CSI: Acalanes

Restriction Fragment Length Polymorphism (RFLP) is the method forensic labs use for DNA profiling.  It gives a unique banding pattern based on the restriction sites that are represented in a person's DNA sequence.  In this lab we will compare band patterns produced by restriction enzyme cleavage of DNA samples when separated on an agarose gel.  We will be trying to find the culprit of a "crime" based on DNA taken from a crime scene in the case.  Restriction enzymes are key in this lab, they are naturally found in bacterium to protect itself from viruses or bacteriophages.  What they do is they find specific sequences of DNA base pairs that it recognizes from the phage and they cut it at that point.  The effect is that the DNA from the invading cell is never sequenced therefore the phage does not succeed in creating a replication plant from the bacteria cell.  If a restriction enzyme notices multiple codes of the DNA it is looking for it will make cuts at both sites, which will then result in differing lengths of DNA fragments.  This is what is key in our lab and in the fingerprinting process.  Then we will use the process known as agarose gel electrophoresis to create a plasmid map of the DNA.  We will place the DNA fragments in a conductive solution.  Then we will shoot a current through the gel and because the DNA is negatively charged it will move towards the positive end of the gel.  But the smaller fragments will be able to travel farther than the large ones, and the large ones will congregate together.  Then we will compare the plasmid map with that of the DNA of the suspect, and the ones that match will be the criminal.

I was not there for the first day of the lab, but we successfully put the DNA fragments into the gel using the pipets.  Then when we charged the solution we saw the fragments moving through the gel.  After the gel was finished moving we were able to analyze the results.  We saw that the gel most closely resembled that of Chloe's, so we believed that she was the culprit in the crime.  It was cool to actually use the process that real crime scenes use and to be able to analyze the results and come up with our own conclusion on who did it.  Unfortunately I was not there on the day when the lab was set up, but I wish I was because I thought it was a really cool lab.

Passing Gas: Finding Ways to Use Biofuels

In this experiment we will be analyzing the effectiveness of cellobiase at breaking down cellobiose to make ethanol. Biofuels are being attempted as replacements for petroleum.  Biofuels are fuels that are derived from the plants around us, a much cleaner and healthier fuel for the environment.  Ethanol is a biofuel that is a possibility in the search to replace petroleum.  Ethanol is already used in some parts of the US at the pump but cannot completely replace the use of petroleum.  The problem with Ethanol is that cellulose must be broken down in order to create ethanol, but that is a very hard task considering that plants have evolved to make their cell walls extremely hard to break down.  To help break the cellulose down we rely on enzymes to help speed up the process.  Enzymes are catalysts that speed up the rate at which a reaction occurs.  In this lab we use cellobiase to break down the cellulose and then cellobiose into two glucose sugars which are then able to be converted into ethanol.  Being able to assess the success that specific enzymes have at converting cellulose into ethanol will help scientists create the most efficient ways for us to produce mass amounts of ethanol to replace petroleum in the long run.  This is very important as our petroleum is a limited resource, but the use of biofuels through plants is a much more reliable source because we constantly grow plants and they are easy to find and harvest.  In the lab we will take the enzyme cellobiase and use it to assess how much it will produce at different time periods.  So for the indicated time periods we will add pnitrophenol to the solution so that the solution will turn yellow to assess the amount of product produced. I believe that the longer the enzyme has to work the more amount of product will be produced.

After the experiment was completed I was correct in thinking that the longer that the enzyme was working the more product it produced.  The most product was produced after 8 min of allowing the reaction to take place.  At first when we measured every minute it was much harder to see the difference in the amount of product produced, but once the enzyme had been working for 5 min it got much more distinct in the amount of product was increasing as time went on.  As the pnitrophenol caused the solution to turn yellow we could clearly see the difference between the time periods after around 5 min.  The solution turned much darker yellow indicating that there was more product in the solution.  One of the variable that I thought of was the unequal amounts of enzyme and solution and substrate poured into each tube.  If there was more or less in one tube then it might have been more indicative of producing more or less product.
Day 2 was an interesting lab.  I expected that the mushroom would produce less that the enzymes because it was a fungus and I did not know wether or not the mushroom would have a faster production rate than the enzyme did.  But after testing the mushroom it seemed to be about equivalent to the enzymes ability.  I was surprised to see how well the mushroom broke down the cellulose and the cellobiose.  It made me think more about just using mushrooms to conduct reactions because there are so many different species of mushroom that I think we could easily find one that has the capability of rapidly breaking down cellulose into glucose and helping us more efficiently create biofuels.  We were using a portabella mushroom which is used more in cooking than anything else so I believe that with the right mushroom the production of biofuels could be greatly helped.

(P.S. Mr. Chugh, the reason I did not post this earlier is because I gave my packet to Nate so he could do the lab because he was absent the whole week and I just got it back.  I'm sorry that it is late but I couldn't do it off the top of my head.  I hope you understand and can still give me full credit. Thanks!)

DNA in a Bottle!

DNA contains the genetic material for all of the cells in our body. It is contained in the nucleus of a cell and codes for all of the proteins made in our body. It is a double stranded helix that is composed of a phosphate a sugar and a base. It has 4 bases. The purpose of this lab is to precipitate our own DNA from our cheek cells and place it in a glass tube. To do this we have to swab the inside of our cheek and place it in a test tube. We had to swish a cup of salt water around in our mouths to help get the DNA, then we poured the lysis buffer into the tube with our DNA. Then we had to invert the tube to mix the lysis buffer with the water and DNA in the tube. Then we added protease to kill the DNAse that kills DNA. Then we had to place the beaker in a hot water bath so the protease would perform at its highest level for 10 min. Once we got the tube out of the water bath we had to place 10 mL of alcohol in the tube. After that we just had to wait and watch the DNA precipitate.
Once we had the DNA precipitated it appeared as white strands floating in the glass tube we placed it in. It was really cool to see what our own DNA looked like and how much there was for only a small amount of cheek cells. I also thought it was weird once Mr. Chugh explained that there is an enzyme that kills DNA strands because I didn't understand why our body would want to destroy DNA. But then I understood more once he explained that it was for foreign DNA like viruses. That also helped explain why DNA doesn't leave the nucleus, because DNAse is not in the nucleus so it would only kill the DNA of invading cells. I enjoyed being able to walk out of the classroom with a tube of my own DNA hanging around my neck and explaining to people what it was.

Yogurt Lab: Bacteria Tastes Good!

Bacteria are present everywhere in our environment.  They are the most abundant lifeforms on our planet and are present in or on everything in this world.  Bacteria are prokaryotes which means that they are single celled organisms with no separate nucleus to hold their reproductive DNA.  Although, humans associate bacteria with disease, actually only a small number of bacteria cause disease.  We understandably associate them with disease because the ones that do have caused some of the most devastating diseases known to man (cholera, bubonic plague, typhus).  We will base this lab off of Koch's Postulates, which provide the steps for identifying a disease caused by a bacteria.
We will work with the beneficial bacteria that allows found in milk which allows for the production of yogurt (which is more nutritious than milk because it contains enzymes that help break down lactose as well as preventing the growth of dangerous spoilage bacteria).  In this lab we will practice the microbial technique and test Koch's Postulates by isolating the bacteria that allows yogurt to be created when it is fermented with milk.
For the procedure we made our tubes of the yogurt plus milk, milk, yogurt plus ampicilin, and ecoli by mixing the ingredients from the tubes of the substances (ecoli was in an agar plate). then we took them over to the vortex to mix it even more effectively. After that we labeled the tubes and placed them in the incubator overnight. The next day we removed the tubes and tested the pH of the tubes to check to see whether or not they became yogurt or stayed milk, if they became yogurt the pH would be slightly acidic while if they did not then it would remain around neutral. We found that the milk plus yogurt bacteria turned into yogurt in the incubator but the ecoli tube did not turn into yogurt it became spoiled milk. Interestingly, the yogurt plus ampicilin was somewhat of a mixture of the two, the ampicilin did not kill enough of the bacteria that the yogurt making process completely stopped but it did kill enough so it did not become totally yogurt. I found it interesting that the yogurt plus ampicilin became a mixture of the two because I expected the ampicilin to be able to kill all of the yogurt producing bacteria but it could not. We as a group had one main source of error in that we accidentaly mixed a tube with a bleached stick, so we had to observe the bleached tube to test what would happen as well as make a new tube with the right ingredients. (The bleach killed all the yogurt producing bacteria as we expected). But it was cool to be able to see the benefits of some bacteria and be able to test the effectiveness of ampicilin at killing bacteria!