Unit 6 Reflection

This unit focused on the field of biotechnology, which is the use and manipulation of living things and their parts for human benefit. There are 4 main applications of biotech: industrial and environmental, agricultural, medical/ pharmaceutical, and diagnostic research. In the industrial and environmental field, fermentation as well as biofuels (using cellulase to break down cellulose into simple sugars as well as fermentation to produce alcohol for the fuel). Medical and pharmaceutical biotechnology includes gene therapy--both germ line gene therapy and somatic gene therapy. Gene therapy is inserting a copy of a healthy gene into a person who has a defective copy of that gene. An example of agricultural biotechnology is classical breeding, where individuals with a certain desired trait are bred several times. GMO, or transgenic organisms, have had recombinant DNA inserted into them. I felt that I understood this overview of biotechnology fairly well. There are also certain ethical questions that are asked of the field of biotechnology, called bioethical questions. Bioethics is the study of decision-making as it applies to certain advances in biology and medicine.

One technology in the field of biotech is recombinant DNA (rDNA). It is taking DNA from one organism and inserting it into another. The first step in this is to identify the gene of interest and the location and the sequence. Restriction enzymes are very specific enzymes which cut DNA whenever they read a specific sequence. They make a jagged cut and create two "sticky ends" that can bond with other DNA. Plasmids, which are circular DNA in bacteria, are naturally resistant to a certain antibiotic. I felt that this topic was one of my strengths, especially after doing the recombinant DNA lab, where we modeled inserting the insulin gene into a plasmid that was resistant to tetracycline, and only the bacteria with the plasmid would survive.

Model of recombinant plasmid

Another technology of biotech is PCR (Polymerase Chain Reaction), which is a procedure that creates millions of copies of a sequence of DNA so that that sequence can be analyzed. The DNA is denatured with heat, primers are annealed to the single-stranded DNA above and below the gene, DNA Polymerase are extended, and the process is repeated. Gel electrophoresis uses electricity to separate DNA fragments based on size, since the larger fragments travel more slowly than the smaller fragments. 

In the pGLO lab , we added a plasmid which contained GFP (Glowing Fluorescent Protein) to E.coli. There were 4 plates: -pGLO LB, -pGLO LB/amp, +pGLO LB/amp, and +pGLO amp/ara. The bacteria on the -pGLO plate formed a carpet of colonies. The -pGLO LB/amp plate had no growth. The +pGLO LB/amp plate had roughly 130 colonies, and the +pGLO LB/amp/ara had 150 colonies and glowed green under UV light. This lab helped me understand the process We also did the candy electrophoresis lab, where we put four reference dyes (Blue 1, Red 40, Yellow 6, Yellow 5) into four wells. Then we extracted dyes off of candies, such as purple skittles, blue m&m's, red skittles...etc and inserted them into remaining wells. We were able to compare the size of the fragments and identify if any of the reference dyes were present in the candies. From this lab, I was able to better understand how gel electrophoresis works and how to analyze the results. 

Candy Electrophoresis Lab 

I want to learn more about gene therapy and the advances in this technology that are being made today that are getting us closer to the "GATTACA" world. I also wonder about the ethical questions that go along with so many of these advances in gene therapy. 

This year, one of my new year's goals was to actively take charge of learning in biology by participating in class and acknowledging what I don't understand of the material. I am definitely putting more thought into writing relate and reviews and I am also including diagrams in my vodcast notes to help me understand the material better. My next steps are to think about what I don't understand and ask specific questions. 

pGLO Lab

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 carpet tan tan - pGLO LB/amp none none none + pGLO LB/amp 130 tan tan + pGLO LB/amp/ara 150 tan green ** In our actual results, the -pGLO LB was accidentally plated on the plate labeled -pGLO LB/amp and the -pGLO LB/amp was plated on the plate labeled -pGLO LB.

2. The transformed bacteria now glow green under UV light because they have GFP (Glowing Fluorescent Protein). They also have resistance to ampicillin. 

3. The carpet of bacteria on the -pGLO LB plate indicates that there were hundreds of bacteria in the 100 uL which reproduced to create thousands.  

4. Arabinose is a sugar that gives bacteria the ability to glow under UV light. The plates which did not have arabinose did not glow under UV light. 

5. If the GFP gene is attached to a protein inside a cell, it can act as a microscope and allow the inside of a cell to be seen. Also, GFP is an indicator of activity inside the cell, especially protein activity. It can monitor gene expression. 

6. Bacteria can be genetically engineered to mass produce a protein product such as insulin, which is very useful in the medical field. 

+pGLO LB/amp/ara plate: bacteria which contain GFP

Candy Electrophoresis Lab

1. Some of our samples produced different color bands than the reference dyes. Some of the shades of the blue and the yellow were slightly different shades than the reference dyes. This could be because some of the candies have other ingredients in their coloring other than just the reference dyes, possibly some natural dyes.

2. Betanin (beetroot red) would probably migrate in a similar way to Blue 1. This is due to the fact that they are larger fragments, and the larger ones move more slowly through the gel than smaller molecules. Citrus red 2 would move in a similar way to Red 40 because they are of similar length.

3. Dog food manufactures might use artificial colors in dog food because it makes the food more appealing to the consumer, or the owner who is purchasing the dog food. Even though the food without the artificial dyes would taste the same, it looks less visually appealing and may dissuade owners from buying it.

5. The size of the fragment of the dye controls how far the dyes migrate away from their well. Also, the positive electrical current attracts the polar fragments and urges them to migrate through the gel.

6.  The positive electric current helps attract the dyes and move them through the gel.

7. The molecules of similar size will travel in groups and the larger molecules will not move as far through the gel as the smaller molecules. Therefore, the results will show which fragments are the smallest and the largest.

8. The molecules that weigh 600 daltons will be the farthest away from their starting point, the 1000 dalton molecules will be the next farthest away, then the 2000 dalton molecules, then the 5000 dalton molecules.

Recombinant DNA Lab: Thinking Like a Biotechnician

In this lab, we wanted to better understand the techniques of recombinant DNA technology and how bacteria can be used to mass produce a protein product, such as insulin for diabetics. We were given paper strips of cell DNA that had to be attached end to end in order to build a model. We also created a plasmid, which is circular DNA that is found in bacteria. Plasmids are naturally resistant to a certain antibiotic; in our case, the plasmid was naturally resistant to the antibiotic tetracycline. 

During the process of transformation, restriction enzymes cut DNA whenever they recognize a specific sequence. It cuts above and below the gene of interest (insulin gene) to "cut out" the gene to be placed into the plasmid. Usually a restriction enzyme makes a jagged cut, creating "sticky ends" which can bond with other DNA. The restriction enzyme also recognizes the same sequence of DNA on the plasmid. In our lab, we used the enzyme Hpa II because the sequence it recognizes matched base pairs on the DNA and one the plasmid and because its restriction site was close to the insulin gene. It cuts the DNA at two sites (above and below the gene) and it cuts the plasmid once (this is where the gene will be inserted. If an enzyme was to cut the plasmid in two places instead of one, part of the plasmid would be removed before the gene was inserted. Now, the enzyme ligase is added, which reattaches sticky ends. At this point, a recombinant plasmid has been created.

Next, you would put the bacteria in a petri dish containing the antibiotic that the plasmid is naturally resistant to. In our case, this would be tetracycline. We wouldn't use any other antibiotic because the plasmid will not be resistant to those. This would test whether the host cells have taken in the plasmid. Only cells with the plasmid will survive. When the bacteria containing the plasmid reproduce, they will begin to produce the gene product (insulin).

This process is vital in our everyday lives for mass producing a protein product that will be useful to us, especially something as important as insulin, which can be used to treat a diabetic patient. Recombinant DNA technologies could also be used for delaying food expiration and making it last longer, as well as resistance to pesticides. 

New Year's Goals

This year, I will actively take charge of learning in biology and by participating in class acknowledging what I don't understand of the material. My action plan to achieve this goal is to take time and put thought into writing the relate and reviews at the end of each vodcast. I will ask specific questions in class based on what topics I didn't understand from the previous night's vodcast.

This year, as a student in general, I will pay closer attention in class and do extra practice on difficult topics before a test. My action plan is to do homework or practice problems carefully and use it to determine my grasp of the topic. I will then go back after the unit is finished and redo problems that I got wrong or that were difficult. I think this will be a good way to study for tests.