CSI: Lafayette-- Restriction Enzymes, Gel Electrophoresis, and One Lengthy Prison Sentence

Only the most talented criminals are capable of committing a crime without leaving a single source of DNA at the crime scene. DNA, the molecular name tag, is found in hair, blood, skin cells, saliva, semen, etc. Just one droplet of blood or a couple cells could help police identify who committed a crime. Science has made it much harder to be a bad guy.
   DNA profiling is done using Restriction Fragment Length Polymorphism (RFLP). The key to this technique is the restriction enzymes. Such enzymes are found in bacteria, where they are an intrinsic defense against bacteriophage viruses. When the bacterial cell detects foreign DNA from a virus, restriction enzymes cut the viral DNA at a specific, palindromic site, destroying the DNA. There are usually several of these sites at different locations on the DNA, varying from person to person. This is where RFLP comes into play. When restriction enzymes are used on human DNA they cut in different places, resulting in fragments of different sizes. This is how we differentiate between suspects.
   In our experiment, we will use restriction enzymes and gel electrophoresis to determine who committed a crime, comparing the five suspects' DNA to DNA recovered from the crime scene. The restriction enzyme we are using is from the bacteria E. coli. Because all the suspects have different DNA, the enzyme will digest the genes at different locations, creating fragments of different lengths (in base pairs).
   In order to actually see the differences in length, we use a process called gel electrophoresis. The agarose gel we will use is a matrix through which DNA can travel. Electricity is added to the gel, and the current is stabilized by a buffer. The DNA starts on the negative end, but because it has a negative charge, it slowly migrates to the positive side. Smaller fragments will travel further because they can more easily squeeze through the matrix. Conversely, larger DNA fragments (more base pairs) will not be able to travel as far. After running the gel, we will stain the DNA so it can be visualized
   We will run the DNA samples of all suspects, as well as the DNA from the crime scene. One of the suspect's DNA profiles should match the profile of the crime scene DNA, and that will let us know who is guilty. There will also be a marker that serves as a control to make sure the gel worked correctly.
   So, the only question is, WHO DUNNIT?

Using Cellobiase to Break Down Artificial Substrate, Mimicing Degradation of Cellobiose to Glucose for Creation of Ethanol

     For years, scientists have researched alternative sources of renewable energy that could eventually replace the dwindling supplies of oil and other natural resources used for feul. One field in which there is great promise is biofeuls, or feuls extracted from natural biomass. Certain biological oils and alcohol feuls such as ethanol could be the future because they have a cyclical, renewable energy process with little to no pollution. Therefore, we could potentially create an infinite supply of energy that is less toxic to the environment.
     To harness the energy in biological products, enzymes are necessary to break the mass down to its smallest components. Enzymes, usually proteins, bind to specific molecules called substrate and catalyze the reactions. Our lab will use the enzyme cellobiase, which simplifies the two-glucose molecule cellobiose to a single glucose molecule that can be harnessed for feul. Cellobiose is a simpler form of the sugar cellulose, which is found in the cell wall of plants, such as corn.
     As I have already said, scientists have obvious reasons for being interested in biofeuls. Glucose, specifically, can be converted to ethanol by fermentation. Ethanol is already being used as an energy source, but researchers are currently trying to perfect it. We are doing this lab to further our knowledge of enzymes and observe the degradation of cellobiose (artificial substrate) to glucose, which can be used to make ethanol.
     This lab is actually very simple. Rather than use actual cellobiose, which would break down into glucose that is invisible without a microscope, we will use an artificial substrate called p-Nitrophenyl glucopyranoside. We will add this substrate to a test tube with cellobiase, causing the substrate to break down into glucose and p-Nitrophenol.
     On the side, we will set up four beakers filled with a strong base. At different times, we will pour out some of our concoction from the test tube into each beaker. The base has two effects on our solution: first, it will kill the cellobiase, thereby ending the reaction immediately; also, the strong base will react with the p-Nitrophenol, turning it yellow. This allows us to have a visible indicator of how much glucose is being produced, and that the reaction was even successful at all.
     Because we are adding base at different times, we would expect the shade of yellow to be stronger the longer the reaction is occurring. The more the artificial substrate is broken down, the more glucose we will have. The variables in this lab are the different timepoints that we add the base, affecting the amount of reaction that can to occur. For our control, we will have one tube of artificial substrate in which we do not add cellobiase, so no reaction should occur.

Conclusion:
     Due to contamination by the previous period, our lab was unsuccessful. Our tube of substrate had been mixed with enzyme, so the reaction had already been completed before we began the experiment. All of our tubes showed the same dark shade of yellow because all the substrate had already been broken down. Also, our control sample was yellow even though we never added enzyme.