Dissections

Starfish Dissection:

Our species of starfish was an Ophiuroidea, which are brittle stars and basket stars. We begin by placing the starfish with it's aboral surface, which is covered with spines, facing upward. Start cutting into one of the arms of the starfish and then peel back the surface to reveal the pyloric caeca and, below that, the tube feet. The bulb like tops on the tube feet are called ampulla. In the center of the starfish is its madreporite, which is on top of the central disk and is where water enters into through and to the water vascular system. 

Grasshopper Dissection

Since it's easier to see the important internal organs and nerves, we had the grasshopper's dorsal side facing us. Taking a pair of scissors, we began cutting at the bottom in an upward motion. We were sure to cut as slow and close to the exoskeleton as possible so not to cut any of the ventral nerve cord. In order to see the brain and dorsal ganglion, we could've continued cutting higher into the head. Once we get to the top near the neck region, we started cutting toward the sides. We did this by cutting from both the top and bottom portion of the prothorax. By spreading the exoskeleton to the sides, we revealed the inside of our grasshopper. We also inspected many of the outer parts such as the tympanic membranes which they use for hearing and the antennas which they use for feeling surfaces and sensing danger around them.  

Perch Fish Dissection:

Turn the perch onto its dorsal side and begin a rectangle shaped incision in the operculum and peel back in order to reveal the organs. Below the operculum are the bony gill rakers. In the gills are the cartilage support called the gill arch along with soft gill filaments that make up each gill. At the posterior end of the stomach are the coiled intestines. The pericardial cavity contains the heart. The upper part of the body below the lateral line is the swim bladder.

Crayfish Dissection:

First step is to lay the crayfish on its side. Locate the cephalothorax and the abdomen region. Turn it with its dorsal side upward and locate the rostrum (beneath it should be the eyes). Locate where the appendages in the head region are and find the antennae. The, study the mouth which have mandibles behind the antennae. The largest part of the appendages is the chelipeds or the claws. Behind each of these are walking legs. Then turn the crayfish central side up to locate the appendages from the ventral side. Lift away the carapace and using scissors cut the case of the tail and lift the parts of the carapace to reveal the gills and other organs. The maxillae passes the food and travels down the esophagus to the stomach. When opened up, one is able to see the intestine, the stomach and the anus of the crayfish.In the tail, we can see the intestines and tail muscles which help crayfish swim through the water.

Electrophoresis Lab

Purpose: The purpose of this lab is to analyze unknown DNA to determine the number of cut sites for each restriction enzyme and the positions of those cut sites relative to one another. The goal was to use electrophoresis to help us visualize the placement of each of the bands compared to one of the master bands in order to find out the size of each.  

Introduction: Restriction mapping is a critical step in analyzing and characterizing a new DNA sequence. A restriction map of a piece of DNA can be compared to a fingerprint, something to identify each piece. In order to set up this lab, we were to use three restriction enzymes, PstI, HpaI, and SspI. The purpose of this lab, as stated before, is to find out the number of cut sites present in the DNA sequence for each restriction enzyme and the positions of those cut sites relative to one another. In order to do this, the DNA should be loaded into each well of the gel and analyzed after gel electrophoresis has taken place. 

Methods: Fortunately, the agarose gel was already pre-made when we did our experiment, so we did not have to worry about casting the agarose gel. Our first task was to simply load the DNA into the gel. We began by drawing samples into the pipet, making sure we expelled any excess air so that air bubbles wouldn’t form in the wells. We steadied the pipet over the gel and dipped the pipet through the surface of the buffer, positioning it over the well, and letting out the liquid. We did this a total of 4 times, one for the comparison or control group, and the rest for each of the three different restriction enzymes. The next task was the actual electrophoresing process. This consisted of an electrophoresis chamber which was connected to a power supply with high voltage. Our teacher monitored the electrophoresis and took it out when he saw fit and then we loaded it onto the staining tray. After being stained, we were given back our trays to be able to examine the movement of each restriction enzyme bands through a lightbox. The lightbox helped see the bands better so that we could label them and compare by size marker. 

Discussion: The movement of the different bands in each well were dependent on their varying sizes. The well that showed the farthest movement down the gel towards its positive end was well Pst I. This can lead us to assume that the DNA strands that were present in that solution had strands that were cut thin enough so as to be able to make its way so far down it's given well. In contrast to that, the well of Pst I also had a marker in it that did not travel far in comparison to the rest, meaning that it's DNA strands were much too large to move much distance at all. When compared to the constant well that was the Marker well, most other wells only conducted of a given 2-3 bands. This would lead us to assume that the DNA we were working the wells with we're relatively basic in the sense that not many bands were present or shown. ThBeing pre-given approximations for the positions where each marker should have ended, accuracies in their placement verified that the correct loading substance was placed into its appropriate well. Furthermore, after constructing the positions of the various recorded bands into a visual circular chart for every well and it's bands, the lengths in which they were recorded straight on the gel came to match with the circular graphs, 

Data:

Conclusion: Through the use of gel electrophoresis, we were able to identify where certain cuts in all restriction enzymes occurred. We were able to label the bands we saw under the lightbox and compared it with the control well in order to find out our efficiency. After taking a close look, we realized not all of the bands that were supposed to be present were visible. This may have been due to an error in loading the wells. We realized after the put away our try to the electrophoresis station that one of our tubes had not been fully pipetted into the well and that may have caused some inaccuracies in our results. We acknowledged our mistakes and realized it could have been avoided by making sure we expelled all excess air from out pipet. However, aside from small mistakes, our data was pretty accurate in reflecting what should have been seen according to our data. Wells 2-4 were all supposed to have the same total size of DNA fragments and as seen in our data, it was within the same range, since it was an estimate of course. As different fragments of DNA moved at different rates and therefore different positions, we used this data to construct diagrams of said fragments.  

pGlo Lab

Purpose: The purpose of this experiment was to oversee the process of genetic transformation. We were trying to see how this procedure accounts for the pGlo plasmid's ability to code for Green Flourescent Protein (GFP)

Introduction: In this lab we were to perform a procedure known as genetic transformation. This protein gives the organism a particular trait. This involves the insertion of a gene into an organism in order to change the organisms trai. We used that procedure to transform becateria with a gene that codes for GFP. The source of this particular gene is from the bioluminescent Jellyfish Aequorea Victoria, which causes them to glow in the dark. Once the procedure is completed, the acquired jellyfish gene will cause the bacteria to glow a green color under ultraviolet light. In this experiment, we were to learn about the process of moving genes from one organism to another with the help of a plasmid. A plasmis contains genes for one or more traits needed for its survival. Transferring of said plasmid occurs between bacteria to allow them to share these beneficial genes and hence, the bacteria adapts to its new environment. In this experiment, the occurrence of bacterial resistance to antibiotics is due to the transmission of plasmids. The pGlo plasmid encodes the gene for GFP and the gene for resistance to the anitiotic ampicillin. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells nutrient medium. Transformed cells will appear white  without the sugar, and glow green with the arabinose. 

Methods: At the start of the lab, we labeled 4 tubes with either +PGlo, -PGlo, (control group), transformation solution, and broth. Then we pipettes some transformation solution to both the negative and positive tubes and then, we put them in ice. After that, we scooped bacteria out of a Petrie dish and spread the E. Coli in both with a sterile loop and mixed well.  However, we put plasmid only in the positive pGlo container.  In order to compare results, we labeled 4 different Petrie dishes. After, we put the tubes back in ice for 10 minutes and then we used heat shock by putting it in water (45 degrees Celsius) for 50 seconds and back in ice for 2 minutes. Using a pipette, we put broth in both tubes. Using a pipette, we put pGlo according to how the Petrie dishes were labeled as such. Then, we spread out the solution with a loop to increase surface area. The last step was to put it in an incubator at 37 degrees Celsius for 1 day. He next day, we took it out and using a UV flashlight, we measured growth in Petrie dish and it's ability to glow under the light. 

Data:

Discussion: After taking it out of the incubator, we were to analyze the growth and ability of the E. Coli bacteria to glow under UV light. We had the most growth with -pGlo LB, no growth in -pGlo LB/AMP, and some growth in both +pGlo/AMP and +pGlo/LB/AMP/ARA. It was clear since before we did the lab that -pGlo LB/AMP would have no growth because all the bacteria cultures that would've grown would have been killed off by the AMP(Ampicillin) which is an antibiotic.  -pGlo LB Was used as a control group because the bacteria was allowed to spread without the plasmid. +pGlo/AMP and +pGlo/LB/AMP/ARA Both had growth because the bacteria was made resistant to the antibiotic and were allowed to spread. However, +pGlo/LB/AMP/ARA was made to resist the antibiotic and the arabinose activated the pGlo gene which causes it to glow under UV light. These results are valid because we discussed what each of the Petrie dishes should have looked like. We were shown expected results of the dishes and out data was compatible. The Petrie dishes showed that the ampicillin killed the E. Coli that did accept the pGlo gene and we proved that the arabinose help the pGlo gene activate after certain genes have accepted the pGlo gene. 

Conclusion: The final results of our pGlo lab yielded an effectively added Aequorea Victoria that is the bioluminescence gene found in jellyfish showed through in the Petri dish of +pGlo with LB broth, Ampecilin and Arabanose sugar. Our hypotheses proved correct because we expected the fourth Petri dish, which held every additive in it, to not only accept the pGlo gene but also to isolate it on the account that we added the Ampecilin to eliminate any remaining genes that had not accepted to bioluminescence gene. As our final results matched the expected results fairly similarly, we can conclude that the lab went relatively successful. For improvements, we could have attempted to cause greater colony growth on the Petri dishes as that would have shown more efficiency in our dishes.