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 lawn yellow yellowish white - pGLO LB/amp 0 NA NA + pGLO LB/amp 62 yellow white + pGLO LB/amp/ara 350 yelow green

2.	What two new traits do your transformed bacteria have?
	The resistance to ampicillin and the GFP gene were the new traits our transformed bacteria had.
3.	Estimate how many bacteria were in the 100 uL of bacteria that you spread on each plate. Explain your logic.
	Each colony started with 1 bacteria. There would have been between 62 and millions of bacteria first. This is because 62 was the least number of colonies and it could have gone up to millions.
4.	What is the role of arabinose in the plates?
	The role of arabinose is to make the bacteria glow green by letting the RNA Polymerase through to the gene.
5.	List and briefly explain three current uses for GFP (green fluorescent protein) in research or applied science.
	The GFP is used because of its ability to generate a glowing color. GFP is used as a active indicator for protease action because then the scientists would know that it is working. Another reason why GFP is used is because it can glow inside an organism so the scientists could see what goes on inside the organisms cell. The third use is GFP is used for Biosensors, which allows you to analyze different conditions, like pH levels.
6.	Give an example of another application of genetic engineering.

In medicine, genetic engineering has been used to mass produce human growth hormones, insulin, monoclonal antibodies, vaccines and many more.

Candy Electrophoresis Lab

Although none of our dyes traveled in the wrong direction or mixed colors, there were some minor differences between the reference dyes and the ones we were testing. For example, our red and orange were darker than the reference colors, whereas the reference dyes for blue and yellow were darker than ours. However, I think this can be attributed to the amount of dye we extracted from the candy. I did not find any major variance between the distance traveled by reference and test.

I think that citrus red 2 will migrate similar to the blue 1, carminic acid will go about as far as our red 40, fast green FCF should go about as far as yellow 6, and betanin will be about the same distance as yellow 5. This is my hypothesis because although they aren't the same colors and size of molecules, the order of dyes that we tested, yellow 5, yellow 6, red, blue will correspond to betanin, fast green FCF, carminic acid, and citrus red 2. In other words, although the chemicals won't go as far as the dyes (due to their size), they should order up in the way specified above.

Dog food manufacturers probably put food coloring in the dog food to entice the dogs to eat it. Most dog food does not consist of things that a dog would naturally be fed, so to get the dog to eat up, they need to use artificial flavoring, coloring and smells.

In my food I found the artificial dyes red 40, yellow 5, yellow 6, and blue 1. I also found 2 natural dyes in cereal: annato extract color, and turmeric extract color. I found most of these dyes in cereals and sauces. It surprised me that I found the exact same dyes that we tested in the lab. I then searched the dyes up and learned that they are four of the seven permitted food colorings in the US.

The 2 factors that control the distance the dye travels is the dye's size, and how long you leave the gel in the electrophoresis box. In addition, I also think that the overall charge of the dye must also play a part in the direction it travels.

The force that moves the dye through the gel is the electromagnetic force. It is propagated through the current, caused by the voltage difference from the red cathode to the black one.

The reason why smaller dyes travel farther than large molecules of dye is because of the porous nature of the gel. Because the dye is inserted into the wells, they travel through the gel rather than on top. Thus, smaller dye molecules find it easier to navigate the cave-like environment found inside the gel.

Because DNA molecules of this size are so much larger than the dyes, I expect them not to travel as far. For this reason, it is necessary to leave the electrophoresis going for longer to see a difference in the distance traveled by each molecule of DNA.

Recombinant DNA Lab

  In this lab, we imitated the process of inserting plasmid into a bacteria plasmid. Our plasmid had a resistance to kanamycin. We found a restriction enzyme that cut a segment out of the DNA and cut the plasmid open. We used an enzyme that was closest to the insulin gene. Then we combined the DNA and plasmid with Ligase(tape). In order to test if bacteria took our plasmid, we would use kanamycin because our plasmid has a natural resistance to it and if the bacteria survived then the insertion was successful. We wouldn't use tetracycline or ampicillin because it would kill our bacteria. Restriction enzymes are proteins that bind to a segment of DNA and cut it. The one we used was Eco R1 since it cut the the human gene in two places close to the insulin gene and cut the plasmid in only one l place. If the restriction enzyme cut the bacteria in more than one place, then the lygase would not know where to attach the insulin gene to.  This is important in every day life, because it is used to create many vaccines and medicines, by making bacteria that can produce these products. This technology can also be used to create GMO foods, such as fruits that spoil more slowly.

New Year Goals

1. I will make varsity soccer next year

• I will drink milk and protein every day to become taller
• I will go to the weight room at least once every week
• I will go for a 4 mile jog at least twice every week

2. I will study for tests more efficiently by doing the following:

• I will make note cards for each main topic
• I will not start studying a few weeks ahead of time instead of the last few days
• I will first study what I am having difficulty with  

Unit 5 Reflection

This unit was all about how DNA and RNA helps our body make proteins that help it function. This process is called protein synthesis. In this process, DNA first replicates itself into a one stranded copy called mRNA, which replaces base T with base U. This process of making RNA is called transcription. Next, the mRNA leaves the nucleus and goes to the ribosomes. Here, the ribosomes take groups of three base pairs called codons, and make amino acids. All the amino acids that the ribosomes make are chained together to form the protein. These proteins help our body work. Here is a visual example of protein synthesis.

 I also learned how mutations in this process affect our bodies. The most dangerous mutations are probably the frameshift mutations, either insertion or deletion. These either change the protein completely, or don't make the protein at all. Any mutation, either frameshift or substitution at the beginning of a sequence really causes damage to the end protein. Here is a visual representation of how mutations affect the protein.

The protein synthesis lab really helped me understand this topic, and I think at first, I was at a 3 on a scale of 1-5, and now I am at a 5. If there was one topic from this unit that I had to work on, I think it would be gene expression and regulation. Was that vodcast just an extension of the protein synthesis vodcast? In the last unit, I took a survey and learned that I learn best with hands on activities. This unit, I did the lab conclusions very carefully and thoroughly, and learned a lot from the DNA extraction lab and protein synthesis lab. In all, I am a better student than I was at the beginning at the unit, both content based and skil based. 

Protein Synthesis Lab

Protein synthesis has three different steps. The first one is transcription where DNA is copied into a mRNA, which is where uracil is substituted for thymine and it is a single strand. The mRNA then leaves nucleus and travels to the cytoplasm. The next step is called translation, where the actual protein is made. In this step, the mRNA attaches to the ribosome, and the ribosome matches each codon to an amino acid. After translation is finished, the amino acids fold up to make a protein!

The mutations that seemed to be harmless or very in affective seemed to be the small substitution mutations. When taking out a letter and replacing it with the other, the protein did not change at all. However when DNA had a letter added the effects were obvious. Even when the strand was deleted the protein turn out to be a disaster. However, I wanted to see what would happen if we affected the beginning of the sequence, so I used substitution at the begging, and there was no start codon, so there was no protein. I think that any mutation, either frameshift mutations (insertion or deletion) or just substitution to the start or end of a sequence would completely change the end result. In the middle, these mutations aren't quite as harmful, especially with substitution. This is why the location of the mutation matters.

To make a mutation with maximum effect I substituted one of the base pairs in the first codon. I chose this because it would change the start codon, which would prevent the ribosome from making a protein at all. Substitution has to occur in the start of the sequence for it to have a major effect, otherwise it's effect will be very minor, like what we saw with our first substitution, where the protein didn't change at all.  

Proteins normally help us carry oxygen in our blood and digest food, so mutations can damage these processes or even stop them completely! And it's not only these things that mutations could affect, it could affect any other phenotype which humans should have. Uner Tan syndrome is a genetic disorder caused by a mutation in the DNA, and it causes people to walk on all fours. These are one in many ways mutations can affect our lives.

DNA Extraction Lab

The question of the lab was, "How can DNA be separated from cheek cells in order to study it?". We claimed that DNA can be extracted through three different steps, homogenization, lysis, and precipitation, which is where the DNA first becomes visible. Homogenization was when we swished Gatorade in our mouth and spit out cheeck cells, and let the Gatorade break them down. Lysis was when we added salt, detergent, and then the pineapple juice as the protease  to destroy the cell membrane or wall. Precipitation was when we added alcohol and the DNA became visible at the top because the Gatorade is nonpolar and the alcohol is polar. After our procedure was completed, we were able to see small strands of white lines in our alcohol solution which was DNA and after completing the procedure twice we got the same result. This shows that DNA can be extracted from cells and can be seen if following the right procedures.

While our hypothesis was supported by our data, there could have been a few errors. First, the alcohol could have mixed with the Gatorade if you poured it too hard. That would cause the DNA to remain unseen because it would not rise to the top. Second, there could have been too much  Gatorade and/or too little cheek cells . That could have caused the DNA to not show up or not float up to the alcohol during precipitation. Due to these errors, in future experiments, I would recommend to have a measured amount of Gatorade and wipe the inside of the cheek with cotton around ten times. To solve the Gatorade and alcohol mixing, you should tilt the Gatorade test tube and let the alcohol trickle in. 

  This lab was done to demonstrate that DNA could be separated from cheek cells in order to study and observe it. From this lab I learned and what an enzyme help create a reaction an eventually some DNA, this helps me understand how a enzyme works on a larger scale. Based on my experience from this lab, I could apply this knowledge to another situation if I were to maybe look at DNA on more of a molecular level, and needed to re-create someones DNA in that kind of form.