Glow Bacteria, Glow!!

       Genetic Transformation will be the primary objective of this lab, which involves the insertion of a gene into another to alter its genetic make-up, usually for a particular phenotype change. Since genes are universal in all organisms, they can be carefully tampered with to transform a particular organism's trait. For example, in agriculture, if you take the gene that codes for drought resistance (maybe from a cactus) and insert it into a corn cell's DNA, then the corn cell will in effect produce baby corn with the drought resistance gene. In the world today, this can be very controversial. This topic goes into depth for a number of reasons but one negative effect of this might include the limited genetic diversity of the certain species. 
     Specific to this lab, we will be using the gene from the sea jelly (Aequorea Victoria) that encodes for the Green Fluorescent Protein, or GFP. After the insertion of the gene into the bacterial plasmid, or circular piece of DNA, the bacteria should hopefully glow a fluorescent green under ultraviolet light, like the sea jellies do. Plasmids in bacteria are essential to their survival. Usually, plasmids contain one or more genes that code for certain traits that can be beneficial to their development. Bacteria in nature can naturally transfer plasmids from cell to cell, which also explains the recent occurrence of antibiotic resistance. Bio-Rad's pGLO plasmid which we will be using has the GFP coding gene, as well as an ampicillin resistance gene. However, when inserted into the bacterial DNA mix, the pGLO doesn't just light up the bacteria. The addition of a control, a sugar aribinose, must be added to "switch on" the GFP gene. Without the sugar, the brilliant glow will not show. 
       Some possible sources of error include interferences from the environment such as spores or fungi for example, or if the setting is right for the proper bacterial growth. 
       The results were extraordinary, yet somewhat depressing as well. As usual, Mr. Chugh's sample is perfect, yet the rest of the classes' bacteria was stunted in growth by the very low temperatures over the weekend. The environment was not ideal for the bacteria to grow, so most of our specimen were destroyed. However, under nothing but ultraviolet light, Mr. Chugh's bacteria were glowing! They were shining brighter than I could've imagined. 



       

Case File: DNA Fingerprinting

     Dna Profiling involves the identification of a person's DNA. How do we do this, if 97 percent of our DNA is all the same? DNA Fingerprinting, or profiling, is the process by which we use to differ one person's identification from another. It involves small segments of DNA called microsatelites, which repeat throughout the DNA numerous times. They are ideal for forensics because they tend to be highly variable. Restriction enzymes, which come from bacteria, cut DNA into multiple fragments if they recognize sites that are repeated in the DNA, and thus creating a unique banding pattern in every peice of DNA. The location of restriction sites decide the length of each fragment. Agarose gel Electrophoresis is how we can measure the length of the bands. The pores in the agarose gel act as small passageways that the smaller segments of DNA pass through more easily than larger ones. The reason the DNA segments travel, that were cut by restriction enzymes, is because they are attracted to the positive pole of the gel. DNA is obviously negative, and opposites attract. The DNA is then stained and can be seen in the gel.

     More specific to the overview of this lab, Restriction enzymes are used to cut the DNA into palindrome sequences. These cuts will produce RFLP's, or restriction fragment length polymorphisms, which are specific patterns that the enzymes cut in someones DNA. The following step involves "running a gel", which involves cutting the gel into a square peice with little divets. The DNA, by pipet, is injected into the divets of the agarose gel and will travel to the opposite end (positive pole). The DNA will be stained and recorded as different patterns in the gel.
     The Crime Scene case file will be solved by running 6 segments of DNA through the agarose gel. One of them is the DNA found at the scene (the victim). The banding patterns of the 6 DNA fragments will be compared. Two of them will match up and we can finally punish the accused suspect that committed the crime! Some sources of error involve getting DNA mixed with other columns before the overnight banding patterns begin to develop. Another might be putting the DNA into the wrong columns.
     My hypothesis on this lab is practically useless, but I will take a wild stab. Number 4 will be the culprit. However, the results will show the real criminal and the reason we will know, if our work was not incorrect in the making, is because the banding patterns in the gel will match up.
     The results have been recorded. Katie Records, a known local person convicted of several felonies, was the criminal. The banding patterns of the original given DNA matches up almost identical to the number 3, Katie Records.This lab has not only increased my knowledge of how modern DNA forensics are practiced, but has led me to conclude that society can have an efficient system of DNA records to help in crimes today.

The Power Tool of the Future: Biofuels

     A basic overview of the process and understanding of biofuels: the Biofuel Enzyme kit measures the activity level and optimal condition cases of the enzyme cellobiase, which breaks down cellobiose, which is crucial to the process of making cellulosic ethanol. This cellulosic ethanol is an alternative replacement for petroleum.

     Enzymes, which speed up reaction rates of various processes, work again and again because they are not actually involved in the reaction. Long live the enzyme! In an enzyme catalyzed chemical reaction, the reactant is called the substrate. The substrate fits into the enzyme at the active site, which is a cleft in the protein and the substrate's chemical groups are attracted to the enzyme's amino acids facing the substrate. Different enzymes have different capabilities of maintaining their internal environment, even with changes in salinity, pH and temperature. Most enzymes have a decreased effectiveness when these are changed. Enzymes usually function best at moderate tempuratures (20-40 C). The concentration of enzymes present and the concentration of substrate present alters the reaction aswell. The more enzymes for that specific reaction, the faster it will take place. The more substrate, the increased amount of product production rises. Conditions to which enzymes preform under different reactions depends on the way they are found in nature. In other words, the behavior of enzymes in a reaction depends on what the enzyme is "used to", if you will, in nature. For example, the enzymes in a person's stomach work best under conditions with a significantly low pH.

     Cell walls in plant cells are very hard to break down. The enzyme callulase, breaks down cellulose like a chram. Cellulose is the main structural component in a plan'ts cell wall. Animals such as cows that eat grass, have to break down cellulose. They contain a bacterium know as Bacteroide Succinogene which digests the plants cows consume, and produces cellulase. Why is it important to break down the cellulose in plants? The breakdown of cellulose, which is catalyzed by cellulase, is converted to glucose, one of the main food molecules for a huge majority of different cells.

     How does glucose convert to a fuel, ethanol, that can power our present day automobiles? The process is quite simple. Cellulose (with cellulase to speed up the reaction) is made up of long strands of glucose, held together by cellobiose. The cellobiose is broken down by cellobiase, which then ultimately leaves single glucose molecules. The energy from the glucose molecules is used to make ethanol, or the alternate source of fuel. However, in the process, glucose is practically invisible, so glucose is mixed with P-Nitrophenol as the alternative substrate. Then, a stop solution (strong base) is added and this kills enzymes (such as cellobiase) and turns the p-nitro yellow, and the glucose is now visible.

     We are doing this lab because it is important in our world today to further study the possibilities of advancement of alternative biofuels. The biofuels will help replace depleting fossil fuels and other unreliable sources. My opinion is that more scientists from around the world should work together and compare results to create an effective method of producing the selective breed of biofuel that is more effective in production than the others.

     DNA, Deoxyribonucleic Acid is the hereditary material for life itself. It is unique in every single person, bacteria, plant, or other cell species. Who we are and why is the fundamental understanding of this nucleic acid. DNA nowadays can be used to help cure diseases, making better products to help people in all different areas from biological sources, and maybe one day to extend life itself. The DNA molecule is constructed of a sugar-phosphate backbone, with the four bases, Thymine with Adenine and Guanine with Cytosine. The sugar, Deoxyribose, and the Phosphates make the backbone strong. The shape of DNA is a double helix and the sides run in opposite directions (figured out by Francis Crick). The bases are organized to make messages that are delivered to cells called genes. These genes contain the information to make proteins, which lead to the body's structures and functions. DNA are arranged in chromosomes, or long strands of DNA tightly coiled around a protein. Chromosomes are split apart and copied in cell replication. All 46 chromosomes in the human body make up one's genome. Even though DNA contains the directions for protein synthesis, it does not carry it out. The templates for protein synthesis is mRNA, which carry the instruction to ribosomes, which manufacture the proteins. The ribosome decodes the information and links amino acids together in specific orders to make up the proteins.
     The purpose of the DNA necklace lab is to understand more about the structure and function of DNA, help explain its importance in life, understand the genetic combinations that could occur within our bodies, and how to extract DNA from cells using chemical reactions and certain steps.
     The purpose of the chemicals and their steps in our lab was to extract the DNA from their nuclei in cells. They broke down the plasma membranes, helped maintain pH, killing DNase, which is found "out in the world" beyond the cells that break down DNA. The use of the cold alcohol is to precipitate the DNA so we can put it in our necklaces, and so its visible. The use of the micropipette allows us to use precise measures when preforming our experiments.
    

Yogurt: Bacteria you can eat

       Microbial diseases have been on the minds of scientists for the past 200 years. Until Robert Koch, studying them has been but a mystery to the many scientists on their tail. Bacteria are probably the most abundant life forms on earth, and come in various sizes, shapes, characteristics and natures. Bacteria as pathogens can cause harmful and sometimes fatal sicknesses, such as scarlet fever, tuberculosis, and brucellosis, but many forms are extremely beneficial to health. Bacteria divide very rapidly and grow their colony size quickly to a visible level. The shapes of the colonies under a microscope can include circular, irregular, or branching. Using enzymes, or large proteins that quicken reactions by lowering activation energy, bacteria break down sugars chemically into other molecules. This process is called fermentation, and is their main food source. Specifically, Yogurt forming bacteria break down lactose to pyruvic acid, then to lactic acid , which lowers the pH of the milk. Lower pH levels allow the milk's proteins to curdle and become denaturized to give it the more solid texture we call yogurt. Like other beneficial Bacteria, yogurt helps break down organic waste material the body digests when it consumes food. Other types, like Intestinal bacteria, synthesize nutrients and break down indigestible material.Bacteria are very important in maintaining a healthy lifestyle, not that we can do anything about the billions that live within us.
       Specifically, yogurt is made by fermenting milk which contains specific strains of bacteria, all under controlled temperatures. The Bacteria feed on the natural milk sugars, passing on lactic acid as a waste product, lowering milk's pH levels. This increased acidity leads to entanglement, or denaturation, of milk's most common protein, casein. This gives yogurt its texture. In our lab, we will be using Koch's postulates to grow and test our yogurt bacteria, as well as ampicillin. Koch's postulates include first, describe the symptoms shown. Second, isolating the pathogen and establish a pure culture, then infect a healthy species to identify if the same symptoms occur. Finally, be sure that the pathogen is the same as the first.
       Our lab will be starting from Koch's third step, since we already know the bacteria causes yogurtness. This basic procedure will summarize how bacteria is used in some common foods we eat today. It will also explain the nature of  some common bacteria.

       My prediction is that, without any error or improper technique, the yogurt bacteria will create a well established yogurt texture in the milk. When working will E. coli, I will have to be very important in using sterile technique because it can be dangerous. I predict the nature of the E. coli will act much differently then the Yogurt bacteria visibly and what the actual bacteria consumes and produces.
       A summarization of the Procedure starts with explaining the key steps of the lab. After all of the test tubes were labeled, we have to make sure we not only stir well the different bacteria into the milk, but that we put the right bacteria into the right test tube. Proceeding the contamination of the bacteria to the milk, we have to place the test tubes into the incubator for  the correct environment for growth of the bacteria types.
       When we mixed the bacteria into the milk, there was no immediate effect, unlike a chemical reaction. As for the results the following day, here is the list. Test Tube 1, the positive control of just milk, smelled like sour milk, and had the same texture as before, liquid. Test Tube 2, the negative control or the yogurt in milk, transformed into a yogurt texture at the bottom half of the tube, and it smelled like regular milk. Test Tube 3, the yogurt + ampicillin, had the same results as Test Tube 1, same smell and texture. The E. Coli in Test Tube 4, had the same results, with a slight alteration in the smell. It didn't smell sour. As for color and pH, the milk, yogurt+ampicillin, and the E. Coli test tubes all were off-white with a pH of around 6-7. The yogurt had a more solid color and a lower pH of about 4.5.
       The reason the yogurt test tube was more solidified, and a more white color was because the bacteria of yogurt fed on the milk sugar, casein overnight and broke down the proteins, causing them to denature and spread apart (curdle). The ampicillin nuetrilized the yogurt bacteria, the E. coli bacteria has its own nature and doesn't feed on milk sugar, and the regular milk just maintained its color texture and pH. The reason the pH drops for the yogurt is because the bacteria can only properly ferment under those conditions, so the bacteria lower the level.
       The possible sources of error could have resulted from many things. These could include mixing the wrong bacteria into the right test tube, not labeling them in a color we would remember so we could find it in the incubator the following day, or mixing up the litmus paper when we took the pH's, which could have confused our results.