained separate pore sizes by the use of hydrostatic pressure. After each experimental run was conducted, the membrane analyses showed that residue from all four solutes was detected on each membrane. (Table 2) This indicates that some solutes did not filter through the membrane. The filtration rate (ml/min) increased as membranes with larger pores were utilized.

This happened because the solute molecules were able to transport through a particular membrane at a faster rate being that the membranes’ pores were larger. The filtrate in the bottom beaker was analyzed and no solutes were detected with the 20 (MWCO) membrane. (Table 3) With the 50 (MWCO) membrane, only NaCl was detected in the filtrate at 4. 81 (mg/ml). (Table 3) The 100 (MWCO) membrane showed to have NaCl at 4. 81 (mg/ml) and Urea at 4. 74 (mg/ml) present in the filtrate. (Table 3) Glucose and powdered charcoal were not present. The last membrane with pore size 200 (MWCO), had the solutes NaCl at 4. 81 (mg/ml), Urea at 4. 74 (mg/ml), and Glucose at 4. 39 (mg/ml) detected in the filtrate. (Table 3) Powdered charcoal was not detected in this filtrate. Table 3) The molecules in powdered charcoal were too large to pass through any of the membranes tested. The 20 (MWCO) membrane pores were too small to allow any solute molecules to pass through. The membranes that contained larger pores allowed the solutes with larger pores to pass through. The amounts (mg/ml) of the same solute detected in the filtrate were the same for each membrane. (Table 3) This is because the pressure that was released into the top beaker remained at 50 (mmHg) for all experiment runs.

References:

1. Marieb, Elaine N., Mitchell, Susan J. (2008). Exercise 5B. Human Anatomy & Physiology Laboratory Manual Ninth Edition (pp. PEx-5 – PEx-13). San Francisco, California: Pearson Benjamin Cummings.

“Respiration” and “Photosynthesis” All life depends on two chemical reactions “Respiration” and “Photosynthesis”. These two processes are quite crucial because they are a source to nearly all life on Earth. Both of these processes are quite similar yet differentiate vastly. In this essay I’ll be comparing and contrasting “Photosynthesis” and “Respiration”. I’ll start by discussing what actually happens in both these processes and how? Moving on to the energy transfers these processes go through and how these reactions relate to each other.

Plants feed using a process called ‘Photosynthesis’. Photosynthesis is the chemical change which happens in the leaves of green plants. It’s the first step towards making food; not just for plants but ultimately every animal on the planet. During this reaction carbondioxide and water are converted into glucose and oxygen. For this reaction to occur both carbon and nitrogen are absorbed from the roots as nitrate and so is carbon as carbondioxide from the air and it also needs energy as the reaction is endothermic, therefore the energy is ‘light’ from the sun.

This is absorbed by a green substance called chlorophyll in the leaf. Therefore, photosynthesis takes place in the chloroplasts which are present in the palisade cells (found near the top of the leaf. ) (fig: 1 ) As chloroplasts contain a green pigment called chlorophyll, which absorbs light energy needed to make photosynthesis happen. The equation for this reaction is: 6CO2 + 6H2O+ light energy —> C6H12O6 + 6O2

Sugars created in photosynthesis can be later converted by the plant to starch for storage, or it can be combined with other sugar molecules to form specialized carbohydrates such as cellulose, or it can be combined with other nutrients such as nitrogen, phosphorus, and sulfur, to build complex molecules such as proteins and nucleic acids. Moreover, ‘respiration’ is reversibly related to photosynthesis because it takes in light energy and respiration gives out energy, which is used by the body as the glucose is converted into starch or stored.

Oxygen is consumed unlike in photosynthesis, although in respiration energy is released in a more controlled and manageable way. During photosynthesis water and carbondioxide are chemically combined to make glucose and oxygen. The equation as follows: C6H12O6 + 6O2 —> 6CO2 + 6H2O In addition, respiration and photosynthesis are both processes which depend on each other on a very wide scale. Fig: 2 explains this. (Fig: 2 ): Shows a cycle and how photosynthesis and respiration are dependent on each other.

As the leaf produces oxygen through photosynthesis and then that oxygen is inhaled by an animal. Later, the animal breathes out carbondioxide, and that is then absorbed by the plants through leaves by the air, in order for respiration to happen; making this ongoing cycle continue. Though photosynthesis only takes place in leaves of plants (inside palisade cells) during the presence of light, respiration takes place in every living organism inside the mitochondria of the cell, with or without light being present or not.

As all cells need energy through the sugar they made in their leaves; oxygen moves through diffusion through the plant from cell to cell. Any oxygen not used by the plant in respiration, leaves the plant through tiny holes under the leaf called stomata. That’s then part of the air we breathe, making the whole process start again. That’s how closely photosynthesis and respiration are linked to each other. Whilst, both respiration and photosynthesis are quite similar yet unlike at the same time. Respiration is a process by which energy is liberated.

On the other hand photosynthesis is a procedure where energy is stored in carbohydrate molecules as in, photosynthesis is an anabolic (constructive) process, resulting in the building up of carbohydrate molecules. While in respiration a catabolic (destructive) process occurs, resulting in the breakdown of carbohydrate molecules. So, respiration results in a loss in dry mass where photosynthesis is totally the opposite, it results in a gain in ordinary mass. Furthermore, there are enzymes involved in the process of respiration.

Enzymes are biological catalysts that speed up a reaction. The organic compounds that are broken down are substrates. Glucose is the most common substrate. The general equation for respiration is: C6H12O6 + 6O2 —> 6CO2 + 6H2O + 38ATP During respiration, glucose undergoes glycolysis forming pyruvic acid. Glycolysis consists of nine separate chemical reactions, each catalysed by a specific enzyme. The key players in glycolysis are the enzymes ‘dehydrogenase’ and a coenzyme (a non-protein helper). So, in photosynthesis is catalysed by chlorophyll.

To summarize, photosynthesis takes the raw ingredients of water, carbondioxide and uses light to make glucose and oxygen. Despite, respiration uses glucose and oxygen to produce carbondioxide and water as waste products. Both these processes takes place in cells except respiration takes place in the mitochondria of a cell. Photosynthesis is catalysed by chlorophyll acting in concert with lipids or sugars, while in respiration ‘dehydrogenase’ is used. Nevertheless, photosynthesis and respiration both are a part of an ongoing cycle and work together in order for plants or animals to survive.

Case – 1 Hostile Mint it’s probably the last place you might expect to find a hostile work environment. First of all, it’s a federal workplace. And even more surprising, it’s heavily guarded against intrusion. But the situation inside the U. S. Mint in Denver was anything but a safe place for 71 women who brought a complaint to the facility’s equal employment opportunity (EEO) officer in 2003. When the organizers of the complaint began to fear that they were the investigation targets instead of the complaints, 32 of the women decided to take the matter to the U. S.

Equal Employment Opportunity Commission (EEOC)

Their contention: The Denver Mint was a hostile work environment. These allegations were the culmination of a number of incidents that had occurred over a long period of time. The Denver Mint, which opened in 1863, has 414 employees, of which 93 are women. One woman who started working at the Denver Mint in 1997 said, “She found the atmosphere completely hostile toward females. ” When she filed an EEO charge claiming discrimination, she was retaliated against by having most of her job duties reassigned and being required to work at home.

Events leading to the current complaint started in 2001, when another female employee who was inspecting a men’s room for cleanliness saw a loose ceiling tile, removed it, and found 40 to 50 sex magazines. Some months later, this same employee was checking for rats in an attic and found a stash of pornographic magazines. Both times she made these discoveries, she was with a male colleague. Later, she would say in a statement given to the main office of the U. S. Mint that to her knowledge no action was every taken to address the situations.

Another female employee filed a claim of retaliation and sexual harassment with the facility’s EEO officer in 2000. It was 2003 before she got a hearing with the EEOC and an administrative judge ruled in favor of the Mint. However, when she filed her claims in federal court in 2005, a jury found that she “worked in an environment hostile to women and awarded her $80,000. ” In 2001, the facility’s new superintendent held a women’s forum attended by the then-director of the U. S. Mint. However, the highest-ranking woman at the Denver Mint—the administrative services chief, Beverly Mandigo

Milne—said, “Nothing changed. ” The final straw that triggered the complaint was the demotion of the mint’s acting EEO manager in February 2003. The month after the demotion, the 71 women filed the petition alleging a hostile work environment. An individual from the San Francisco Mint was assigned to investigate; however, the women claimed that the investigation never focused on the facts, but on Milne. One of the women said, “They believed that Beverly coerced everyone into filing the petition. ” That was when 32 of the women took the matter to the EEOC.

Despite the filed petition, hostile situations still continued. One woman said that in 2004, a male co-worker offered to pay her for sex. Another woman said that after she returned after a short bereavement leave following her husband’s death in 2005, a male supervisor propositioned her. On March 31, 2006, the U. S. Mint and the female employees who had filed the class complaint reached a proposed settlement. The terms of the settlement included a payment of $8. 9 million for damages, fees, and costs.

FILTRATION “Filtration may be defined as a process of separation of solids from a fluid by passing the same through a porous medium that retains the solids, but allows the fluid to pass through. ” The suspension to be filtered is known as slurry. The porous medium used to retain the solids is known as filter medium. The accumulated solids on the filter are referred to as filter cake, while the clear liquid passing through the filter is filtrate. When solids are present in a very low concentration i. e. , not exceeding 1. 0% w/v, the process of its separation from liquid is called ‘clarification’.

Process of filtration: The filtration operation is shown below in the figure * The pores of the filter medium are smaller than the size of the particles to be separated. * Filter medium (for eg: filter paper or muslin cloth) is placed on a support (a sieve). * When slurry (feed) is passed over the filter medium, the fluid flows through the filter medium by virtue of a pressure differential across the filter. * Gravity is acting on the liquid column. Therefore, solids are trapped on the surface of the filter medium Figure 1: filtration Once the preliminary layer of particles is deposited, further filtration is brought about wherein the filter medium serves only as a support. * The filter will work efficiently only after an initial deposit. * After a particular point of time, the resistance offered by the filter cake is high that virtually filtration is stopped. For this reason, a positive pressure is applied on the filter cake (upstream) or negative pressure (suction) is applied below the filter medium (downstream). Factors affecting the rate of filtration:

The rate of filtration which depends on various factors can be written as: Rate of filtration = Area of filter X Pressure difference Viscosity X Resistance of cake and filter The rate of filtration depends on the following factors: 1. Pressure: * The rate of filtration of liquid is directly proportional to the pressure difference between the ‘filter medium’ and ‘filter cake’. * Thus, the rate of filtration can be increased by applying pressure on the liquid being filtered or by decreasing the pressure beneath the filter. 2. Viscosity: * The rate of filtration is inversely proportional to the viscosity of the liquid undergoing filtration. Liquids which are very viscous get filtered slowly in comparison to liquids with low viscosity. * Reduction of viscosity of a liquid by raising the temperature is frequently done in order to accelerate filtration. eg: syrups are more quickly filtered when hot and cold. 3. Surface area of filter media: * The rate of filtration is directly proportional to the surface area of filter media. * Pleating the filter paper or using a fluted funnel increases the effective surface area of filter paper for filtration. Filter press also works on the same principle. 4. Temperature of liquid to be filtered: Temperature plays an important role in the rate of filtration. * Viscosity is reduced by a rise in temperature and the filtration of viscous oils, syrups etc is often accelerated by filtering them while they are still hot. 5. Particle size: * The rate of filtration is directly proportional to the particle size of the solid to be removed. * It is easier to filter a liquid having coarse particles than that having finely divided particles because coarse filtering medium can be used to filter liquid having coarse and hence it increases the rate of filtration. Therefore before filtration, some method should be adopted to agglomerate the finely divided particles into coarse particles or to increase the particle size by precipitation. 6. Pore size of filter media: * The rate of filtration is directly proportional to the pore size of the filter media. * The liquid having coarse particles requires a coarse filtering media to remove them. So, the rate of filtration is increased when a coarse filter medium is used for filtration. 7. Thickness of cake: * The rate of filtration is inversely proportional to the thickness of the filter cake formed during the process of filtration. As the filtration process proceeds, the solid particles start depositing on the filter medium, and thus, it increases the thickness of the cake and decreases the rate of filtration. 8. Nature of the solid material: * The rate of filtration is directly proportional to the porosity of the filter cake. * The porosity of the filter cake depends on the nature of the solid particles to be removed from the liquid. * Filter aids are sometimes added to the filtering liquid to make a porous cake Theories of filtration

The flow of a liquid thorough a filter follows the basic rules that govern the flow of any liquid through the medium offering resistance. The rate of flow may be expressed as: Driving force Rate = ——————– (equation 1) Resistance The rate of filtration may be expressed as volume (lit) per unit time (dv/dt). The driving force is the pressure differential between the upstream and downstream of the filter. The resistance is not constant.

It increases with an increase in the deposition of solids on the filter medium. Therefore filtration is not a steady state. The rate of flow will be greatest at the beginning of the filtration process, since the resistance is minimum. Once the filter cake is formed, its surface acts as filter medium and solids continuously deposit adding to the thickness of the cake. The resistance to flow is related to several factors as mentioned below. Length of capillaries Resistance to movement = ———————————————————— Poiseuille’s Equation:

Poiseuille’s considered that filtration is similar to the stream line flow of a liquid under pressure through capillaries. Poiseuille’s equation is ? pr4 V = —————– 8L? Where, V= rate of flow, i. e. , volume of liquid flowing in unit time, m3/s(1/s) p = pressure difference across the filter, pa r = radius of the capillary in the filter bed, m L = thickness of the filter cake (capillary length), m = viscosity of filtrate, pa s If the cake is composed of a bulky mass of particles and the liquid flows through the interstices (correspond to a multiplicity of capillary tubes), then the flow of liquids through these may be expressed by poiseulle’s equation. Darcy’s Equation: Poiseuille’s law assumes that the capillaries found in the filter are highly irregular and nonuniform. Therefore, if the length of a capillary is taken as the thickness of the bed, correction factor for radius is applied so that the rate equation is closely approximated and simplified.

The factor influencing the rate of filtration has been incorporated into an equation by Darcy, which is: KA P V = ——————– ? L Where, K = permeability coefficient of the cake, m2 A = surface area of the porous bed (filter medium), m2 p = pressure difference across the filter, pa L = thickness of the filter cake (capillary length), m ? = viscosity of filtrate, pa s

The term K depends on the characteristics of the cake, such as porosity, surface area and compressibility. Permeability may be defined quantitatively as the flow rate of a liquid of unit viscosity across a unit area of cake having unit thickness under a pressure gradient of unity. This model relates not only to filter beds or cakes but also applies to other types of depth filter. Equipment is valid for liquids flowing through sand, glass beads and various porous media. Darcy’s equation is further modified by including characteristics of K by Kozeny-Carman. Kozeny-Carman Equation:

Poiseuille’s equation is made applicable to porous bed, based on a capillary type structure by including additional parameters. Thus the resultant equation, which is widely used for filtration is Konzeny- Carman equation. A p ? 3 ?S2 KL (1- ? )2 V = —— ——- ——– Where, ? = porosity of the cake (bed) S = specific surface area of the particles comprising the cake, m2/m3 K = Konzeny constant p = pressure difference across the filter, pa L = thickness of the filter cake (capillary length), m ? = viscosity of filtrate, pa s

The Konzeny constant is usually taken as 5. The effect of compressibility of the cake on flow rate can be appreciated from equation (1), since the flow rate is proportional to ? 3/ (1- ? )2. A 10 percent change in porosity can produce almost 3-fold change inn V. Limitations of Kozeny Carman equation: Kozeny Carman equation does not take in to account of the fact that the depth of the granular bed is lesser than the actual path traversed by the fluid. The actual path is not straight throughout the bed, but it is sinuous or tortuous Mechanisms of filtration:

The mechanism whereby particles are retained by a filter is significant only in the initial stages of filtration. Some of the mechanisms are: Straining: Similar to sieving i. e. , the particles of larger size cannot pass through the smaller pore size of the filter medium. Impingement: Solids having momentum move along the path of streamline flow and strike (impinge) the filter medium. Thus, the solids are retained on the filter medium. Entanglement: Particles become entwined (entangled) in the mass of fibres (of cloth with a fine hairy surface or porous felt) due to smaller size of particles than the pore size.

Thus the solids are retained on the filter medium. Attractive forces: Solids are retained on the filter medium as a result of attractive forces between particles and filter medium, as in case of electrostatic precipitation. FILTER MEDIA AND FILTER AIDS Filter media: The filter medium act as a mechanical support for the filter cake and is also responsible for the collection of solids. Filter medium should have the following characteristics: 1. It should have sufficient mechanical strength. 2. It must be inert; it should not show chemical or physical interaction. 3.

It should not absorb the dissolved material. 4. It should allow the maximum passage of liquid, while retaining the solids. It means that it must offer low resistance to flow. The magnitude of the resistance of the filter medium will change due to the layers of solids deposited earlier, which may block the pores or may form bridges over the entrances of the channels. Therefore, the pressure should be kept low at the beginning to avoid the plugging of the pores. The usual procedure is to filter at constant rate by increasing the pressure as necessary. When normal working pressure is reached, it is maintained.

On continued filtration, the thickness of the cake further builds up and hence the rate of filtration decreases. When the rate is uneconomical, filtration is stopped. The filter cake is removed and filtration is restarted. Materials: The following materials are used as filter media: 1. Woven materials such as felt or cloth: * Woven material is made of wool, cotton, silk, glass, metal or synthetic fibres (rayon, nylon etc. ) * Synthetic fibres have greater chemical resistance than wool or cotton, which are affected by alkali and acid respectively. * The choice of the fibre depends on the chemical reactivity with the slurry. . Perforated sheet metal: * For eg: stainless steel plates have pores which act as channels as in case of meta filter (edge filter). 3. Bed of granular solid built up on a supporting medium: * In some processes, a bed of graded solids may be formed to reduce the resistance to the flow. * Typical examples of granular solids are gravel, sand, asbestos, paper, pulp and keiselguhr. * The choice of solids depends on the size of the solids in the process. 4. Prefabricated porous solid unit: * Porous solids prefabricated into a single unit are being increasingly used for its convenience and effectiveness. Sintered glass, sintered metal, earthenware and porous plastics are some of the materials used for the fabrication. 5. Cartridge filter media: * Cartridge units are economical and available in pore size of 100µm to even less than 0. 2 µm. * These can be used either as surface cartridges or depth type cartridges. a) Surface type cartridges: * These are corrugated and resin treated papers. These are used in hydraulic lines. * Ceramic cartridges are advantageous in cleaning for reuse by back flushing or firing. * Porcelain filter candles are used for sterile filtration. ) Depth type cartridges: * These are made of cotton, asbestos or cellulose. * These are disposable items, since cleaning is not feasible. Filter Aids: Filter aid forms a surface deposit which screens out the solids and also prevents the plugging of the supporting filter medium. The important characteristics of the filter aids are: 1. Chemically inert to the liquid being filtered and free from impurities. 2. Low specific gravity, so that filter aids remain suspended in liquid. 3. Porous rather than dense, so that previous cake can be formed. 4. Recoverable Justification:

The object of the filter aid is to prevent the medium from becoming blocked and to form an open, porous cake, hence reducing the resistance to flow of the filtrate. a) Usually low resistance is offered by the filter medium itself, but as layers of solid built up the resistance will be increased. The cake may become impervious by blocking of the pore in the medium. Flow rate is inversely proportional to the resistance of the solid cake. b) Slimy or gelatinous material and highly compressible substances form impermeable cakes. The filter medium gets plugged and the flow of filtrate stops.

Disadvantages: * The filter aids remove the coloured substances by absorbing them. Sometimes active principles such as alkaloids are absorbed on the filter aid. * Rarely, filter aids are a source of contaminants such as soluble iron salts, which can provoke degradation of sensitive ingredients. * Liquid retained in the pores of the filter cake is lost in the manufacturing process. Example of filter aids: * Keiselguhr, Talc, Charcoal, Asbestos, Paper pulp, Bentonite, Fullers earth * Activated charcoal is used for removal of organic and inorganic impurities. Keiselguhr is a successful filter aid and as little as 0. 1% can be added to the slurry. The rate of filtration is increased by 5 times or more, at the above concentration, though the slurry contains 20% solids. Handling of filter aids: Filter aids are mostly used for clarification processes, i. e. , where solids are discarded. Different flow rates can be achieved depending on the grade of the aids. * Low flow rate (fine solids) – fine grade filter aids –mainly intended for clarity. * Fast flow rate (coarse solids) -coarse grade filter aids –acceptable filtrate.

The filter aid can be employed in either one or both ways. a) Firstly, a pre coat is formed over the medium. For this purpose, a suspension of the filter aid is filtered to give a coating up to 0. 5/m2. b) Secondly, a small proportion of filter aid (0. 1-0. 5% of total batch weight) is purposely added to the slurry. So the filter cake has a porous structure and filtration can be efficient. The filter aid of 1-2 parts per each part of contaminant is mixed in the feed tank. This slurry is re circulated through the filter until a clear filtrate is obtained. Filtration then proceeds to completion.

The body mix method minimises equipment requirement and cross contamination potentials. Sterile Filtration: Sterile filtration is carried out for removal of microorganisms from fluids. It is a cheap and satisfactory method for sterilizing heat-sensitive (thermolabile) materials. The method implies the use of membrane filters which do not impart any particulate matter, fibers, or chemical reaction to the filtrate unlike unglazed porcelain candles, asbestos pads and other filters. In addition, no pretreatment is required, cleaning is no problem and the filters can be autoclaved or gas sterilized after assembly in its holder.

Even when sterility is not warranted but ‘polishing’ (removal of particulate matter including live or dead bacterial cells in order to obtain high purity and clarity) is desired in products like oral or topical antibiotic preparations, membrane filters are the best choice. The following filters are used for bacterial filtration: 1. Candle filter 2. Seitz filter 3. Edge filter 4. Sintered glass filter 5. Membrane filter Candle filters: Candle filters are made of unglazed porcelain and are available in various porosity grades, either cylindrical or in the shape of the flanged test tube.

Normally the filtration is so carried out that the liquid flow is from is from outside inwards and greater filtration surface is available to the incoming liquids. Candle filters can be sterilized by steaming, by hot moist air, or by autoclaving. Cleaning may be affected by drawing a large volume of distilled water through the candle filter thereby completely washing the previous solution from the pores. Thus the surface of the filter should be gently scrubbed with a soft brush, rinsed well with water and finally ignited in a muffle furnace. The main disadvantage of such filters s that the pores become plugged with organisms and debris which necessitate a very thorough cleaning. Sietz filter: It consists of an asbestos pad. The pads are available in several porosities that make them valuable for ‘polishing’ of solutions as well as removal of bacteria. Unless however the filter is backed with nylon mesh or sintered stainless steel: fibers occasionally get into the solution. The lower edge is fitted with a broad flat flange and the upper part is cylindrical. A perforated plate fitted into a lower part of the funnel supports the asbestos pad.

As the pads are meant only for single use, the cleaning of filter media is no problem. Each time a fresh pad is to be used. The apparatus is simple in operation but suitable mostly for small quantities of liquids. Sintered glass filters: These are made of borosilicate glass. Borosilicate glass is finely powdered, sieved and particle of desired size are separated. It is then packed in to a disc mould and heated to a temperature at which adhesion takes place between the particles. The disc is then fused to a funnel of suitable shape and size. The sintered glass filters are available in different pore size.

Hence the funnel with a sintered filter is numbered according to the pore size. The filtration is carried out under reduced pressure. These funnels are used for bacterial filtration. Sintered filters are also available in stainless steel which has a greater mechanical strength. However these are very much liable to attack by the solutions passing through them. Edge filters: In edge filters a pack of the filter media used and filtration is done edges by passing the liquid or slurry between and not through the media. Such filtration must be conducted under pressure or under partial vaccum system.

Meta filter and stream line filter are two types of edge filters but the former is of greater use in pharmaceutical industry. Meta filters: Meta filters are useful in those manufacturing processes where filter presses are not frequently suitable. It requires no cloth, gauges, paper etc. and may be used at any pressure and temperature and for any liquid. It can be thoroughly cleaned after each operation. In its simplest form, meta filters consists of a grooved drainage rod or guide tube on which a series of rings are packed. On keeping the pack and finds its way along the grooves in drainage rod and ultimately to the receiver.

These may be operated with pressure or under vaccum system. The rings are usually of stainless steel, of about 15mm inside diameter, 22mm outside diameter and 0. 8mm in thickness, with a number of semicircular projections on one surface. These pressure filters can be used for the filtration of very viscous liquids such as syrups or oils by fitting a steam jacket and rendering the liquids less viscous. They are also useful in the clarification of injection solutions and products such as insulin liquids. This type of filter can be cleaned easily by back-flushing with water or steam.

Because of the shape of the pores in the ring, back-flushing will wash away the filter bed completely. Meta filters are very economic in use. Streamline filters: Operation wise and also geometrically, the streamline filter is similar to meta filter but the cylindrical filter pack consists of compressed paper discs. The liquid flow takes place radially inwards through the small space between individual papers and through the papers themselves. Membrane filters: * Ultra filtration methods have become popular in recent years mainly due to increased refinement of various membranes. Cellulose and cellulose derivatives are mostly commonly used materials for these filters. They are available in a wide range of pore sizes, ranging from 8µ down to 0. 22µ. * However, for sterile filtration, membranes with pore size of 0. 22 to 0. 45µ are usually specified. * As such fine porosity of membranes may get clogged rapidly, a prefilter is used to remove colloidal matter in order to extend the filtration cycle. * The filter primarily acts as a simple screen and retains on its surface all particles of size greater than the pore size of the filter (resembling sieving action). Due to an enormous number of very fine pores, the pore volume approximates 80% of the total volume of the membrane. * The action of the filter is mainly due to the combined forces of gravity and van Der Waals forces. * Membrane efficiency can be predicted in terms of its bubble point which is a characteristic function of porosity. It is defined as the pressure required to push air through a liquid saturated filter. Filter pores retain liquid until this point is reached. * Each membrane has specific bubble point which depends on the liquid wetting the membrane. An obvious disadvantage of membrane filter is their brittleness when dry and this makes handling difficult. The use of filters in cartridge form, overcomes this problem. * Apart from the small laboratory models, large models are available for pilot plant and small scale production to handle up to about 25litres/minute of liquid through a 0. 45µ pore size membrane. * Membrane filters find extensive use in filtration and sterilization of a variety of pharmaceutical products such as ophthalmic and intravenous solutions, other aqueous products, biological preparations, hormones and enzymes. In conjunction with a suitable pipette syringe, it is very useful in dispensing measured volumes of sterile fluids. * This assembly is often utilized for handling of pharmaceutical, biological and bacteriological preparations which can be damaged by metallic contact. Centrifugation Centrifugation is a unit operation employed for separating the constituents present in the dispersion with the aid of the centrifugal force. Equipment used for centrifugation is centrifuge. Centrifugal force is used to provide the driving force for the separation. It replaces the gravitation force in the sedimentation.

Centrifugation is particularly useful when separation by ordinary filtration is difficult. Centrifugation provides convenient method of separating two immiscible liquids or solid from liquid. * Centrifugation is a separation process which uses the action of centrifugal force to promote accelerated settling of particles in a solid-liquid mixture. * If particles size in the dispersions is 5 micro meter or less, they undergo Brownian motion, hence they do not Sediment under gravity, therefore a stronger force, centrifugal force is applied in order to separate

Two distinct major phases are formed in the vessel during centrifugation: The sediment Usually does not have a uniform structure. The centrifugate or centrate which is the supernatant liquid. Process of centrifugation: The centrifuge consists of a container in which mixture of solid and liquid or two solids is placed and rotated at high speeds. The mixture is separated into it’s constituent parts by the action of the centrifugal force on their densities. A solid or liquid with higher specific gravity is thrown outward with greater force & it is retained at the bottom of the container leaving a clear supernatant liquid.

The speed of the centrifuge is commonly expressed in terms of number of revolutions per minute. Theory of centrifugation: If a particle (mass = m kg) spins in a centrifuge (radius r, m) at a velocity (v, m s-1) then the centrifugal force (F, N) acting on the particle equals m v2/r. The same particle experiences gravitational force (G, Newton) = m g (where g = acceleration due to gravity) Centrifugal force = f = mv2/r Centrifugal effect (C) = F/G = mv2 /mgr (v = 2 ? r n ) c = (2? r n)2/ g r = 4 ? 2r n2/ g (d= r/2) = 2 ? 2 d n2/ g (g = 9. 807) C = 2. 013 d n2 Centrifugal effect, C= 2. 013 n2d n= speed of rotation( revolution per second of centrifuge) * d= diameter of rotation So * Centrifugal effect is directly proportional to diameter of rotation * Centrifugal effect is directly proportional to (speed of rotation)2 There are two main types of centrifuge used to achieve separation on an industrial scale, * Filtration centrifuge: Those using perforated baskets, which perform a filtration-type operation (work like a spin-dryer) and * Sedimentation centrifuge : Those with a solid walled vessel, where particles sediment towards the wall under the influence of the centrifugal orce Perforated basket centrifuge: Figure: Perforated Basket Centrifuge In this type of centrifuge, a basket is mounted above a driving shaft. This type of centrifuges are used for batch processes. Principle: Perforated basket (bowl) centrifuge is a filtration centrifuge. The separation through a perforated wall based on the difference in the densities of solid and liquid phases. The bowl contains a perforated side wall. During centrifugation, the liquid phase passes through a perforated wall, while solid phase is retained in the bowl.

The solids are removed after stopping the centrifuge. Construction: It consists of a basket, made of steel (sometimes covered by vulcanite or led) or copper. The material of construction should be such that it offers greatest resistance to corrosion. The basket may have diameter of 0. 90 meters and capacity of 0. 085 meter cube. The diameter of perforations must be based on the size of crystals to be separated. The basket is suspended on a vertical shaft and is driven by a motor using suitable power system.

Perforated basket is kept in a casing which collects the filtrate and discharges it through outlet. Working: The material to be separated kept in the basket. The loading of material must be done to give an even distribution. The power is applied to run the basket at speed of 1000 rpm. During centrifugation the liquid passes through the perforated wall and solid phase retaind in the basket. Uses: * Perforated basket centrifuge is extensively used for separation of crystalline drugs (aspirin) from mother liquor. Sugar crystals are separated using the perforated basket centrifuge. * Precipitated proteins from insulin can be separated. Advantages: * The process is rapid * The final product has low moisture content * It cam handle slurries with high proportion of solids even those having paste like consistency * Dissolved solids from cake can be separated. Disadvantages: * On prolonged operation solids may form hard cake. * It is a batch process. Non-Perforated Centrifuge: Principle This is sedimentation centrifuge.

The separation is based on the difference in the densities of solid and liquid phases without a porous barrier. The bowl contains a non perforated side wall. During centrifugation, solid phase is retained on the sides of the basket and liquid remains at the top removed by skimming tube. Construction: It consists of a basket, made of steel (sometimes covered by vulcanite or led) or copper. The material of construction should be such that it offers greatest resistance to corrosion. The basket is suspended on a vertical shaft and is driven by a motor using suitable power system Working: The feed is continuously introduced into the centrifuge while the liquid (centrate) is continuously removed from an overflow weir inside the centrifuge * Solids build up during centrifugation forming a cake that must be periodically discharged Figure: Non-Perforated Basket Centrifuge * After the basket becomes filled with solids the centrifuge slows down and “skimming” (the removal of the top semi-liquid soft cake layer) takes place * Skimming typically removes 5 to 15% of the bowl solid volume * The bulk of the cake is discharged using a ploughing knife moving into the slowly rotating cake * The solid is discharged centrally at the bottom of the centrifuge * Solid accumulation is typically up to 60 to 85% of the maximum available depth * This type of centrifuge is typically operated at low centrifugal forces and has a relatively low solid handling capacity. The imperforated basket centrifuge is the only basket centrifuge commonly used for typical sludge dewatering applications. * High solid recovery can be achieved with this centrifuge even without chemical additives. Uses: Non-perforated basket centrifuge is useful when deposited solids offer high resisttance to the flow of liquid. Conical disc centrifuge: Principle: It is a sedimentation centrifuge. The separation is based on the difference in the the densities between phases under the influence of centrifugal force. In this a number of cone shaped plates are attached to the central shaft (which has provision for feed) at different elevations.

During centrifugation, the dense solids are thrown outwards to the underside of cone shaped casing. While lighter clarified liquid passes over bowl and collected from top of the cone. Construction: It consists of shallow form of bowl containing series of conical discs attached to the central shaft at different elevations. The discs are made up of thin sheet of metal or plastic separated by narrow spaces. A concentric tube is placed surrounding the central drive shaft. Working: The feed is introduced into the concentric tube surrounding the drive shaft. The feed flows down and enters the spaces between the discs. The solids and heavier liquids thrown out ward and move underside of the discs.

Low speed and short time of centrifugation is sufficient to give high degree of clarification. Uses: * Two immiscible liquids can be easily separated by continuous process after liquid-liquid extraction in manufacture of antibiotics. * Precipitated proteins in manufacture insulin can be clarified. Advantages: * Conical disc centrifuge is compact and occupies very less space. * By controlling speed of rotation and rate of flow, particles are separated into two sizes. * Separating efficiency is very high. Disadvantages: * Capacity of conical disc centrifuge is limited * Construction is complicated * Not suitable if sediment of solids form hard cake. Figure: Conical Disc Centrifuge Tubular bowl centrifuge: The tubular bowl centrifuge has been used for longer than most other designs of centrifuge. It is based on a very simple geometry: it is formed by a tube, of length several times its diameter, rotating between bearings at each end. The process stream enters at the bottom of the centrifuge and high centrifugal forces act to separate out the solids. The bulk of the solids will adhere on the walls of the bowl, while the liquid phase exits at the top of the centrifuge. * As this type of system lacks a provision of solids rejection, the solids can only be removed by stopping the machine, dismantling it and scraping or flushing the solids out manually. Tubular bowl centrifuges have dewatering capacity, but limited solids capacity. Foaming can be a problem unless the system includes special skimming or centripetal pumps. Figure: Tubular bowl centrifuge * This type of centrifuge can also be used to separate immiscible liquids. * Rate of sediment can be control by controlling the inlet rate. * The uses of centrifugal sedimenters include liquid/liquid separation, e. g. during antibiotic manufacture and purification of fish oils, the removal of very small particles, the removal of solids that are Compressible and which easily block the filter medium, The separation of blood plasma from whole blood (need C =3000).

Bacterial cells are a common choice for in vivo replication of DNA of interest, and in this study, the heat shock method was employed for bacterial transformation. Plasmids, which are DNA molecules themselves, were used as expression vectors for the DNA of interest, the GAP gene. Because only transformed cells exhibit antibiotic resistance, trans armed cells survived on plates containing inclining. Only those cells that took up plasmid s containing the GAP gene fluoresced in IV light.

By restriction enzyme analysis and gel electro prophesiers, the relationship between genotype and phenotype was observed using isolated p zamias from the bacteria. Because the presence of the GAP gene codes for fluorescence, it is expected t hat a genotype coding for the protein would express the glowing phenotype. Regular action of GAP gene expression was observed in samples that were grown with rabbinate, inimical in, and varying amounts Of glucose. Our results Suggest that the presence Of glucose in the s rounding environment inhibited transcription from the rabbinate bad promoter. Age 3 INTRODUCTION This study examined the transcriptional regulation Of the rabbinate Oberon pr emoted found in Escherichia coli (E. Coli). To facilitate this study, the Green Fluorescence t protein (GAP) was utilized as a reporter gene with the rabbinate promoter. The KEEP gene w as 772 base pairs (BP) long and was extracted from Quarrel Victoria. This gene was implanted into plasmids, which were inserted into the E. Coli through bacterial transformation (3). In order to obtain enough copies of these DNA samples in a reasonable ammo NT of time, two methods can be used.

The first is considered in vitro , or in glass, (such as a test tube) and is known as polymerase chain reaction. This method, in which a machine heats t he DNA sample ND Tag polymerase clones the DNA, is expensive and less convenient, so it is not always used. The other method is In Vivo , or in life, and is called transformation. This method was used in this experiment by shocking E. Coli bacteria with heat in order for them to take in plasmids that were transformed to contain the GAP gene. Not all bacteria were transformed, and not all transformed bacteria contained the GAP gene.

To differentiate between transformed and n untransformed bacteria, they were grown on inclining, as the transformed bacteria were rest assistant to impact Olin but the untransformed bacteria were killed by it. The plasmids with and with out the GAP gene were differentiated by visualization under IV light (4). The genotype of the remaining plasmids, both transformed and nontransparent med, were then tested to determine the relationship between the genotype, or plasmid c imposition, and phenotype, or presence of fluorescence.

The plasmids were removed from the e bacteria, with some samples left whole as controls and others cut into pieces by restriction enzymes. In this case, doll, originating from Hemophilia influenza , was the enzyme used to cut the plasmids Page 4 at their respective Hind doll sites, where the GAP gene would have been inserted. This was done to determine whether or not the KEEP gene was taken in by the plasmids when it was electrophoresis, as the difference in size of the pieces was observed in the gag arose gel (4).

Even though a transformed bacterium may have had the GAP gene in its insert Ted plasmid, it needed the promoter bad (consisting of genes Arab, area, and award) and t he GAP gene in the right direction and position in order for the bacterium to have the potent al for fluorescence. It may still not have fluoresced if there was not enough rabbinate present for the bad promoter to run, or if a high concentration of glucose inside the cell was present to rep as the bad promoter.

With all the necessary genetic coding for fluorescence, the amount that was visible depended on the amount of glucose present, as no glucose caused it to floorer see brightly, and a low concentration made it glow dully (4). Each step of this experiment was vital in analyzing the transcriptional regulate on of the rabbinate Oberon promoter. Through bacterial transformation, recombinant DNA and cloning methods were used in order to insert the GAP gene into the plasmid.

The pellet of cells was then resume need using a pipette and vortex mixer. This allows for a XX concentration to be obtained. After spreading, the four plates were incubated upside down (overnight at 370 C) in a microbial incubator. Following incubation each plate was placed upside down n a IV box and photographed. Restriction Analysis In restriction analysis, two restriction enzyme digestion reactions (one uncut b Y enzyme, one cut by enzyme) occurred for each of the two DNA samples (nonresistant and non page 6 fluorescent, resistant and inflorescent, and resistant and fluorescent).

For the uncut samples, components were added in the following order: Pl XIX Buffer ( supplied by environment by life technologies”‘ containing: 100 mm Trisect, pH 7. 5 100 mm Magical mm Theoretical 500 mm Nasal), 1 Pl Water, and 5 Pl DNA For the uncut samples, components were e added in the allowing order: Pl XIX Buffer, Pl Water, pi DNA, and III Handbill NZ. (sup plied by invitation by life technologies””). After all components were added they were mixed by overexerting and collected at the bottom of the tubes by using the microelectronic gem The tubes were then incubated for 30 minutes at 37 co.

Following incubation, Pl of XIX loading g buffer (1% (w/v) SD (sodium decoded sulfate) 50% (v/v) glycerol 0. 05% (w/ v) bronchiole blue) was added to each tube. The samples were mixed by overexerting and collected at the e bottom of the tubes using the microelectronic- Agrees gel electrophoresis was prepared by ding Pl of 1 KGB plus ladder ( supplied by environment by life technologies””) into the first and final well. 12. Pl of each sample was then loaded into remaining wells on the gel and the electro prioress was run for 1 hour at 1 VIVO.

The gels were then placed in a IV box and photographed. Using the photo of the gel, the genotypes of each sample were verified. This was done by finding the size of fragments through comparison with the DNA standard ladder. Gene Expression TO allow for observation Of phenotypes gene expression, bacteria were transfer erred to three types of media: inducing, introducing, and repressing. Five plates were used: LB,’Amp, LB/Marry, LB/Amp/AR/Glue 0. 2%, LB/Amp/AR/Glue 0. 5%, LB/ Amp/AR/Glue 2% (xx).

On each plate, bacteria with empty plasmids and therefore no GAP gene for flour essence were spread on one half, and bacteria with plasmids containing the GAP insert were e transferred to the Page 7 other. Both of these types of cells came from a master plate. Using a sterile to toothpick, each type of bacteria was patched in the appropriate area of each plate. GAP+ bacteria were patched in a “+” shape, while GAP bacteria were patched in a shape. Plates were labeled properly and Leary and were placed Poseidon in a ICC incubator overnight.

Plates were e observed for fluorescent bacteria on the IV transformational box after 24 hours, after 72 h ours, and again after 96 hours. Page 8 RESULTS Bacterial Transformation In order to investigate GAP gene expression, it was first necessary to obtain co pies of the DNA of interest through bacterial transformation, which allowed for plasmid ( and sometimes GAP) uptake by numerous bacterium. Phenotypes results from this procedure can suggest possible genotypes. Transformation plates that were prepared after heat shoo KC transformation can be Seen in Figure 2.

Individual colonies were visible on each AMP+ plate, while a lawn of bacteria had formed on the AMP plate and individual colonies were not discern enable. Under IV light, fluorescence was observed in those colonies expressing the GAP gene. If guru E highlights the difference observed between glowing and knowing bacterial colonies. The fraction of colonies that appear fluorescent and are assumed to be GAP+ is noted in Table e 1 along with complete results of this bacterial transformation (4).

Structural Analysis by Restriction Analysis and Gel Electrophoresis TO confirm the structure Of DNA plasmid genotypes, samples were run through h gel electrophoresis after being treated with a restriction enzyme specific for cleave ins the gene of interest (GAP). Standards were run along with each of the components on the electrophoresis gel. In order to determine size in base pairs of fragments of interest, a graph of the e relationship between the size and migration of the bands in the 1 KGB plus DNA Ladder was assembled (Figure 4), and a line of best fit was determined.

The relationship between the base 1 O log of size and migration is linear, and graphing them together gave a trend line with an ex. action useful in determining the size of experimental fragments with known migration values. These were the fragments obtained by cleaving the plasmids with the Handbill restriction enzyme. Table 2 page 9 organizes the sizes and migration distances for the fragments of the standard included during electrophoresis. These values were used to construct the calibration curve m mentioned before (Figure 4).

Figure 3 shows the agrees gel obtained by gel electrophoresis. Ta able 3 lists all sizes determined based on comparison with the calibration curve generated from migration standards (Figure 4). Sizes are noted for both the vector and the insert (4). Analysis of Reporter Gene Expression Investigation of gene regulation and interaction of environmental rabbinate a ND/or glucose with genotype required GAP+ cells to be spread on various plates, an d fluorescence to be observed over time.

Table 4 summarizes the observations of the phenotype o f patches streaked onto AMP plates containing or lacking rabbinate and/ or glucose. Glowing patches suggest expression of the GAP gene. Observations show that the rabbinate sample pop site for the GAP insert fluoresced brightly as time went on. The sample with rabbinate and 0. 2 % glucose increasingly fluoresced over time, while plates higher in percentage glucose c imposition did not fluoresce. Cells that were GAP were also spread in order to serve as a surrogate et marker.

These cells do not contain the gene for GAP, so they will not fluoresce under IV light . This gives a comparison, making it easier to determine if cells are expressing GAP fluoresce once or not page 10 DISCUSSION Regulation of Gene Expression: The samples that were grown with rabbinate, inclining, and varying amounts of glucose showed that the presence of glucose in a bacterium’s surrounding environment NT can affect the ability of its rabbinate bad promoter. The plate with no glucose added flour cede brightly, while the plates with glucose added showed very little to no fluorescence.

The only plate with glucose added that fluoresced in the end was the plate with the least glucose added These results are due to the glucose inhibiting the rabbinate Oberon from trap inscribing the bad promoter DNA. When glucose is present in a bacterium, the cell metal likes the glucose instead of the rabbinate, and the rabbinate Oberon is not utilized. However, when the cell is lacking glucose, it reaches a state of “hunger” and begins producing cyclic adenosine Mephistopheles (CAMP). This reacts with the CAMP receptor protein (CROP), who chi allows the cell to use rabbinate to induce the transcription of the rabbinate bad prom otter.

This promoter contains the genes Arab area , and award, which are part of the rabbinate Oberon. This system can only function if rabbinate is present in the cell; otherwise the gene arc will prevent the rabbinate Oberon from carrying out transcription by forming a “knot,” or loop in the DNA The rabbinate bad promoter reacts with the GAP gene to show when the Arabian SSE Oberon is in use and how strongly it is induced by rabbinate (5). The plate with the least glucose added began to glow over time, as the cell be an using up the glucose in its environment by metabolize it for energy.

It started to FL recurrences dully once the concentration of glucose was not high enough to fully repress the AR baboons Oberon, showing that there is a range of repression and induction, not just a state of ” on” and “off’ for page 11 these function. The more the bacteria used the glucose, the less of it was arrow ND to repress the Oberon, which is why its fluorescence strength grew over time. If the study WA s to be continued past the 96 hour mark, all of the plates would have eventually fluoresced as t hey used up their loses resources and began activating the rabbinate Oberon (4).

The fungal species about 100 stains which were previously isolated at the laboratory of biology department king khalid university will be used in this study. Fungi will be cultured on potato dextrose agar pda medium for 7 days at 27 °c. Identification of these species will be done basically on their microscopical and cultural characteristics. The identity will be confirmed by amplification of its gene using universal primers.

The fungal genomic dna extraction will be carried out using the qiagen dneasy plant/fungi mini kit protocol according to the nstructions. The its region of fungal dna will be amplified using the fungal specific-primer set: Its1-f cttggt cat tta gag gaa gta a and its4 r tcc tccgct tat tga tat gc as described by white et al.

1990 pcr reaction will be performed in a final volume of 50 ?L containing 10 mm tris-hcl 50mm kcl 1.5 m m mgcl2 each dntps at a concentration of 0.2 mm and 1.25 iu of taq polymerase. The amplification will be carried out by pcr. The initial denaturation temperature is 95 °c for 5 min followed by 40 cycles at 94 °c for 1 min 55 °c for 1 min 72 °c for 1 min; final extension at 72 °c for 10 min and holding at 4 °c.

The amplified products will be examined by electrophoresis in 1.5% agarose gels in tae buffer. Then the pcr product will be purified and will be sent for sequencing at macrogen company korea. The its sequence of fungus isolate will be used for blast search in the embl genbank database.

The sequence of the isolate will be further aligned and compared to publish its region sequences searched with the taxonomy browser of the national center for biotechnology information ncbi http://www.ncbi.nlm.nih.gov and retrieved from genbank. Screening for mycogenic biosynthesis of ag-nps all the identified fungal species will be screened for the biogenic synthesis of ag-nps. For the biosynthesis of silver nanoparticles the biomass of each isolated fungal species will be grown aerobically in cazpeks broth medium the inoculated flasks will be incubated on orbital shaker at 27 1 °c and with agitation at 150 rpm for 5 days.

The fungal biomass then will be harvested after incubation by filtering using filter paper whatman no. 1 followed by three times of washing with distilled water to eradicate the residues of the medium from the biomass. Ten g fresh weight of mycelia will be added to 100 ml of sterilized double distilled water for 48 h at 27 1 °c in a 250 ml erlenmeyer flask with shaking again at 150 rpm. After the incubation the cell filtrate will be obtained by filtration through filter paper whatman no. 1.

The filtrates will be inoculated with 1 mm silver nitrate agno3 solution and incubated at room temperature in dark abdel-hafez et al. 2016 the production of the nanoparticle will be checked visual by the changing the color into brown color. Cell-free filtrate without addition of silver nitrate will be severed as control. Purification of silver nanoparticles after formation the silver nanoparticles the agnps solution will be centrifuged at 10.000-14.000 rpm for 15-20 min.

The supernatant will be excluded and the pellets will be dispersed with distilled water. This dispersion will be again centrifuged. The procedure will be repeated 3 times to clean agnps the free entities and unbound biological molecules. The purified formed pellets will be dried at 50-60 °c and stored in a brown-glass container for further characterization. Characterization of the biosynthesized silver nanoparticles the obtained silver nanoparticles will be characterized using different advanced tools including uv -visible spectroscopy at absorption curve range between 410-480 nm.

Determining of size and shape of agnps by electron microscopy sem will be carried out. Particle sizing experiments will be carried out by means of laser diffract meter using zeta sizer nano-series nano zs the crystallinity of agnps will be confirmed by their xrd pattern. Ft-ir spectra will be recorded in the range 4000–500 cm?1. Uv-visible spectrometry measurement: Biotransformation of metal ions will be affirmed by uv–visible spectroscopy measurement.

Labomed uv–vis double beam within the wave length ranged from 200 to 600 nm will be used mourato et al. 2011 x-ray diffraction xrd measurement: Xrd technique will be used for examination of quality of the prepared nanoparticles. Xrd pattern of the obtained nanoparticles on glass material will be estimated in wide selection of bragg angles 2? At a scanning rate of 20 min-1. Fourier transform infrared ft-ir spectrometry analysis: Sample containing nanoparticles will be scanned by ft-ir spectrometry using a spectrophotometer. Ft-ir spectra will be scanned in rang 4000–400 cm–1 in ftir spectrometry at a resolution of one cm-1.

Transmission electron microscopy tem the morphology and size of produced nanoparticles will be determined using tem. Antimicrobial activity of the characterized nanoparticles antimicrobial activities of ag-nps will be performed by the agar well diffusion method in muller hinton agar plates selvamohan et al. 2012 human pathogenic bacterial species such as escherichia coli pseudomonas sp. Proteus mirabilis klebsiella pneumoniae and staphylococcus aureus will be used for the assay.

The bacterial species will be grown in muller hinton broth at 37 oc for 24 h. The bacterial growth will be prepared on agar medium and wells will be cut using sterile cork borer. In to the wells agnp will be applied at different concentration and incubated at 37 oc. The plates will be examined for appearance of inhibition zone and then their diameter will be measured and will be compared with standard antibiotic such ciprofloxacin.

Optimization of silver nanoparticles for large scale production and stable mycofabrication of agnps using fungi it is necessary to investigate the ideal physical and chemical parameters required for the production of effective and small sized agnps mishra et al. 2014 different parameters such hydrogen-ion-concentration ph temperature t °c concentration of silver nitrate agno3 and time t of reaction will be studied.

The absorbance of the resulting solution after color change will be measured using uv–vis spectrophotometer. For each condition respective controls will be maintained.the length of the text: 6244 (no spaces: 5243)get new reportthe uniqueness of the text: 65.1 %we strongly recommend not to use this text for academic purposes

What is the difference between an oxidizing agent and a reducing agent? The oxidation number (overall charge of the atom) is reduced in reduction and this is accomplished by adding electrons. The electrons, being negative, reduce the overall oxidation number of the atom receiving the electrons. Oxidation is the reverse process: the oxidation number of an atom is increased during oxidation.

This is done by removing electrons. The electrons, being negative, make the atom that lost them more positive. When first learning to balance equations, we learned that the number of atoms of each element in the products and reactants must be equivalent. What are some additional factors that must be taken into account when balancing equations for redox reactions?

Some additional factors that must be taken into account when balancing equations for redox reactions are: dividing the equation into an oxidation half-reaction and a reduction half reaction, multiplying each half-reaction by an integer such that the number of electrons lost in one equals the number gained in the other, and combining the half-reactions then cancel. What are half reactions? A half- reaction is simply one which shows either reduction OR oxidation, but not both. What two aspects of the half-reaction equations must be balanced?

Oxidation and reduction charges 5. For the equation Ag + NO3 – ? Ag + + NO (Note: This reaction takes place in an acidic solution. ) Step 1: What substance is reduced? NO3 Step 2: What substance is oxidized? Ag Step 3: What is the half reaction for oxidation? Ag ? Ag+ + 1e- Step 4: What is the half reaction for reduction? (NO3)- +4H+ +3e- ? NO + 2H2O Step 5: What is the net balanced equation? 3e- + 3Ag + 4H+ + NO3? 3Ag+ +NO+ 2H2O+ 3e- Step 6: What is the reduced equation? 3Ag + 4H+ + NO3 —> NO + 2H2O + 3Ag