Unit 6 Reflection

This unit was all about biotechnology. The introduction of biotechnology discussed how it is known as the study and manipulation if living things in order to benefit mankind. This large field focuses on understanding DNA, proteins, and inheritance. There is a long timeline of biotechnology.

•  It began in 4,000 BCE in the classical biotechnology time period, where dairy farmers developed in the middle east and Egyptians used yeast to bake and learned to make wine through fermentation. 
• In 3,000 BCE Peruvians selected and cultivated potatoes.
•  In 2,000 BCE Egyptians, Sumerians, and Chinese develop techniques of fermentation: brewing and cheese making. 
• In 1500 CE acidic cooking techniques led to sour kraut yogurt- 2 examples of using beneficial bacteria to flavor and preserve food. Aztecs make a cake from spirulina algae.
• In 1861 french chemist Louis Pasteur develops pasteurization: preserving food by heating it to destroy harmful microbes.
• In 1910 American biologists Thomas Hunt Morgan discovers that genes are located on chromosomes
• In 1953 James Watson and Francis Crick Describe the double helix of DNA using x-ray diffraction patterns of Rosalind Franklin and Maurice Wilkins.
• In the early 1970's Paul Berg, Stanley Cohen, and Herbert Boyer develop ways to splice DNA, introducing recombinant DNA techniques.

A big emphasis in the introduction to biotechnology was the 4 real-life applications of the science and its effect. 

• Industrial and Environmental: Fermentation- the use of bacteria or yeast in an oxygen-free environment to convert sugars into acids, gasses or alcohol. (ex: food like yogurt, cheese, beer, wine, bread etc)
• Medical and Pharmaceutical: Gene therapy-putting a healthy copy of a gene into cells of a person whose copy is defective, Germ Line gene therapy- insert the gene into germ cells(sperm/egg), Somatic gene therapy- insert the gene into specific cells w/defective expression of the gene. 
• Agricultural: Classical breeding- individuals with desired traits are bred over many generations. GMO/Transgenic organisms are the product of recombinant DNA(Foreign DNA inserted in them)
• Diagnostic: Genetic testing- search for genes or DNA segments indicating risk for various diseases or disorders.

An interesting infographic about GMOs 

We also talked about the "technologies of biotechnology" and the different steps and procedures commonly used. We talked about Polymerase Chain Reactions (PCR) first, and then we talked about Gel Electrophoresis. We had already learned about gel electrophoresis when we did our Candy Electrophoresis Lab and our virtual lab.
   The lab was long, but it was lots of fun. It really helped me understand how gel electrophoresis really works. Although some errors got in everyone's way(as mentioned in the link), I feel like I have a better understanding of why each step in the procedure was there. Also, I learned how to use a micropipette to fill the wells of the gel after extracting the colored dyes from candies.

PCR: A procedure to amplify a specific DNA region that yields millions of copies of a sequence of DNA. It is the first step in preparing DNA for many experiments like gel electrophoresis and any other form of analyzation. The process makes millions of copies of the small DNA fragment so it is easier to study.
Check out this video that explains PCR: https://www.youtube.com/watch?v=3XPAp6dgl14

Steps of PCR:

1. Denature the double stranded DNA with heat
2. Anneal the primers to single stranded DNA above and below the region of interest. *A primer is a small fragment of DNA that bonds with a specific sequence.
3. Extend primers with DNA polymerase yielding new double stranded DNA. This cycle repeats steps 1-3 usually 20-40 times. The copies from PCR occur in exponential amplification which means after 30 cycles, DNA is amplified over a billion-fold. 

Helpful diagram displaying PCR

Gel Electrophoresis is the use of electricity to separate DNA fragments based on size. Large pieces travel slower than small pieces, and the results of distance traveled by the unknown lengths are compared to those of known lengths. It has many different applications such as forensics, biochemistry, genetic diagnosis, etc.

Sequencing was also discussed in the vodcast. Sequencing by definition is determining the exact order or sequence of a given DNA strand. DNA polymerase, primers, extra bases, and fluorescent dyes are all used to create copies. These copies are one base longer and contain fluorescent dyes attached. They are then electrophoresed and analyzed with a computer. The result is an electropherogram and the colored bases represent 1 of 4 bases. Finally, the sequence of bases is recorded.

Recombinant DNA was also discussed, where DNA of one organism is inserted into the DNA of another organism. Recombinant DNA or rDNA is also known as genetic engineering. The result of rDNA is GMO's (genetically modified organisms) or transgenic organisms. The steps followed to use rDNA technology in bacteria is as follows.

1. Gene of interest: know the location and sequence of your gene(above and below it)
2. The restriction enzyme cuts DNA wherever it reads a specific sequence. It latches onto the restriction site. Each restriction enzyme leaves a sticky end at the cut, which allows DNA to bond other DNA. 
3. Plasmids: circular DNA found in prokaryotes contains a replication gene that tells the plasmid to be copied. Typically it contains genes with antibiotic resistance that are small enough to be passed through pores of the cell membrane.
4. Ligase: an enzyme that re-attaches base pairs. 

The process of transforming bacteria to mass produce a protein product:

1. Isolate the DNA by finding the gene of interest and organism to insert the gene into
2. Get the plasmid, and know what antibiotic it is naturally resistant to
3. Digest  the DNA by finding a restriction enzyme that will cut the plasmid once above and below the gene
4. Mix the digested DNA(plasmid + gene)
5. Add ligase to attach sticky ends
6. Mix recombinant plasmid with the bacteria
7. Plate bacteria on agar with the antibiotic mixed in (only those with the plasmid(antibiotic resistance) will survive)
8. Grow the transformed bacteria and transfer to broth(liquid agar) to make many bacteria expressing the gene
9. Extract and purify the protein the inserted gene produced. 

Recombinant DNA technology has an interesting history. It was discovered when Herb Boyer and Stanley Cohen met at a conference. With their combined knowledge of restriction enzymes and isolating genes, they were able to work on the toad/bacteria rDNA together in the March of 1973. They discovered how DNA could be transferred between different species, and the world of genetic engineering was born. 

Another topic was the pGLO lab, which elaborated on the previous vodcast. It was about the bacterial transformation of an e.coli bacteria, using the presence of plasmid for a fluorescent glowing reaction.  My pGLO Lab Analysis and this video do a great job of explaining the process. Without doing the virtual lab, and the pGLO lab, I would have little understanding of how the procedure occurs, and why every step does what it does.  https://www.youtube.com/watch?v=OZyFX9megs8
   I learned how to predict which bacteria would glow, grow, or have nothing at all. For example, the plate with just LB, or lysogeny broth which makes the bacteria grow, would have bacteria on the plate. The plate with LB/amp (ampicillin, the antibiotic which the plasmid is resistant to) without the plasmid would not grow. The plate with LB/amp with the plasmid, would grow. Lastly, the plate with LB/amp/ara (arabanose is the sugar that acts as a promoter of the GFP gene, controlling its expression) would grow bacteria and glow with green flouresecnt protein. 

A picture of the results of another group in the class.

The last, most unique topic explored was about bioethics, and how we should answer not only ethical but bioethical questions. I feel like it has really opened my eyes to what my morals and values are, which will help me make ethical decisions in the future. The answer to ethical questions come from your morals(your justification of what is right or wrong), and your values(what is most important based off of your unique, personal experiences). The same approach is taken when you are faced with a bioethical question. You must first clarify your values, then identify the issue. Find as many options possible, and then list the pros and cons of each decision . Rank your ideas from best to worst, and once you've come to a decision, defend it. 

After reading articles in class, I wonder about all the infinite possibilities and ideas people can come up with to apply biotech. After listening to summaries of other classmate's articles, I was intrigued by the various fields and forms biotech took. I did further research on my own article about gene editing tools used to cure genetic diseases caused by mutations which you can find on my blog: Bioethics Reading. That's why it was a bit of a struggle for me to get started on my 20-time project, because I wasn't used to such vast boundaries. Usually, there is a set goal, but I think this free- thinking has really reignited my creativity. 

Lastly, I have been checking in on my New Year Goals, and I have found that I've been improving my studying skills. I can identify the forms of studying that are best for me, much quicker for each topic, but I still struggle with time management. As for drinking more water, I have bought a new water bottle, and I have been motivated to stay hydrated. I think overall, I am making good progress for both of my goals, but there is always room for improvement. 

Works Cited:

GMO infographic. Digital image. Kids Right to Know. N.p., n.d. Web. 31 Jan. 2017. <http://www.kidsrighttoknow.com/wp-content/uploads/2010/05/What-is-a-GMO.jpg>.

Polymerase Chain Reaction - PCR. Digital image. Wikimedia. N.p., n.d. Web. 31 Jan. 2017. <https://upload.wikimedia.org/wikipedia/commons/thumb/9/96/Polymerase_chain_reaction.svg/835px-Polymerase_chain_reaction.svg.png>.

pGLO Lab Analysis

pGLO Observations, Data Recording & Analysis

1.

Obtain your team plates.  Observe your set of  “+pGLO” plates under room light and with UV light.  Record numbers of colonies and color of colonies. Fill in the table below. Plate Number of Colonies Color of colonies under room light Color of colonies under   UV light - pGLO LB 0 tan tan - pGLO LB/amp N/A N/A N/A + pGLO LB/amp 1 tan tan + pGLO LB/amp/ara 4 tan fluorescent green

2.	What two new traits do your transformed bacteria have?
	The bacteria are now resistant to ampicillin(antibiotic), and glow fluorescent green under UV light.
3.	Estimate how many bacteria were in the 100 uL of bacteria that you spread on each plate. Explain your logic. Because each plate has 100 microliters of bacteria spread on it, the number of bacteria is the same for each plate. The volume of 1 E. Coli bacteria is about 1 micrometer cubed, which is equivalent to 0.001 microliters. 1 microliter(the volume of each bacteria) is equal to 1, 000 micrometers (or 1,000 bacteria). Since there are 100 microliters of bacteria spread on each plate, and 1,000 micrometers of bacteria 1,000x100 gives the answer of 100,000 bacteria on each plate. 100,000 bacteria(micrometers) is equivalent to the 100 microliters spread.
4.	What is the role of arabinose in the plates?
	The role of the arabinose sugar is the key component for making the bacteria on the plate (which were resistant to ampicillin, and contained the plasmid) glow. It is a promoter of the gene, allowing it to be expressed.
5.	List and briefly explain three current uses for GFP (green fluorescent protein) in research or applied science. GFP is a protein extracted from a jellyfish gene, which is commonly used to tag genes and cells of interest with its fluorescent properties. It can be used to identify expressed genes, making it easier for research scientists to observe their results. Some common applications are GFP being used to track and label cancer cells that can travel throughout the body. Some genetic engineers and breeders are also incorporating GFP into the cells of living things like fish(i.e. glo-fish).
6.	Give an example of another application of genetic engineering. The use of transgenic organisms, or creating GMOs (genetically modified organisms) in foods. A specific example is genetically modified foods. Some corn with GMOs can produce a poison to kill insects without pesticides. This contributes to the agricultural industry, making it a lot easier to produce mass amounts of crops without the conflict of naturally occurring environmental factors. Photos: Icing the tubes with transformation solution during the lab. Transferring the cold tubes to the heat bath. Incubating the tubes with and without the plasmid(-pGLO and +pGLO) Results after two days (under normal light0 Results after two days under UV light

Bioethics Reading

 After reading the article, "CRISPR gene editing tool used to treat genetic disease in an animal for the first time," by Peter Dockrill on January 4, 2016,  I was intrigued by the biotechonological advancements that were displayed. The article talks about how the CRISPR gene editing tool was used to treat a genetic mutation in a mouse for the first time.
   The acronym CRISPR stands for: clustered regularly interspaced short palindromic repeats. This tool uses pieces of prokaryotic DNA with "short, receptive base sequences," along with pieces of spacer DNA which are exposed to foreign DNA like plasmids or viruses. The reason behind it is because the CRISPR gene is really an important component of the immune systems of bacteria and other unicellular organisms. For now, this tool is currently the simplest and most precise method of genome editing. 
   The article explained how CRISPR was used to treat the genetic disorder of DMD or Duchenne Muscular Dystrophy. DMD is caused by a genetic mutation which, according to the article, affects 1 in 5,000 human males. 
   Researchers from Duke University used an adult mouse model of DMD to carry out the experiment. The CRISPR system was programmed to cut out the dysfunctional exon on the protein of the gene, which urges the body's natural repair system to attach the remaining gene together.  This results in a functioning version of the gene that is shortened. 
  The technique was applied when the gene therapy was applied directly into the leg muscle of the mouse. It had been restored in strength because it had a new supply of dystrophin.
  According to the article, "They injected the CRISPR/AVV(virus) combination into the animal's bloodstream. This resulted in partial dystrophin corrections in other muscles throughout the body including the heart- which is significant, as heart failure is a common cause of death for patients of DMD."
  Overall, the results were promising, and with further research and development, the goal is to start clinical trials. This breakthrough shows the potential of many benefits to the many people with genetic disorders caused by mutations.  If other experiments conducted in the future are as successful, this system could be a game-changer. 
   This article doesn't show any potential risks from the study at the moment, but I believe some risks may appear. Overtime, there is a possibility that other diseases may develop if the important, though DMD causing gene is cut. 
  Although there are some risks, CRISPR is a great way to treat a disease like DMD, and in my opinion, the benefits outweigh the costs. Although it can cause inherited diseases, or restrict the function of genes that can kill cancerous cells, a way to stop DMD directly seems more beneficial. The constant suffering of  people with the muscle deteriorating diseases is more of a moral issue to debate. When someone is in pain, the instinct is to stop it as soon as possible. If it were me, I would like to live happily and live present, and focus on tackling the current issue rather than worrying about future risks. 

Diagram of how the CRISPR system functions

Visual aid to understanding the effects of the muscle deterioration caused by Duchenne Muscular Dystrophy

Effects of Duchenne Muscular Dystrophy

Works Cited

"CRISPR: A Game-changing Genetic Engineering Technique." Science in the News. N.p., 31 July 2014. Web. 26 Jan. 2017. <http://sitn.hms.harvard.edu/flash/2014/crispr-a-game-changing-genetic-engineering-technique/>. 

"CRISPR." Wikipedia. Wikimedia Foundation, n.d. Web. 26 Jan. 2017. <https://en.wikipedia.org/wiki/CRISPR>. 

CRISPR/CAS9 diagram. Digital image. Advanced Analytical Automating Genomic Discovery. N.p., 2015. Web. 26 Jan. 2017. <https://www.aati-us.com/instruments/fragment-analyzer/crispr/>. 

Duchenne Muscular Dystrophy Effects. Digital image. Genetics Home Reference. N.p., n.d. Web. 26 Jan. 2017. <https://ghr.nlm.nih.gov/condition/duchenne-and-becker-muscular-dystrophy>. 

"Gersbach Lab." In Vivo Genome Editing Improves Muscle Function in a Mouse Model of Duchenne Muscular Dystrophy. | Gersbach Lab. N.p., Jan. 2016. Web. 26 Jan. 2017. <http://gersbach.bme.duke.edu/publications/vivo-genome-editing-improves-muscle-function-mouse-model-duchenne-muscular-dystrophy-0>. 

Muscular Dystrophy Diagram. Digital image. Pintrest. N.p., n.d. Web. 26 Jan. 2017. <https://www.pinterest.com/explore/muscular-dystrophy-symptoms/>. 

Skerrett, Patrick. "Experts Debate: Are We Playing with Fire When We Edit Human Genes?" STAT. STAT, 09 Mar. 2016. Web. 26 Jan. 2017. <https://www.statnews.com/2015/11/17/gene-editing-embryo-crispr/>. 

"What Is CRISPR-Cas9?" Facts. The Public Engagement Team at the Wellcome Genome Campus, 19 Dec. 2016. Web. 26 Jan. 2017. <http://www.yourgenome.org/facts/what-is-crispr-cas9>.

   

Candy Electrophoresis Lab

Focus Questions:

1. We found that our experimental samples did not match the color of all four reference samples. We used yellow m&ms, which was close to the yellow 5 color. The yellow Reese's pieces were similar to the yellow 6 reference dye. The purple skittles did not match the Blue 1 reference dye in any way, and the pink Mike and Ike were slightly similar in color. But the blue reference dye traveled the slowest, behind all the others on the gel. The light pink pigment was much softer and clearer than the bright, thick Red 40 reference dye. It was difficult to compare the bands of each dye because there was an assembly when we ran our gels, and most groups in our class struggled due to the time limitations. Some dyes ran off the gel, while some were still making their way across it. Unfortunately, the experimental error affected our test a lot, because we had to leave the electrical current running through the gel for too long. 
2. The fast green FCF will most likely migrate similarly to the Blue 1 reference dye because they have the exact same structure and chemical composition. The citrus red 2 color is also close to the Red 40 dye because of similar structure, but the chemical composition is slightly different, therefore it may migrate similarly. 
3. Dog food manufacturers might put food dyes into their products because it makes it more appealing the dog, and human. Like the ABC news article by Susana Kim, "11 Food Ingredients Banned Outside the U.S. That We Eat" mentioned, people will only eat something that is good for them if it smells or looks good, regardless of the taste. Food dyes that are not as dangerous are said to be mostly used for the influence of perception, source Michael Pariza mentioned. "Taste, appearance, and smell all go together. You can have the most fantastic, nutritious thing in the world, but if it looks bad and smells bad, you're not going to eat it," he said. All in all, dyes are placed to make the food more desirable. 
4. Natural food dyes that can replace artificial dyes include; red beet juice as an alternative to Red 40, spirulina extract for Blue 1, turmeric powder for Yellow 5, and paprika for Yellow 6. Usually, artificial dyes are used over natural dyes because they last longer, are more vibrant in some cases, don't affect taste/smell, and are much cheaper to have access to. 
5. The level of voltage in the electric current affects the dyes ability to migrate. We were on a time crunch so the voltage and power were on the maximum setting. The higher powered electrical currents made dyes move faster. Another important factor is the size of the dye and how much the wells in the gel were actually filled. The dyes with higher amounts moved slower than those with less, which moved across the gel faster.
6. The negatively charged dyes are attracted to the positive electrical current, so the dyes are pulled across the gel in the direction of the positive electrode. 
7. The agarose gel acts like a filter, and molecules are separated by size through the pores. Smaller molecules can travel through the gel quickly and with ease, while the opposite is true for larger molecules.
8. I would expect the molecule with the lowest molecular weight(6000-dalton) to separate the fastest because of its small size. Following, 1,000 and then 2,000 dalton molecules. Last, I would expect the 5,000-dalton molecule, the heaviest and largest of all to be the slowest to separate. In conclusion, the 6,000-dalton molecule would travel the farthest and fastest because of its smaller size, and the 5,000-dalton molecule would travel the slowest, and least amount because of its large size

Photos:

Our final gel

The dyes extracted from our candy

Our candy selection

Electrode being hooked up to gel

New Year Goals

   One of my goals for this semester is to start studying for tests ahead of time instead of procrastinating. This will be beneficial to my learning process because it will allow more time for me to identify specific forms of learning based off of my multimodal learning type. Also, it will force me to stop procrastinating and help me manage my time better for all of my classes. This way I will retain a lot more information over a longer period of time, making tests and finals seem a lot easier. The most challenging part of this goal is to make time for extra studying because before, procrastination was my only motivation. To achieve this goal, I will be more mindful and stay focused in school and at home and spend less time on my phone. 

  I will also drink more water. To do this, I need to carry around my water bottles more and challenge myself sip throughout the day. In the past, I've often been dehydrated so this goal will help my personal health as well as my learning process. Being able to incorporate certain goals into my daily routine is a great skill to have, and I think this is a good way to start. I will start by setting reminders on my phone by the hour to keep myself on track. Also, I will need to practice mindfulness to keep my goals in check.