How To Make Your pGLO; Transformation Of Fluorescent Sea-jelly Genes Into Bacteria

A) Transformation is a genetic process which means change caused by genes, and involves the insertion of foreign DNA into an organism to change its trait. Transformation is carried out by restriction enzymes to cut plasmid DNA and insert new code in its place, and heat shock (rapidly heating and cooling cells with calcium chloride) which will move the plasmid (DNA which contains one or more genes which would be beneficial to a bacteria's survival) into the cell. It also is a key instrument of biotechnology, and is used in numerous ways, such as, bioremediation (the genetic manipulation of bacteria to digest oil from spills), and medicine (use of gene therapy to correct defective genes). Also, another use of Transformation is to make bacteria glow by inserting the Green Fluorescent Protein (from Seajellies) into bacterial cells.

B) The purpose of this experiment is to insert the GFP gene into a plasmid, and move the plasmid into bacterial cells. This will cause the cells to produce Green Fluorescent Protein, and they will glow brilliantly. We hope to have a flourishing fluorescent colony by the end of this experiment.

C) In this lab, we will have four petri dishes: +pGLO and LB/amp, -pGLO and LB/amp, -pGLO, and LB, and the +pGLO and LB/amp/ara. We will use Transformation, which is carried out by restriction enzymes to cut plasmid DNA and insert new code (GFP gene) in its place, and heat shock (rapidly heating and cooling cells with calcium chloride) which will move the plasmid (DNA which contains one or more genes which would be beneficial to a bacteria's survival) into the cell. The controls of this experiment are the -pGLO, and LB petri dishand the -pGLO and LB/amp. The variables will be +pGLO and LB/amp,and the +pGLO and LB/amp/ara. I predict that the +pGLO and LB/amp will be the only bacterial colony to exhibit fluorescent characteristics. This is because it contains the ampicillin resistance gene, and the plasmid containing the GFP gene.

Discussing The Strategic Use Of Microarrays In The Study Of Lung Cancer Genes

A) Results: From the gene chip our lab group was able to determine that: Genes 1, 2, and 5 were active in in the cancer cells, Genes 2,3, and 6 were expressed in cells obtained from healthy tissue, and also that Gene 4 was express in neither the healthy cells nor the cancerous cells. We were able to tell this becauseof the colors of each gene on the slide. Genes 1 and 5 were pink (pink means expressed only in cancer), Genes 3 and 6 were blue (blue means only expressed in healthy cells) , Gene 2 was purple (this represents expression in both healthy and cancerous), and Gene 4 was clear (expressed in neither).

B) Sources of Error:
- Not enough of the cDNA was added, and the true color of the gene was not shown.
- The genes from the gene chip were not accurate, and we tested the expression of the wrong gene.
- We pipetted too much of the cDNA, and the slide overflowed, thus we were unable to get an accurate reading of the expression of the certain gene.
- We left the gel in the pipette for too long so it hardened and less than 20 microliters was exposed to the slide.

Translating Diseases; The Strategic Use Of Microarrays In The Study Of Lung Cancer Genes

A) Microarrays have simplified how scientists study gene expression in thousands of cells. This new technology detects patterns in transcription and translation, and aids in the understanding of the normal and abnormal aspects of cell function. The process of using Microarrays to analyze/study genes involving lung cancer include: creating a DNA chip, and using the DNA chip to analyze complementary DNAs isolated from both cancerous and noncancerous tissue (the same tissue). The Microarray will then indicate the differences between the two corresponding genes. This will be an indication of the extent to which transcription occurred. Some scientists have indeed observed that some genes are not transcribed as much in cancer cells as in healthy cells, and that some genes are not transcribed as much in healthy cells as in cancer cells.

B) The purpose of this experiment is for our lab table to use Microarrays to study Genes Involved in Lung Cancer. This will hopefully give our group a better understanding of the causes of the disease on a genetic level, and thus allow us to speculate with possible methods to prevent or cure it.

C) In this experiment, our lab table will be studying six genes using a Microarray. We will prepare the Microarray by spotting six different gene sequences onto a glass slide. Then we will obtain DNA tubes from a 70 degree water bath, and spot the appropriate gene solution onto the correct areas of out slide. To hybridize the Microarray, our group will add fifteen microliters of hybridization solution to each spot. We will then wait and observe our results when they appear.

D) Although we have the six genes that we are to study, making predictions is not possible because we do not have the background knowledge to hypothesize our results. However, I am quite certain that our group will accomplish something worthwhile, and we will learn valuable facts about gene expression and the effects of cancer on this.

The Sticky Ends Justify The Means Discussion

A) During Day one of the lab, our lab table acquired the DNA of the five suspects, and the DNA found at the crime scene. The purpose of the experiment was to identify the culprit by matching the DNA found at the scene with the DNA of the five suspects using Restriction Fragment Length Polymorphism. After acquiring the DNA, we added restriction enzymes to the tubes, flicked/tapped them, and placed them in a hot water bath for 30 minutes. On Day two we ran the newly cut DNA through a gel, and visually compared the samples using loading dye to stain the DNA (make it visible), and attracting the fragments to the positive end through Gel electrophoresis. Gel electrophoresis functions by pulling fragments of DNA (negatively charged) through the gel matrix towards the red end (positive), run to red. On Day three, our group was able to compare the DNA of the five suspects with the DNA found at the crime scene. We were able to positively identify Chloe Krey as the culprit in this crime scene.

B) Possible Sources of Error:
- We did not add enough loading Dye so the DNA fragments were not visual.
- Accidentally scraped off a skin cell into a test tube, and my DNA was analyzed instead of a suspect's DNA.
- We did not add enough DNA to the tube, and thus not enough substance was present for DNA to become fully visible.
- Accidentally added the wrong suspects DNA into the wrong well, and the wrong suspect was 'proven' guilty by the DNA evidence.
- Accidentally added the food coloring in place of the Loading Dye, and the DNA was not properly stained thus leading to an inaccurate reading.

The Sticky Ends Justify The Means; The Strategic Use of Restriction Enzymes In DNA Fingerprinting

A) DNA fingerprinting has revolutionized how crimes are solved, and has also aided in other applications. Since described in 1985, Restriction Fragment Polymorphism (RFLP) has acted as the factotum for DNA fingerprinting/profiling, and currently Polymerase Chain Reaction (PCR) is in use. The Restriction Enzymes are vital to the functionality of DNA fingerprinting. Restriction Enzymes are obtained from E-Coli, and the enzymes act as a natural defense against viruses. Restriction Enzymes work by cutting DNA at palindromes. To compare the desired strands of DNA, it is ran on a gel. The DNA differences are the fragments of different lengths. Running DNA on a gel produces different results, because gel electrophoresis is the use of electricity on a gel, giving it both a positive and a negative end. DNA has a slightly negative charge, so the fragments run to red. In the case of DNA, the smaller fragments go farther and the larger fragments remain closer to the original well.

B) The purpose of this experiment is to match DNA from a crime scene. During the experiment, our lab table will be attempting to match the DNA of the suspects with the DNA found at the crime scene. We hope to analyze the DNA correctly and receive experience in the field of crime solving.

C) The use of Restriction Enzymes in this experiment is vital. To analyze the various sample of DNA, and to successfully determine the culprit we will be using six samples of DNA: one from the crime scene, and five other tubes which contain DNA samples from suspects 1-5. We will be pipetting the restriction enzymes into the different tubes. We will then make a gel  (add correct ingredients, and mix it) and run the various samples with a dye to make the fragments visible on the gel. The smaller fragments will move farther, and the success of the lab will depend on the similarities between the crime scene DNA and the DNA from one of the five sample tubes. The Variables of this experiment will be the five DNA samples, and the control will be the DNA from the crime scene. We will attempt to visually compare and identify the controls with the variables to figure out which variable most matches the control.

D) The hypothesis cannot be produced at this time, because I have no knowledge of the suspects or the crime. However, I do predict success for my lab group and I as we use DNA fingerprinting in our special CSI lab.

Prolonging Life with Life Discussion

A)  Throughout day one the lab, our group attempted to record the effectiveness and efficiency of cellobiase on an artificial substrate which represented cellobiose. One day two of the lab, our group measured the effectiveness and efficiency of edible mushroom in turning cellobiose into glucose. We measured the rate of conversion from the artificial substrate (acts as cellobiose) to glucose by adding a strong base. The strong base turns the artificial substrate yellow, and stops cellobiase. To measure reaction rate, we labeled five tubes and added a fixed amount of artificial substrate and cellobiase (day 1) and a fixed amount of artificial substrate and our mushroom (day 2)., and added the strong base at set points in time. This allowed our group to discover that both were approximately even when it came to efficiency. Overall, my hypothesis was correct. The cellobiose was converted into glucose efficiently by both the cellobiase and the mushroom solution on both day one and day two of the lab.

B) Possible Sources of Error:
- When adding the strong base, we could have added it at the wrong time messing up the correct coloration of yellow.
- Failed to grind the mushroom up enough, thus the majority of the enzymes remained unable to produce glucose.
- Not enough cellobiase was added, thus a slower conversion occurred and inaccurate readings were obtained by our group.
- The centrifuge was not spinning for long enough to fully separate the mushroom solution, not enough of the mushroom was added to accurately convert the artificial substrate.
- Too much cellobiase was added, so the artificial substrate was converted too rapidly for our group to notice a change after three minutes.

Prolonging Life With Life; Extending The Future By Exploring Possibile Benefits Of Biofuels

A) The current fuel system is subject to change. The global economy is dependent on petroleum, which is a polluting natural resource which is destined to run out. Although the future seems bleak for the global economy, there is hope in alternate fuels. Some of the most promising of the prospects to replace gasoline are biofuels. Biofuels are the classification for fuels derived from biomass. In the wall of plant cells lies a polymer with a lot of potential named cellulose. Cellulose cannot be broken down naturally by animals, and this is the reason why it's importance for energy has not become relevant until now. Cellulose is broken down by the cellulose enzyme which is present on the inside of Protists. Enzymes are beneficial to reactions because they speed up chemical reactions and are not consumed with the reaction, so they can be used again and again. The majority of enzymes are proteins, and have a specific 3-D structure. For a chemical reaction to take place, the active site (site of chemical reaction) binds to the substrate (the reactant of the enzyme catalyzed reaction). The process is sped up because the substrate is positioned in a way which allows the transition state of the reaction to become stabilized, and the activation energy is lowered to allow quicker reaction rate. However, the conditions of enzymes must be kept within an optimal range, (the right pH, the right temperature, and the right level of salinity). For centuries, termites have broken down cellulose and used its energy as fuel, and not until recently, scientists discovered how the termites were able to accomplish this feat. The termites have a protozoan called Trichonympha living inside their abdomen, and the trichonymphba has a bacterium named Rs-D17 that lives inside it and produces cellulase enzymes, including cellulase. Cellulase enzymes convert the cellulose into cellobiose, which is converted into two glucose molecules by the enzyme cellobiase. Biofuels work by using glucose (energy) obtained from cellulose to power their engines.

B) For our class, the purpose of this experiment is to measure the enzymatic activity of cellobiase, and to identify the optimal conditions for the enzyme. This also acts as a real world example of the possibilities behind enzymatic studies, and the relevance in the possible future. For industry, the research of Biofuels can offer an alternative energy which would be more sustainable than petroleum. The biofuels offer a new future for industry, one which could make life less polluting, more sustainable, and more reliable.

C) The lab will start with a one milliliter pipette of cellulose. Cellulose is broken down by bacteria, protists, and fungi, so we will use enzymes derived from these sources to convert cellulose into cellobiase. Cellobiase is composed of two glucose molecules, and will be the substrate of the experiment. Cellobiose will be placed in a test tube along with Cellobiase, an enzyme which breaks down cellobiose into single glucose molecules. To measure the effect of the cellobiase on the cellobiose, we will substitute the cellobiose with an artificial substrate. The artificial substrate will be composed of Glucose and P-Nitrophenol. At various time points, we will add a strong base to the test tube which kills cellobiase, and turns P-Nitrophenol yellow. This will allow documentation of the coloration of the test tube, and we will be able to drw a conclusion from this data. For extended exploration in this experiment, we will bring in mushrooms to test which mushrooms are better decomposers. We will grind up the mushrooms, and add them in place of the cellobiase (add them with the Glucose and P-Nitrophenol). This part of the experiment will give us a greater understanding of the cellobiase content in the foods that we eat, and also in some of the mushrooms that we see.

D) I believe that the cellobiase in Day 1 will make glucose disappear faster than the ground mushrooms which we will add in day 2. The controls of this experiment are the Cellobiase and the artificial substrate (Day 1), while the variables of the experiment will be the ground mushrooms and artificial substrate (Day 2). The cellobiase acts as the control, because cellobiase will definitely separate the artificial substrate at a quicker pace with its higher concentration. The ground mushroom acts as the variable in this case, because we are unaware of the cellobiase concentration within the organism, and we are charting the difference between cellobiase and the mushroom.