Lab Report N*7 Molecular Evolution PDF

Preview

Summary

Laboratory reports for Spring 2018 Cell & Molecular Biology Laboratory by Professor. Henri P. Antikainen and Cervantes. ...

Description

Molecular Evolution: Separation of Fish Proteins Using Polyacrylamide Gel Electrophoresis Introduction: From the theory of Evolution shown by Charles Darwin, it was concluded that living organisms were derived from a common ancestor. In other words, the theory “Descent with Modification”, discusses the hereditary changes that occur over many generations. These modifications happen as a result of DNA changes cause by framed shift and points mutations. These mutations create alterations of the structure of DNAencoded proteins. While DNA is our fundamental unit of life, it’s most important as it accounts for the variation that can lead to genetic diversity. Another aspect of diversity or change in the gene pool is evolution. These changes in the gene pool are produce by natural selection, a process by which some individuals and not others are given traits that may enhance their survival, reproductive rate, adaptation, or metabolic processes. However, we must understand that DNA’s essential role is to determine protein amino acyl sequences, their structure and function. Proteins are the molecules that perform the function programmed by the genome. In this experiment, we will accomplish protein analysis by SDS-PAGE or polyacrylamide gel electrophoresis, which is a method of separating molecules based on the difference of their molecular weight. Polyacrylamide gel electrophoresis, is the separation of macromolecules in an electric field in electrophoresis. It’s most commonly used in the separation of proteins as it uses a discontinuous polyacrylamide gel as a support with help of the SDS to denature proteins. The gel electrophoresis is able to achieve separation by size because of the SDS detergent. SDS (Sodium Dodecyl Sulfate) is a strong detergent that is best known to denature proteins. Sodium Dodecyl Sulfate is used to denature polyacrylamide gel electrophoresis for the determination of protein molecular weight. SDS is able to denature proteins as proteins are first dissolved in a solution of SDS as it disrupts all noncovalent interactions by applying a negative charge to each protein in proportion to its mass. Objective: While studying the means of evolutionary relationships among several groups of fish through analysis of the protein composition of their muscles, we will determine the common phonetic relationship among the fishes through the SDS-PAGE technique. Method: a) Sample preparation b) Electrophoretic separation of fish muscle proteins c) Staining

d) Imaging a phylogenetic tree to find common ancestry In order to start the experiment a sample will be created by setting up the electrophoresis rig and adding TGS electrophoresis buffer to the chamber. SDS is a detergent that works as a denaturing agent by facilitating the separation of the protein as it gives off electrical charge. The separation which will take one hour will be run at 200 V in one TGS buffer. After the separation has been completed samples will be distained to find common relationships in a phylogenetic tree, which can be complete by several methods. Result: Lane Volume (uL) Sample 1 Empty 2 10 KS Markers 3 10 Catfish 4 10 Trout 5 10 Hake 6 10 Bass 7 10 Swordfish 8 10 Flounder 9 Empty 10 Empty Table 1.1: Shows the sample fishes used, and volume used to create our band gel using the GelAir Cellophane. Protein

Molecular Weight kDa

Distance migrated from well (mm) Myosin-Heavy chain 203.0 7 B-Galactosidase 135.0 12 Bovine Serum 86.0 20 Carbonic Anhydrase 41.5 36 Soybean Trypsin inhibitor 33.4 40 Lysozyme 19.5 48 Aprotinin 8.0 56 Table 1.2: Shows the identified proteins and their distance migrated from the well in millimeters. Semi-Log Graph–Graph 1.1: Shows the standard curve on a semi-logarithmic paper with three cycles by which is constructed by plotting size of standards in kilo-daltons versus migrated distance (mm).

Molecular Weight (kDa) vs. Distance (mm)

Molecular Weight kDa

250 200

f(x) = 374.21 exp( − 0.52 x ) R² = 0.98

150 100 50 0

0

2

4

6

8

10

12

Distance traveled (mm)

This photograph shows our polyacrylamide gel stained with coomassie blue, revealing the protein bands from fish muscle samples. Discussion & Questionnaire: From the theory of Evolution shown by Charles Darwin, it was concluded that living organisms were derived from a common ancestor. In other words, the theory “descent with modification”, discusses the hereditary changes that occur over many generations. These modifications happen as a result of DNA changes cause by framed shift and points mutations. These mutations create alterations of the structure of DNA-encoded proteins. Proteins are the molecules that perform the function programmed by the

genome. In this experiment we first create a sample preparation of the types of muscles from different fish. As we continued, the electrophoretic separation of fish muscle protein took that for 1 hour at 200 V in a TGS buffer. Then we stained our gel after electrophoresis, by removing the gel from the cassette and transferring the gel to a container with 40 mL of Coomassie Blue Stain. Finally, we create an imaging factor to our experiment, meaning our gel was dry by using the GelAir Cellophane. For clearly results, a picture under a transparent surface was taken. Overall, we were able to find the common phenotypes and genotypes between these fishes as they are all related to one common ancestor: Euteleost best known as Teleost fishes. 1. The name of the fishes whose muscle proteins we studied were: Catfish, Trout, hake, Bass, Swordfish, Flounder. 2. According to our phylogenetic tree, the Flounder, Swordfish, and Bass are most closely related because of their common ancestor: Acanthopterygii (bone fish) class. The least related is the catfish to the Flounder by a factor of age, but its most closely related to the Trout. 3. Sample 2 (Trout), sample 5 (Swordfish), and sample 3 (Hake) shared the most protein banding similarity, while sample 1 (Catfish) and sample 6 (Flounder) shared the least banding similarity. 4. We distinguished the protein profiles of different species from each other by putting them in sample order when conducting the experiment. 5. The most possible and logical explanation for this variation in the species is because different phenotypes and genotypes. The environment could also be a factor to their change through natural selection and evolution. 6. Before conducting our experiment, we thought the most related two fishes were the Hake and Swordfish because of how similar they appear to be. 7. There is one similar muscle protein found in both the Hake and Swordfish. 8. I was able to detect two different muscle proteins in both the Hake and Swordfish. 9. ½ times 100% = 50% of the proteins are found to be common for both the Hake and Swordfish species. 10. Prior starting the experiment the least two related species were Catfish and Flounder. 11. There are two similar muscle proteins to be found in both the Catfish and Flounder 12. There are two different muscle proteins to be found in both the Catfish and Flounder 13. 50% were common to these least similar species because 2 muscles proteins from the catfish were similar to the Flounder whose had 4 total muscle proteins.

14. Yes, my protein size confirms the hypothesis about the relationship of the fishes we worked with because the thickness, size, or dimensions matters in similarities within the species. 15. The relative position of the bands on the gel indicates the similarities between the proteins found in each specie. 16. No, not all the bands are of equal thickness 17. The thickness of the band could be explained by the amount of protein or different proteins clustered together in the same bone or phenotype of a specific specie. 18. Actin and myosin form muscle fibers, which is the biochemical machinery that causes muscle to contract. Therefore, these two proteins make up the structure and function of muscle that is common to all animals. In another words, all fishes and other animals contain actin and myosin that correspond to develop semisimilar structure or function. 19. Approximate molecular weight of protein = number of amino acids (A.A) x 10 daltons/amino acid; therefore, 43 = A.A x 110  43/110 = Amino Acids = .3909 kDa/amino 20. The data of this experiment corresponds to Darwin’s theory of Evolution. The data support evolution as a change in the gene pool of a population. Due to this evolution different fishes appeared to have different phenotypes, yet nearly similar genotypes as they share common bones found within each other. Conclusion: During our studies of this experiment, we found the means of evolutionary relationships among several groups of fish through analysis of the protein composition of their muscles. We were able to determine the common phonetic relationship among the fishes through the SDS-PAGE technique by using a GelAir Cellophane, so that we could dry our gel bands and compared them later. Overall, amongst all fishes, their bands were mostly alike and similar in shape, distance, and size due to common ancestry....