GMO: Get More Output (testing genetic modified organisms)

Background:
With the rise of the world's population, more food is needed to satisfy the human race. Not only do our crops need to yield more, but they must fight infection and pests, last in extreme weather, and grow faster to meet demand. This is where genetic modification has its role. People opposed to GM foods have concerns that geneticists can create a super-weed or super-bugs that are resistant to toxins. Also, the concern about the general populations having individuals that can be allergic to the new proteins that are added. Although valid points, GM foods supply more benefits than harms, such as helping the environment by lowering chemica use and utilizing land that does not have enough nutrients for normal crops.GMOs are made by taking an organism that has plasmids that can be manipulated. Using a tumor inducing plasmid with a gene that expresses a desired trait, the plasmid is placed in an agrobacteria to grow. Then, the bacteria is placed into the plant cell, which will grow into a genetically modified plant. GMOs can be identified through the enzyme-linked immunosorbent assay, or ELISA, which identifies proteins. This process is very specified according to crop, but it is inexpensive, accurate, and easy to use. The other process, which will be used in this lab, is the polymerase chain reaction, which identifies sequences of DNA that have been inserted into GM plants. PCR tests can be applicable to many crops rather than one. The PCR process uses a primer to target the specific genes. Genetic modification is a controversial subject because on the opposing side, the argument is made that genetic modification can create "superweeds" from cross-pollinating normal plants with GM plants. "Superbugs" could evolve and become resistant to chemical toxins in GM crops as well. If genes are taken from other organisms, some fraction of the population will be allergic to the GM crop. On the side for genetic modification, GM production lowers pollution through pesticide and herbicide use and utilize land that would be previously insufficient to sustain crops.
Purpose:
Our purpose for this lab is to test if the foods that we consume are genetically modified. These tests can be used to see if a farmer's crop is really organic.
Procedure:
We must first get to the DNA within plant cell by grinding up the food with a mortar and pestle to break down the thick cell walls. Eukaryotes do not want foreign DNA within the cell, so they have DNAse that kills them. Since we want to protect our 'broken out' DNA, we add the instaGene matrix which kills DNAse. Then, we put the slurry of our food into a hot water bath to break open the nuclear membrane. The lab will put non-GMO material as a control and a variable food that could or could not be a GMO. Guarding against contamination, non-GMO food will be provided that gives false positives results. If it tests positive, that means the sample was contaminated. We will use a red primer to detect GMO-specific sequences and a green primer that detects all plant DNA. This way, we either get two results, the food is not a GMO or that DNA was not successfully extracted.
Hypothesis:
Our variable is our food that we bring in for testing. My hypothesis is that our food and most foods in the classroom will be GMOs
Results:
After viewing our gel, we found that our test food (broccoli) was genetically modified. We know this is true because the brocolli with gmo primers had the same band width as the GMO control and the genetically modified corn starch. If it was not a GMO, then lane 1, having plant primers, would have a band instead. The plant primers are looking for specific DNA codes that are unmodified, therefore, our broccoli was genetically modified.
Sources of Error:
The hot water bath was left open for some time because of other lab tables. Pipetting into the lanes proved to be messier than anticipated, but we still put a sufficient amount in each well. Otherwise, all the controls seemed to do their jobs, and since they showed positive results, we executed the lab sufficiently.

How Low Can You GLO? (pGLO Transformation)

Background:
    Genes are pieces of DNA that code for certain traits. By definition, genetic transformation is when an organism's traits are changed by way of an inserted, manipulated gene. Agriculture, bioremediation, and medicine, are all fields for which genetic transformation is applicable because plants can be modified for better harvest, bacteria can help clean oil spills, or transform a sick person's cells to healthy ones. For the genetic transformation in this lab, we will transfer a gene to the circular pieces of DNA in bacteria called plasmids. These plasmids contain beneficial genes for the bacteria's survival, so when a new environment is introduced, it can adjust by transferring DNA with its environment. The gene desired for expression in the bacteria is the Green Fluorescent Protein, or GFP, found in bioluminescent sea jellies.
Objective:
     The objectives are to genetically transform bacteria to express the GFP gene. If we are successful, the bacteria should glow in the dark.
Procedure:

• Label one test tube +pGLO and another =pGLO, label with table number
• Open the tubes and transfer 250 microliters of CaCl2 (100-1000 microliters)
• Place the tubes in ice
• Pick up a visible clump of bacteria with a sterile loop, immerse the loop in the transformation solution in the +pGLO tube, no chunks!, Place the tube in ice. Repeat for -pGLO
• Add 10 microliters of plasmid DNA directly to liquid in +pGLO tube only, flick tube to mix, close tube and place in ice. DO NOT ADD PLASMID TO -pGLO tube
• incubate the tubes on ice for 10 minutes, make contact with the ice
• Labels: LB/amp plate: pGLO; LB/amp/ara plate: +pGLO; LB/amp plate: -pGLO; LB plate: -pGLO
• place the + and - pGLO tubes into the water bath at 42 degrees Celsius for 50 secs. Place tubes back on ice after. Incubate tubes for 2 minutes.
• Add 250 microliters of LB nutrient broth to the tube and reclose it, repeat for each tube, incubate for 10 minutes
• Tap closed tubes with finger to mix. Pipet 100 microliters of transformation and control suspensions onto the appropriate plates.
• Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the agar by skating across back and forth.
• Stack up the plates, tape them together, put table and period, place in incubator upside down!
• Clean up and return all supplies

Results:
Due to the colder environment or the insufficient plasmid, the bacteria failed to glow. The bacteria grew slightly.

DNA Chips: Genes to Disease

Introduction


Background:
     In order to fully appreciate the contributions of present, microarray analysis, the collaboration of genomics, computer science, and nanotechnology has expanded what was known about genetics. What would have taken centuries to map and decipher, long codes of genes can be compiled into a microarray chip for analysis. Each chip starts as a solid matrix, like a glass slide, and is imprinted with a specific pattern of designated zones, all containing a specific oligonucleotide representing part of a genome. Granted that this technology can be used to compare different species or families, the more practical use is for cancerous and healthy tissue comparison. By dying the different DNA strands, geneticists can compare which genes are active in cancerous tissue and which are turned off, the same applies for healthy tissue. By synthesizing this data, they can find which genes may be responsible for the cancer, which may lead them to the cure or specified treatment.
Objective:
     The objective for this lab is to familiarize students with how six particular genes are expressed in healthy and cancerous tissue, in essence, the basics of microarray technology. By using lung cancer tissue this opens the door to comparing several diseases and possibly their causes
Procedure: the following steps are to prepare a microarray slide

1. Take 6 tubes of different genes from 70 degrees Celsius water
2. Pipette 20 micro-liters of each of the gene samples onto the slide
3. Then add 20 micro-liters of hybridization solution (cDNA) to each of the spots
4. After solution is added, the color of the gene samples, will change to different colors 

Hypothesis:

     I think that there will be varied colors, and genes expressed in healthy and/or cancer tissue, but results can't be predicted

Results:

    Questions summarized the results of the experiment. As seen in the video, the microarray we created contained not only solid pink and solid blue, but purple as well, suggesting both healthy and cancerous tissue have this as an active gene.

No discussion for this lab.

CSI: AHS

Introduction

Background:
Current forensic study involving criminal activity is now heavily dependent on the practical use of DNA identification and comparison. It is important to rule out subjects who are innocent. Because they are all different people, so is the size of their DNA, thanks to restriction enzymes. These restriction enzymes act as molecular "scissors", that cut specific sequences of base pairs. Geneticists and criminal detectives both rely on this DNA sequence length to differentiate specific DNA from others that may be found. Developed by geneticist, Alec Jeffries in 1985, the practice of Restriction Fragment Length Polymorphism, or RFLP, has become the forefront for genetic profiling. The best application of this process can be found in agarose gel electrophoresis. Electrophoresis means to carry with electricity, which is key because DNA is negatively charged. When placed in the agarose gel, the DNA will travel toward the positively charged anode of the electric field. The speed in which specific DNA travels through to gel is inversely proportional to the size of the sequence of base pairs. After being stained, the template of the resultant DNA can be compared with the subjects that may have executed the crime. Since DNA is present in all cells of the human body, skin, blood, and other tissues can be used for comparison, making it easier to catch the culprit. Other uses include: food purification, identifying human remains, proving convicted inmates innocent, human relation to other species, ancestral relation, identifying lethal traits in organisms, paternity testing, etc.

Purpose:
The purpose for this lab is to have a better understanding of how DNA is fragmented, how DNA can be used for profiling and how to perform the process ourselves.

Procedure:

1. Place the restriction enzyme mix in ice
2. Label 6 tubes, first is CS, and the other 5 are S1 through S5. Give each an independent color, name, date, lab period. 
3. Take 10 microliters of the DNA of each suspect and place accordingly. Be sure to use a fresh tip every time
4. Add 10 microliters of enzyme to each and mix well. 
5. Close each tube and place them into the centrifuge
6. Incubate the tubes overnight
7. Pour 1% agarose gel
8. Refrigerate samples

Day 2:

1. Take samples from the refrigerator and put them into the centrifuge
2. Put 5 microliters of of loading dye into each tube and use the centrifuge once more
3. Take the agarose gel from the refrigerator and place it in the electrophoresis apparatus
4. Do a double check to see that the electrodes are touching the gel
5. Load the samples into 7 wells

They are designated in the following

• Lane 1- M, DNA size markers, 10 microliters
• Lane 2- CS, 20 microliters
• Lane 3-7- S1-S5 in order, 20 microliters each

Hypothesis

• Lizzie's eyes have been really shifty since day one. I'm afraid... I think she's the killer.

Due to a malfunctioning lab computer, our video was not loaded but shall be handled soon. I apologize for the wait.

Results:

After comparing the sets of DNA to the DNA in lane 2 from the crime scene, Lane 5 is the closest match to the crime scene DNA. That DNA belonged to Chloe (much to my surprise). Justice was pleased that day. 

Discussion:

This lab was awesome in the fact that it is more of a real world application than any other lab I have done in this class or previous classes. It intertwined the use of the class and proper technique for the pipets and the electrophoresis machine. I have a better understanding of how DNA is sorted based on the different lengths of the cut strands. More crime solving will be accurate if DNA becomes a standard for all organizations. Although I was gone for Thursday, It was still fun to guess the killer. Although Lizzie was innocent, I will be more cautious. 

Biofuels Lab

Introduction:
     In this lab, we will discover how to make biofuels. Biofuels are substitutions for the world's current fuel source, crude oil. Using the biomass of plants, scientists can synthesize cleaner fuel for the environment. By converting to biofuel, the plants that grow can clean the air of the carbon dioxide that is released from combustion. Granted that the harvesting of some products, like corn, for other than food, can create scarcity or market competition, the benefits outweigh the costs. If scientists genetically modify a plant that is specifically grown for this need, then competition will be less of a problem, and countries can more thoroughly fight crude oil dependency. After engineering said plant, scientists need to convert the biomass into usable fuel. In nature we have mushrooms that decompose material with their enzymes. These enzymes can be used for the genetically engineered plant to break down the cellulose and convert it into glucose. Through the process of microbial fermentation, ethanol can be harvested. The various types of fuel can be the new source of power in the future.
Purpose: Students, like us, become familiar with terms such as substrate, enzyme function, catalysts, and biofuels. Learning how enzymes are used to break down organic substances can be key for future in a career working in labs.
Procedure:
     Normally, the video would show the procedure of the lab, but since there are technical problems, i will summarize the significance of each step.

1. We will use a stop solution, an enzyme, and a buffer.
2. We will take 5 cuvettes to show the enzyme's product increase over time
3. Using the pipette, we put 2 ml of 1.5mM substrate into two conical tubes, one being the enzyme reaction and the other a control. 
4. We then took 500 microliters of buffer solution and mixed it with the control test tube.
5. Then we pipetted 1 ml of enzyme into the enzyme reaction tube.
6. After several intervals of time, we took 500 microliters of the enzyme reaction and put them in chronological order in the cuvettes.

Day 2 proved to be similar, except mushroom paste was added to increase the reaction rate of the reaction so that more products were made in less time. The natural enzymes in the mushroom used for decomposition represent the natural materials that can be used to create biofuels.

Results:
     The results were as predicted with the increased darkening over time of the yellowish color of each progressive sample of product. The mushroom extract in day 2 aided in the production times. We learned that the chemical reaction would not continue forever because eventually the production rate would plateau, due to  a lack of resources, therefore the reaction has limited reactants.

Conclusion:
     Overall, the lab gave a good review of chemical reactions and proper lab measurements. Biofuels are an important subject to teach early because future generations may not have the fossil fuel option that is currently being used. Nature presents a ready solution for an energy problem, so taking the opportunity for a clean, progressive movement is vital.

DNA Precipitation Lab

Introduction:
    The genetic building block of all cells in the human body is deoxyribonucleic acid or DNA. DNA is composed of the deoxyribose sugar, a phosphorous group, the four bases adenine, cytosine, guanine, and thymine. The basic molecular components of cells, besides nucleic acid, are lipids, carbohydrates, and proteins.The cells themselves contain the entire set of DNA, but certain cells express different genes within the DNA. The expression of genes gives humans variation in their attributes, like body type, pigmentation, eye and hair color, etc. This D.N.A. is located in the nucleus, so many of the macromolecules stand in the way of retrieving said D.N.A. for observation.

Purpose:
    The lab teaches students how to precipitate DNA, which in normal terms means, more or less, solidify. Precipitating DNA allows studying the DNA and its traits, comparing other DNA, mapping it or sequencing the DNA, cloning, and testing for diseases.

Procedure Reasoning:
    First the student chews a little bit of the inside of their cheek to loosen the cells so the cells can be harvested for their DNA. The student then rinses out with a 0.9% saline solution which is an ideal salt concentration for the cells so the DNA does not break apart. Then the lysis buffer is added to break open the cell membrane, which is made of lipids. Lysis buffer breaks the membrane because the membrane is soluble in other other liquids besides water. Protease is then added, which is an enzyme that kills the enzyme DNAse. DNAse must be gone when the DNA is released because DNAse kills all DNA, due to the cell's evolution of its defense against foreign DNA. Adding table salt to the DNA solution shifts the proteins from hydrophillic to hydrophobic, which will break them away from the DNA in the nucleus. Placing the solution in a hot water bath denatures the protein, therefore speeding up the process. Finally adding the cold ethanol solidifies the DNA and it becomes much more visible than before.

Overall the lab was entertaining and we learned about DNA, some enzyme functions, and necklace assembly all in one.

Unfortunately, a video could not be constructed due to technical problems and fragmented content

The Yogurt Lab

Introduction:
   In order to understand this lab, it is important to understand yogurt. Yogurt is the product of a certain bacteria that changes the properties of milk into a more viscous form. Bacteria in itself is a single-celled organism that reproduces by binary fission. This means that it splits apart to copy itself and can do it quickly with the right temperatures and food supply, which makes it a very durable organism. Although bacteria does not have DNA like humans, it is found all over the planet and can be considered one of the most successful organisms. Bacteria can survive so well that scientists believe that bacteria would be able to live on other planets or moons with harsh conditions. Over the years, bacteria has been associated with harmful disease, when in fact a small amount is harmful to people. With the help of antibiotics, modern medicine took a leap forward in fighting disease, but the new 'clean' world has created super strains of bacteria that are much harder to kill, like MRSA. Without bacteria, we would not have digestion aid, waste decomposition in nature, or foods such as cheese, some breads, and of course, yogurt.

Purpose:
First, the two main objectives are to understand bacteria's role in yogurt making and to test Koch's postulates.
They are the following:

• Distinguish the "infected" population from the non-infected. Diagnose the infected and recognize differences
• Discover what bacteria is causing the abnormal change
• After discovering the cause, re-introduce said bacteria with a "pure" culture
• Reevaluate the culture for the same abnormalities that were observed, to confirm the bacteria as the source

Procedure:
The following procedure was done at home using materials that were readily available

1. Take about 50 ml of milk and heat to 80°C to kill any bacteria that may be in the milk
2. Add one eight of a teaspoon of yogurt when the milk has come to room temperature so that the bacteria is not killed in the hot temperature. 
3. Cover the milk so it is air tight and wait until it becomes thick.
4. Keep in a warm environment to help the process, an incubator at best, but a laundry room works as well. 

Controls and Variables

Having plain milk in the fridge was the control, the yogurt was testing the bacteria's effects, and the E. coli that  I observed from a friend was to test whether all bacteria makes yogurt.


Hypothesis:
The milk will not change unless there is a sudden change within the fridge. The yogurt added to the milk will convert the milk into yogurt by changing the lactose to lactic acid. The E. coli will not make yogurt.

Results:
The milk with yogurt thickened substantially, but it is not at correct "yogurt" consistency. The milk stayed the same, and other groups said that the E. coli made the milk spoil and did not thicken.

Discussion:
Overall, doing the lab at home was very informative. I did not try the yogurt yet, but I'm sure that it is fine. One source of error may include possible bacteria from the air when mixing ingredients. Through the evidence of others and mine, not all bacteria makes yogurt, and there are many benefits for bacteria in everyday life.