pGLO Lab

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 carpet carpet - pGLO LB/amp 0 none none + pGLO LB/amp 130 pale yellow/grey grey + pGLO LB/amp/ara 35 light yellow neon green glow

2.	What two new traits do your transformed bacteria have?
	First of all the transformed bacteria grew in colony size by a lot because we only used one colony to begin with. While some grew, other bacteria without the pGLO plasmid were killed and some colonies glowed because of the addition of Ampicillin with the pGLO.
3.	Estimate how many bacteria were in the 100 uL of bacteria that you spread on each plate. Explain your logic.
	There were 35 bacteria on the +pGLO LB/amp/ara plate because 35 colonies were created, same with 130 bacteria on the +pGLO LB/amp plate because each colony grows from a bacteria.
4.	What is the role of arabinose in the plates?
	Arabinose causes the bacteria to glow.
5.	List and briefly explain three current uses for GFP (green fluorescent protein) in research or applied science.
	Detect Cancer: In animals, every cell that contains actin will glow green but the cells with cancer will glow red Transcription Reporter: placing GFP into a promoter will help track that organism’s gene expression Cell marker: GFP is used to see which cells have taken up a plasmid
6.	Give an example of another application of genetic engineering.

Another example of genetic engineering is plants that can fight pollution. Trees developed by scientists and the University of Washington can absorb polluted water through their roots and clean it before water is used and released in other forms.

Candy 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

During the Recombinant DNA lab, we modeled recombinant DNA technology to show how bacteria can be used to mass produce a protein product. We were given paper strips that represented cell DNA once we attached them into a long string. We also created a plasmid, which is circular DNA that is found in bacteria, by attaching a strip to itself. Plasmids are naturally resistant to a certain antibiotic; in our case, the plasmid was naturally resistant to the antibiotic kanamycin. 

During transformation, restriction enzymes cut DNA in a staggered fashion (above and below) whenever they recognize a specific sequence. After cutting out the specific gene, it will be placed into the plasmid by bonding their sticky ends(ends of cut gene that will attach to the plasmid). The plasmid also has the same sequence, which is cut out and replenished by the cell DNA. In our lab, we used the enzyme Eco RI because there were two matches on the cell DNA, close to the gene, and another match on the plasmid. The plasmid is cut in only one place because if an enzyme was to cut in two places, part of the plasmid would be removed before the gene was inserted. After all this, the enzyme ligase is added, which reattaches the sticky ends. At this point, a recombinant plasmid has been created.


Now, you would put the bacteria in a petri dish along with a naturally resistant antibiotic. In our case, this was kanamycin. We couldn't use  tetracycline or ampicillin because the plasmid wasn't resistant to those. This would test whether any uninvited cells have taken in the plasmid because only the cells with the plasmid would survive. When the bacteria containing the plasmid reproduce, they will begin to produce the gene product.

This process is vital in our everyday lives for mass producing a protein product that will be useful to us, such as insulin for diabetics. Recombinant DNA technologies could also be used to delay food expiration by making it last longer, as well as resistance to pesticides. 

New Years Goals

Biology goal: 

I will take extra time to understand the materials in vodcasts when they are assigned instead of cramming right before the test. 

Plan:

Instead of watching the vodcast with my main goal to be finishing the notes, I will watch the vodcast with my main intention being to absorb the material.

Personal goal: 

I will be more flexible and have more stamina while dancing.

Plan:

I am doing this for my solo dance performance, which is happening in 2017. I want to improve my flexibility by stretching, my stamina by running or dancing a lot everyday. 

Unit 5 Reflection

Unit 5, named Walking the Dogma, described and went in depth to the function and processes surrounding the DNA in each and every one of our cells. The unit started when we learned about the structure and function of DNA and its code- which was very easy to grasp for me because it was extremely practical. The steps were logical and I felt like watching the videos of the RNA making proteins helped me see what I was learning. DNA expression and regulation, since I could relate it to our bodies, were easy to understand. I think of expression like “that is why it is there” for example having two eyes, and regulation like the opposite, why we don’t have 37 eyes. Though this is a simple example, it helped me relate the science to life. For me, the mutations vodcast was confusing because there were a ton of mutations and things that could go haywire in a DNA. I didn’t know that these all things could happen so it was a bit overwhelming! But, after doing the mutations lab, the concepts made perfect sense and were easy because I tried doing the mutations myself. What made this unit easy was that it was very convenient to try things out and figure out the processes like base pairings and mutations ourselves. And doing the DNA extraction lab towards the end of the unit was also cool and informative to another DNA process. Overall Unit 5 was short and sweet!

Protein Synthesis Lab

To start making a protein, the DNA is transcribed to RNA in the nucleus. Then it is converted into messenger RNA (mRNA) which is sent out of the nucleus to a ribosome. RNA Polymerase pairs the corresponding nucleotides with a RNA strand. In the ribosome, the RNA reads three bases at a time to form codons which code for amino acids. These amino acids are joined together to form a protein. 

In this lab we tested different kinds of mutations that could potentially occur while DNA is being transcribed and their affect on the protein structure. The mutation that caused the least damage to the protein was substitution. This mutation had the least effect on the protein because though the amino acids changed, it was still a complete protein.  The frameshift mutations caused much more damage to the protein because most amino acids were changed and there were extra incomplete codons in the end.


In the lab, after trying different mutation, we were asked to chose the one that would be the most harmful to the protein. I chose deletion, because not only does it change entire amino acids, the end codon is left incomplete. I decided to take out the first C in the DNA sequence, and was left with a completely altered RNA, as expected. 

A mutation that I didn't know stemmed from the genes is sickle cell anemia. It is the result of a point mutation, where one nucleotide is changed for hemoglobin. This causes the hemoglobin in red blood cells to change into a distorted shape and clog the capillaries, cutting off circulation.

DNA Extraction Lab

Conclusion

     In this lab we asked the question, "How can DNA be separated from cheek cells in order to study it?" We found that this procedure involved three steps including homogenization, lysis, and precipitation. We carried this out by mixing our cells in a polar liquid. This breaks down the cell and nuclear membranes of the cheek cell, homogenizing it. Afterwards we added soap to the mixture, initiating lysis which disintegrated the membrane. We used pineapple juice to break down histones found in DNA, causing it to uncoil. This was possible because pineapple juice, like a few other liquids, has catabolic proteases and  enzymes, that help to break down the histones. Finally, we poured cold isopropanol alcohol into our test tube and due to its non-polarity combined with the polarity of the DNA, the DNA became a precipitate and rose to the top of the isopropanol alcohol layer.  This data supported our procedure because, to put it simply, it worked!
     While our hypothesis was supported by our data, there was one vital error that we carried out. After adding pineapple juice and soap, we were supposed to gently invert the test tube 6 times and we chose to do this step after adding the alcohol, so the two layers got mixed. Out of the four people in our group, two people's DNA were ruined because of this. This error happened because we created our own procedure without a more educated approval to tell us when to put each step. Another hypothetical error could have been the time factors. If one gargled gatorade for more or less than thirty seconds or observed for more or less than five minutes, results might be changed though the effect isn't drastic. To minimize these rather small to extremely important details, get a teacher's approval before starting and keep a timer handy!
      This lab was done to demonstrate the process of extracting DNA from a cell. From this lab I learnt how to remove DNA from its original position in a cell which helps me understand the concept of DNA and its structure. Based on my experience from this lab I applied the concepts around DNA and I now have a
 sample of my own!