Protein Transformation Lab Report

this means in order for the new strands of DNA to be accepted by the cell it must be fixed inside a plasmid. The same restricting enzyme is used on the plasmid. This allows the DNA strand and plasmid to stick and join together. Once joined the phosphodiester bonds are formed by the DNA ligase on either edge of the strand. Transformation is the next stage as the newly formed plasmids are taken in by the host bacterial cells. The cells are now a DNA library and can be administered to the required organism.

Enzymes are catalysts that function to speed up reactions; for example, the enzyme sucrose speeds up the hydrolysis of sucrose, which breaks down into glucose and fructose. They speed up reactions but are not consumed by the reaction that is taking place. The most important of the enzyme is the shape as it determines which type of reaction the enzyme speeds up. Enzymes work by passing/lowering and energy barrier and in doing so; they need to bind to substrates via the active. Once they do, the reaction speeds up so much more quickly than it would without the enzyme. Coenzymes and cofactors aid the enzyme when it comes to binding with the substrate. They change the shape of the active site so the substrate can bind properly and perform its function.

Introduction: The biological membranes are composed of phospholipid bilayers, each phospholipid with hydrophilic heads and hydrophobic tails, and proteins. This arrangement of the proteins and lipids produces a selectively permeable membrane. Many kinds of molecules surround or are contained within

The optimum pH level would be pH 7. This is because this is where the highest amount of enzyme activity is taking place.

This lab is about moving genes from one thing to another using plasmids. Plasmid has the ability to replicate, so it replicates independently, and separately from the chromosomal DNA. Plasmid are one or more small piece of DNA and they enter cells as a double strand DNA. When they enter the cell as a doubke strand they do not invade he chromosomal DNA. We will also transform bacteria into GFP which is mainly from the jelly fish Aequorea Victoria. The GFP causes the the jelly fish to fluorescent and glow in the dark. After the transformation, bacteria starts to make the GFP which causes them to glow a green color under a ultraviolet light.

An association between enzyme production, gene copy number, and gene evolution was explored by conducting analysis of the salivary amylase enzyme, AMY1A gene copy number, and the ancestral starch consumption in Homo Sapiens (Tracey 2017, p.22). It was hypothesized that the relative amount of starch consumption was very high for my personal ancestral diet, thus my AMY1 diploid gene copy number in my genome and salivary amylase concentration would be significantly higher than the population mean. With a population of 28 subjects (n=28), individual saliva samples were collected and compared to a calibration curve to determine the approximate amylase concentration by analyzing absorbance values. Individual samples of buccal cheek cells were

This experiment was designed to test and observe the transformation efficacy of the pUC18 and lux plasmids in making E. coli resistant to ampicillin. Both plasmids code for ampicillin resistance, however, the lux plasmid codes for a bioluminescence gene that is expressed if properly introduced into the bacteria’s genome. The E. coli cultures were mixed with a calcium chloride solution and then heat shocked, allowing the plasmids to enter the bacteria and assimilate into the bacterial DNA. The plasmids and the bacteria were then mixed in different test tubes and then evenly spread onto petri dishes using a bacterial spreader, heating the spreader between each sample to make sure there is no cross contamination. Each of the dishes was labeled and then incubated for a period of 24 hours. The results were rather odd because every single one of the samples grew. Several errors could have occurred here, cross contamination or possibly an error in preparation as every single sample in the class grew, meaning all samples of the bacteria transformed and became ampicillin resistant.

Green fluorescent protein (GFP) comes from the jellyfish Aequorea Victoria is rare proteins with high fluoresce and absorbance. The purpose this experiments is to purify and express a His2-tagged recombinant from of GFP (rGFP) from the E. coli strain BL21(DE3)< pRSETA-GFPUV > through a series of experiments by using Ni+2 agarose affinity chromatography technology. The GFPuv gene (UV-optimized GFP) was over expressed in the E. Coil strain BL21 (DE3) (pLysS) as an n-terminal His6/Xpress epitope tagged bind protein. Then using Ni2+ Agarose affinity chromatography to obtain purification of the crude extract. Then observe under the long wavelength UV light, the activity of the rGFP in the column fraction. Bradford assay was performed to obtain the total protein amount. When calculating the

In the pGLO Bacterial Transformation lab, Escherichia coli is transformed with a gene encoding green fluorescent protein by inserting a plasmid containing the GFP gene, beta-lactamase, and arabinose into the bacterium. Successfully transformed bacteria will grow in the presence of ampicillin and glow a bright green color under ultraviolet light. The sugar arabinose is responsible for switching on the GFP gene in the transformed cells, without it, the gene will not be expressed.

The sixth lab I completed in Biology 101 taught me how autotrophs (self-feeders) and heterotrophs (other-feeders) make organic food molecules by using photosynthesis. Photosynthesis uses the energy from the sun and it is captured and stored in the chemical bonds of organic molecules. The sunlight consists of different wavelengths of light. In plant chloroplasts, they have different pigments that capture different wavelengths of light. Light capturing pigments in green plants are called chlorophylls and these absorb all the colors of light except green, which is mostly reflected. To separate molecules from each other according to their solubility in a particular solvent is done by the process of chromatography. This basically means that polar

** The Low vs. High column shows how each protein band was influenced by the salt. If band was present in both conditions, it was noted what condition had and increase or if they were equal.

Protein purification is a process that can be employed to separate a single protein from a larger starting material which may be anything from an organ to a cell. Isolating a purified protein from a larger fraction enables further analysis such as determination of amino acid sequence, potential biological function, and even evolutionary relationship. (Cuatrecasas 1970) In this experiment, the enzyme lactate dehydrogenase will be purified, this enzyme is found extensively in human cells and catalyzes the conversion of lactate to pyruvate, an essential part in energy production. LDH is a key part of anaerobic energy production especially within glycolysis in which LDH catalyzes the conversion of the reverse reaction, pyruvate to lactate, generating NAD+ from NADH, reproducing the oxidized form of the coenzyme which can be used for oxidative respiration. (Markert 1963) Due to the fact that number of purification steps correlates with the purity of the protein multiple purification techniques will be used to isolate a pure form of LDH. LDH will be isolated from a larger “cytosol” fraction collected from a homogenized rat liver in a previous fractionation exercise. Of the procedures that will be used to isolate and purify proteins from a larger fractionate are a set of techniques collectively known as chromatography. These techniques all have the same premise, in that they consist of a stationary phase, also known as the

Transcription is where DNA is transcribed into RNA which then can be pass to the ribosome’s to act as a template for protein synthesis. Before transcription can begin DNA must unwind and the two halves of the molecule much come apart so exposing the base sequence. This process begins when a region of a two DNA strands is unzipped by enzyme called RNA polymerase attaches to the DNA molecule at the imitation site.

Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial

The second stage of the process is complementary base pairing. In this stage, new complementary nucleotides are positioned following the rules of complementary base pairing: adenine (A) to thymine (T) and guanine (G) to cytosine (C). Then, the binding of free nucleotide with complementary bases is catalyzed by DNA polymerase.