Enzyme-Linked Immunosorbant Assay, It's protein, not DNA

Intro:
 Since early mankind, humans have had two constants, pregnancy and illness. It wasn't until 20 years ago that ELISA was created to test and determine both. In order to combat antigens, antibodies must be made using this process. First, the target antigen is placed at the bottom of a well. Then the primary antibody is added through a serum. If the antibodies match with the specific antigen, then they will attach, if not, they will remain unbound. After washing the wells with serum, the secondary antibody enzyme is added with an enzyme attached to the antibody which acts as an indicator. Adding substrate to the well will determine whether or not it is our target antigen because it will activate the enzyme to give a color change.

Purpose: 

     The purpose is to expand our knowledge on disease identification and how the ELISA process works. 

Procedure: (Review from the Intro)

1. Insert the antigen into the well.
2. Primary antibody is added, (Wash and rinse)
3. Secondary antibody is added, (Wash and rinse)
4. Substrate is added, check for color change

Discussion:

     At our lab table, we discovered that Ayan and I were not infected, but Lizzie and Elizabeth were infected. Overall the class had a healthy balance of infected and non-infected individuals. Some possible sources of error could include leaving some floating antibodies in the well even though it was washed or reusing pipette tips. Floating antibodies would give off a color change and reusing pipette tips would redistribute infected fluid to non-infected wells. After narrowing the results using correct timeline deciphering, we got four candidates and it was revealed that it was Taylor and Chloe. 

Sushi, Sashimi, It's All Protein to Me...

Background:
     Geneticists have discovered that the Human Genome is comprised of 22,000 genes. At first, they were puzzled as to why so few genes could create the complex being that is human. The answer was in protein formation. The role of each protein can be determined by when and where a protein is manipulated or expressed. Seeing this complexity gave scientists a new field of study, proteomics. Proteomics covers the structure and function of proteins, creating an even more complex field than genomics. Proteins are exponentially more difficult than genes due to cell location, cell environment, cell life cycle, or cell type. Varying protein types can come about by the following processes: alternative splicing of exons, use of different promoters, posttranscriptional modification, translational frameshifting, posttransitional modification, and RNA editing. Proteomics is practical for mapping organisms' evolution and interspecies relations.

Purpose:
      To compare the different protein makeup of varying organisms so that we can better understand the evolutionary process. By tracing similar proteins, we can trace similar ancestry.

Hypothesis:
      My hypothesis is that they will all be similar according to family, but there could be variance. I think the crustaceans and fish will vary.

Procedure:
      Since all muscles of organisms are made of myosin and actin, we can break them down, or denature, by a heating process. After being broken down and stained, it can be run through an electrophesis gel so that they can be compared under a white light. By looking at the bands, we can determine ancestry and further extend the evolutionary tree

Results:
      Looking at the gel, we could tell that the crustaceans were more similar than the fish, but because the crab was cooked, it showed deformity in the gel as a huge smear. This is obvious because the protein was already denature from being cooked beforehand. The fly DNA did not show up because there wasn't enough fly protein to create a sufficient smear.

Who's Your Momma?...Mitochondria

Background:
     Since the first signs of life, single-cell organisms have contained DNA that gave the origin of their existence. Mitochondria is an example of one of those organisms. It is believed that the mitochondria used to be free-living cells that were captured by eukaryotic cells. This created a symbiotic relationship for the eukaryotes and mitochondria, one gaining nutrients and the other gaining a mechanism to use oxygen for energy. Mitochondria have a unique set of DNA (37 genes) that result from single nucleotide polymorphisms (SNP), which can be used to trace ancestry. In fact, they did. At UC Berkeley, they discovered by tracing generations to a common ancestor, modern humans came from Africa, 200,000 years ago. They realized that the mtDNA is only transferred by the mother to the egg cell. This mtDNA is very important to analyze because it can identify unknown human remains, trace generations, and relate different species to one another. Some disorders may  occur when some mitochondria in an organism's cells do not match up with other cells in the body, called heteroplasmy. These disorders occur in adulthood, as opposed to homoplasmy, which stops embryonic development in the first place.

Purpose:
The purpose of this lab is to understand and better understand mitochondrial DNA and its purpose.

Procedure:
For this lab, we extract our DNA the same way we did for the Disease Gene lab, involving saline solution, instagene matrix, and PCR. The new material to this lab is the mitochondrial DNA. We will use primers to bracket the mt  control region, which will only copy the the specific section of DNA. Afterwards, we will do the same gel electrophoresis that was done in the previous lab to tell us how similar our mitochondrial DNA is.

Results:
Based on our gel results, we saw that the mitochondrial DNA had almost identical bandwidth. This is not a surprise because 99.9% of human DNA is identical. We will send the rest of our DNA samples to the lab for sequencing so that we can compare our DNA even further.

Discussion:
Mitochondrial DNA is important for tracing our origins and studying evolutionary patterns. Doing this lab allows us to understand our development and have us realize how we are connected to others. Because DNA is universal, it is easy to map out our DNA history and at the same time, see ways that we could possibly develop. Minute details in our DNA have been found to cause significant problems, but if we could learn the different aspects as a whole and eventually head off future problems.

The Disease Lurking In Your Genes

Background:
The concept of DNA testing has been around since the discovery of DNA's composition, but has only been exercised recently in the 21st century. DNA testing in its simplest form is comparing sets of DNA in order to identify their differences and analyze how they are expressed in organisms. DNA testing is relevant to every day life because it is used in paternity testing, forensics, species comparison, migration studies, and, in our case, genetic disease testing. The process of DNA testing involves extraction of DNA, through bodily secretions, skin cells, blood, fingerprints, or hair etc. After DNA is broken out of its cell, the DNA is copied multiple times through processes like PCR (polymerase chain reaction). Gel electrophoresis is the most common process for DNA to be compared through. If the DNA is positive for whatever is being tested, it will align correctly with the bands of the positive control. For this lab, we will use these examples in our procedure.
Purpose:
The purpose of this lab is to fully understand the process of DNA testing. Whether our fields of study will include genetic testing, or if we choose to be tested for genetic diseases in the future, it is important to be aware and knowledgeable.
Procedure:
Day 1: After rinsing our mouths with saline solution to remove foreign particles, possibly containing DNA, we swab the inside of our cheeks to take our epithelial cells that contain DNA. We gain access to the DNA inside by breaking the cell and nuclear membranes with a hot water bath at 95 degrees centigrade. Doing so, leaves the DNA exposed to DNAse which will destroy it, unless we add the Instagene Matrix Beads, which will kill the DNAse.
Day 2: Our next step is PCR, so that we can make multiple copies for research. This involves the following - DNA template (original DNA), deoxynucleotides (raw material), DNA polymerase (enzyme to put them together), Magnesium Ions (catalyst to create the chain), oligonucleotide primers (specify the specific place to begin replicating), and a salt buffer (creates the perfect environment for PCR).
Day 3: The last step is gel electrophoresis. To review, we take the replicated DNA sequences and loading dye and pipette the mixture into the agarose gel. Add the control results for comparison. Now targeting this specific gene, not an actual disease gene because that would have ethical dilemmas, can yield 3 results. Either the sample can be homozygous dominant or homozygous recessive (long strand/long strand or short/short), meaning they are the same, or the third option, heterozygous (long/short).
Results: With the overall class, there were three times as many diseased individuals who tested homozygous recessive. At our lab table, Lizzie was the only one who had definitive results (heterozygous), because the other three of us must have punctured holes through the bottom of the gel, causing it to leak and not give us definitive bands. If we were to get results, we would simply compare the bands of the controls and see with which set our DNA would match.
Discussion: As previously stated, our largest source of error was the gel puncturing. This was extremely disheartening because we are left hanging with the results, because we no longer have our DNA samples. If we had failed somewhere else, we could have done one of the following: insufficient amount of DNA from the cheek cells, using the centrifuge instead of the vortex, or gathering matrix that would have killed the DNA in the PCR machine. After having these results, this will drive us to be more careful next time.

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.