Unit 6 Reflection

Unit 6 was about biotech and its various applications. It involves changing the living world by manipulating the DNA of living organisms. There are four applications of biotech, industrial, agricultural, diagnostic, and medical.

Some biotech practices are controversial, and that brings up some people's bioethics, which is what they feel is wrong and right in the biotech field. If faced with ha bioethical question, one should consider the pros and cons of each side, and try to come up with another solution if they can.

One type of biotech is recombinant DNA, which is DNA that has been altered. To do this, you need the gene that you want to insert, a restriction enzyme to cut the gene out of the DNA, and to cut the plasmid once, which is another thing you need, and you need ligase, which reattaches the genes. Recombinant DNA can be used to make large amounts of a protein like insulin. First you put the gene of interest into the organism, get a plasmid, digest the DNA, then add ligase to reattach them, mix the recombinant plasmid with the bacteria, grow the bacteria on a plate with the antibiotic it is naturally resistant to, and then extract and purify the protein, according to my notes.

Some other technologies of biotech are polymerase chain reaction, gel electrophoresis, and DNA sequencing. Polymerase chain reactions are used to make lots of copies of DNA. To do this, you need a strand of DNA that you want to duplicate, and you must denature it with heat, then add primers above and below the region of interest, and then you use DNA polymerase to extend the primers, according to my notes. Gel electrophoresis is used to identify the approximate length of strands of DNA. DNA is put into wells at the end of a gel, and a current is run through it, and the DNA is drawn towards the positive charge. Known DNA strands are used as a template to measure the length. Sequencing is determining the sequence of DNA. Special bases with dyes attached are used do replicate the DNA strand one base at a time. Then a computer analyzes it to find the order of the base pairs.

The set-up for gel electrophoresis

We did a lab on recombinant DNA in E. Coli bacteria, which you can find here: http://markbiologyblog.blogspot.com/2016/01/pglo-lab.html
to summarize what we did, we added modified plasmids to E. Coli bacteria that made them glow green when in contact with arabanose sugar, and let the multiply.

Mice modified to glow due to the pGLO gene. It is sometimes used as an indicator to make sure that a gene was taken in by an organism.

pGLO Lab

In this lab, we inserted modified DNA into E.Coli bacteria, which put the DNA into the plasmid, to make them resistant to the antibiotic ampicillin and glow green under UV light. Each plate started out with 100 micro liters of bacteria, which is about 90 bacteria per plate, because each one is about 2 micrometers long. The bacteria multiplied, resulting in many more. One of the plates contained arabinose, which attached to the promoter on the DNA and allowed the protein green fluorescent protein to be made. There are various uses of GFP. It can be used as an indicator for other transformed organisms by attaching it to the gene you are trying to insert into an organism. It is also used to study the growth of cancer in mice, as well as other diseases, such as blindness is dogs. GFP is also used to track fruit fly sperm to see how sex cells maximize their chances of fertility. There are many other ways to genetically modify organisms, such as making crops resistant to herbicides and parasites.

Plate	# of Colonies	Color: Room Light	Color: UV Light
-pGLO LB	carpeted	gray	gray
-pGLO LB/amp	0	nothing	nothing
+pGLO LB/amp	104	gray	gray
+pGLO LB/amp/ara	64 + partial carpet	gray	neon green

Upper Left: no pGLO gene with LB (helps bacteria grow).
Upper Right: no pGLO gene with ampicillin and LB.
Bottom Left: pGLO gene with ampicillin, LB, and arabinose sugar
Bottom Right: pGLO gene with ampicillin and LB

The transformed E. Coli glowing under a UV light.

Candy Electrophoresis Lab

In this lab, we ran dyes from different candies through the process of electrophoresis. The bands of our dyes were fairly normal, all dyes moving in the correct direction. Some bands left a trail behind them, the orange one on the far right leaving a blue trail. One dye that might move like this is fast green FCF, because many parts of it are negatively charged, like these dyes. Some dog foods and treats. contain dyes. I think this is because in nature, some colors symbolize something can be poisonous, like brightly colored animals, so the food is colored in a way that it won't be seen as poisonous. Artificial food colors could be preferred over natural food colors because natural food colors may affect the taste of what is being dyed, while artificial dyes can be engineered to have little to no flavor. There is also no foods that are naturally blue, even blueberries, which are a dark purple. Some dyes migrated farther than others, which is caused by the size of the molecule, and the strength of the charge. Smaller molecules, like the yellow gel, move faster, and also more strongly charged molecules are more attracted to the positive charge coming from the current, which is what makes the dye molecules move at all. The larger molecules move more slowly through the dye, such as the two blue colored dyes in the gel. The process of electrophoresis is commonly used to separate different lengths of DNA. If a DNA molecule had a weight of 600 daltons, it would go a lot farther than a DNA strand with a weight of 1,000 daltons, because it is a bigger molecule. If it had 2,000 daltons it would go even slower, and if it had 5,000 daltons, all the other molecules would move much farther.

Recombinant DNA Lab Summary

In this lab, we simulated creating recombinant DNA. Recombinant DNA is DNA from one organism that is inserted into the DNA of another organism. DNA is usually inserted into the plasmid of a bacteria. To do this, you need to gene you want to insert, the bacteria, and the antibiotic it is resistant to, a restriction enzyme, and the enzyme ligase. The restriction enzyme you need has to be an enzyme that matches the restriction site. We used the enzyme Hpa II, because it cut the plasmid once, and it cut the insulin gene above and below the gene as close to the gene as possible. It should only cut the plasmid once, because if it cut it twice, then the plasmid would turn into 2 separate pieces of DNA. When the DNA is cut, it will create sticky ends which can attach to the sticky ends of the other piece of DNA.

Sticky Ends on a sequence of DNA.

The enzyme ligase attaches the sticky ends of the gene to the sticky ends of the plasmid to make the plasmid a circle again. After you re-insert the new plasmid into the bacteria, you put it in a petri dish with the antibiotic it is resistant to, in our simulation, out bacteria was resistant to the antibiotic tetracycline. This ensures that the only bacteria  in the dish is the one that you put the recombinant DNA into. The bacteria will multiply, and all the bacteria will have the gene and create insulin. This is how insulin is created for people with diabetes. Recombinant DNA has also been used to make certain fish glow, and is a part of many other parts of our lives, such as being in many foods we eat. Many crops are resistant to herbicides and insects, which comes from recombinant DNA. Scientists have recently found a way to create self healing materials from jellyfish DNA.

Goals For 2016

This year, I will write better relate and reviews for vodcasts and chapter notes. To do this, I will go back to older vodcasts and see what I left out of the relate and reviews that is important. Then I will write all important information in the R&R, while leaving out the less important details, making them more informative and concise. I will know I have completed this when I can study for a test using only R&Rs and understand everything I need to know.

I will also make better food from leftovers in my kitchen. Whenever I make lunch, it usually involves me taking random things out of the refrigerator and putting them into a sandwich. I will start making more creative and tasty things from the leftovers without having to prepare anything apart from using the microwave. I will do this by trying new things that and choosing different methods of making them into a meal that isn't just a pile of food. I will know my goal is complete when I no longer get stumped of what to have for lunch when looking in the refrigerator.