Biochemistry 2280A Chapter Notes - Chapter 157-168: In Vitro Fertilisation, Protein Domain, Lifesaving

Enzyme X is a highly pigmented protein that imparts thecharacteristic color to certain blue-green algae. It alsofacilitates a reaction necessary to the survival of this species;we can follow the kinetics of this reaction by measuring theconversion of Substance X to Substance Y at various times duringpurification.
1. Given a pure preparation of these algae, and the requiredsupplies and equipment, devise and outline an empirical procedurefor purifying Enzyme X.
2. What is a good indication of purity in yourpreparation?

What I have:

The first step to determining protein identification andpurification is to isolate the protein of interest from thestarting material. This process is initiated by determining theproper assay to be used. An assay is a termed used to describe thetechnique that one uses in protein purification. It describesmethodology, protocol, and detailed technique unique to certainexperiments. In more detail, unique to certain experiments,describes the chemical properties of the desired protein. Theprotein of interest must be removed from the cell. Its associatedenzyme is a useful tool in determining its specific activity. Thisis a quantitative measurement that can assess the progression ofprotein purification. Proteins can be purified based on size,charge, solubility, and even binding affinity.

Given a pure preparation of algae and purifying Enzyme X, I wouldbegin by
extraction of all proteins via salting out technique. Then removethe supernatant and centrifuge, followed by column chromatographyto separate all the proteins based on size, followed by labelingvials according to size. Next, remove a small amount of what is ineach vial and add to it substance X. Because Y is unknown, I dependon the rate of substance X's depletion to detect the presence ofthe enzyme. After recording the change in concentration of X(through fluorescence change) per certain amount of time that isfixed regardless of what protein is being tested, I then identifythe vial that has the mix of proteins that has the most enzyme X,and run SDS-page electrophoresis. This provides an idea about thesize of the different proteins. The difference in their sizes isassessed via the column chromatography. Finally, I run just thatvial through the column and retest with substance X. This indicatespurity in the preparation.

my teachers reponse: what you have submitted is a start howeverthis is general protocol. The purpose of the assignment is tomake sure you the student understands the various proteinpurification techniques discussed in class and in the textbook andwrite a general protocol. The M1 assignment is being returnedto you again to refer back to topic section 1.2 within the classand see what is discussed. It specifically states in the commentary%u201CTo isolate a compound completely, the above methods, and manyothers not described, are usually employed sequentially, and thespecific activity is measured after each step. Only the fractioncontaining the highest specific activity is then passed on to thenext step.%u201D It is telling you the reader that all discussedneed to be done at a minimum to purify a protein and more steps areneeded that would be determined by first doing the%u2018general%u2019 steps as described. Also note that as writtenthe protocol submitted started purificaiton with whole cells - thatis not possible. The protein of interest is inside the cell - thefirst step is to get the protein of interest out of the cell, howis that done?

HELP ASAP; Please help me understand my lab results, so be detailed to clearly understand

Then , Please include answers to the question below to include in your response

1. what role does ampicilin (amp) and arabinose (ara) play in the experiment.

2) Because GFP was isolated from jelly fisht, what modification to the GFP DNA had to be made to result in expression in E. coli?

BACKGROUND INFORMATION

Transformation of GFP and the pGLO Plasmid GFP is a commonly used reporter protein in research labs, as the fluorescence creates a marker protein that can be used in many types of cell biology and biochemical studies. In basic research, GFP is often fused to a specific target protein of interest, creating a chimeric reporter protein. GFP has been used as a reporter protein to study blood vessel and tumor progression in mice, brain activity in mice, and malaria eradication in mosquitoes for instance (see website mentioned in notes). In the first week you will use a procedure to genetically transform bacteria with the gene that codes for Green Fluorescent Protein. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light. During the second week you will purify the protein away from all of the other proteins produced by the bacteria using chromatography, and during the third week you will evaluate the success of the purification procedure and estimate the size (molecular weight) of GFP by using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fundamental to the process of genetic transformation of bacteria are plasmids. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The occurrence of bacterial resistance to antibiotics is due in large part to the transmission of plasmids which contain antibiotic resistance genes. The pGLO plasmid contains the gene for GFP, and also contains the gene for the enzyme β-lactamase (bla), which provides resistance to the antibiotic ampicillin. The β-lactamase protein is produced and secreted by bacteria that contain the plasmid. β-Lactamase inactivates the ampicillin present in the LB nutrient agar, allowing bacterial growth. Only transformed bacteria that contain the plasmid and express β-lactamase can survive on plates that contain ampicillin. Only a very small percentage of the cells take up the plasmid DNA and are transformed. Untransformed cells cannot grow on the ampicillin selection plates. pGLO also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells’ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green when arabinose is included in the nutrient agar medium.

The transformation procedure involves three main steps. These steps are intended to introduce the plasmid DNA into the E. coli cells and provide an environment for the cells to express their newly acquired genes. 1) Cells are first treated with a solution of CaCl2, which is thought to neutralize the repulsive negative charges of the phosphate backbone of DNA and the phospholipids of the cell membrane, allowing the DNA to adhere to the cells. This is referred to making the cells ‘competent’ (i.e. the cells are capable, or competent, to take-in exogenous DNA). 2) Cells are then subjected to a heat shock, which is thought to increase the permeability of the cell membrane, allowing the cells to take-in the plasmid DNA. 3) Cells are allowed to grow in a ‘recovery’ phase in the absence of ampicillin. During this time the cells begin to produce the β-lactamase protein, which will allow them to survive when they are subsequently placed on agar plates containing ampicillin.

For the first week section of lab

To eac

h of two microfuge tubes, add 250ul of transformation buffer. Keep these tubes on ice.

Using a sterile loop, pick one colony of bacteria from the LB plate. Transfer this colony to one of the microfuge tubes.

Gently vortex the tube until the colony is dispersed. Repeat for the second tube. Add 0.08ug of DNA to one of these tubes (+DNA tube).

Incubate both tubes on ice for 10 minutes. Incubate both tubes at 42°C for 50 seconds.

Incubate both tubes on ice for 2 minutes. Add 250ul of LB broth to each tube and incubate at room temperature for 10 minutes. Flick each tube gently. Add 100ul of t

his transformation mix to the appropriate plates.

Results For plates:

+DNA: Has the E. Coli pGlo plasmid and - DNA plates do no have EColi pGLo plasmid .

Amp means ampiclin and Ara means arabinose on agar plates

+ DNA Plate LB/amp/ara--> white 9 colonies, Glow: yes

+DNA LB/amp--> 7 colonies, GLow: no

--DNA LB/AMP- 1 colony so no colony growth and no glovw

-DNA: LB--> many colonies, Glow: no

Please explain significance of result or thand the reason for the results in scientific terms based on the background information provided and your own knowledge to be detailed. Would these results be expected as wll.

HELP ASAP;

Please help me understand my lab results, so be detailed to clearly understand . INclude answers to these questions what role does ampicilin (amp) and arabinose (ara) play in the experiment. Because GFP was isolated from jelly fisht, what modification to the GFP DNA had to be made to result in expression in E. coli?

BACKGROUND INFORMATION

Transformation of GFP and the pGLO Plasmid

GFP is a commonly used reporter protein in research labs, as the fluorescence creates a marker protein that can be used in many types of cell biology and biochemical studies. In basic research, GFP is often fused to a specific target protein of interest, creating a chimeric reporter protein. GFP has been used as a reporter protein to study blood vessel and tumor progression in mice, brain activity in mice, and malaria eradication in mosquitoes for instance (see website mentioned in notes).

In the first week you will use a procedure to genetically transform bacteria with the gene that codes for Green Fluorescent Protein. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light. During the second week you will purify the protein away from all of the other proteins produced by the bacteria using chromatography, and during the third week you will evaluate the success of the purification procedure and estimate the size (molecular weight) of GFP by using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Fundamental to the process of genetic transformation of bacteria are plasmids. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The occurrence of bacterial resistance to antibiotics is due in large part to the transmission of plasmids which contain antibiotic resistance genes.

The pGLO plasmid (Fig. 5, not a naturally occurring plasmid) contains the gene for GFP, and also contains the gene for the enzyme β-lactamase (bla), which provides resistance to the antibiotic ampicillin. The β-lactamase protein is produced and secreted by bacteria that contain the plasmid. β-Lactamase inactivates the ampicillin present in the LB nutrient agar, allowing bacterial growth. Only transformed bacteria that contain the plasmid and express β-lactamase can survive on plates that contain ampicillin. Only a very small percentage of the cells take up the plasmid DNA and are transformed. Untransformed cells cannot grow on the ampicillin selection plates. pGLO also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells’ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green when arabinose is included in the nutrient agar medium.

for the first week section of lab

To each of two microfuge tubes, add 250ul of transformation buffer. Keep these tubes on ice.

Using a sterile loop, pick one colony of bacteria from the LB plate.

Transfer this colony to one of the microfuge tubes. Gently vortex the tube until the colony is dispersed. Repeat for the second tube.

Add 0.08ug of DNA to one of these tubes (+DNA tube).

Incubate both tubes on ice for 10 minutes.

Incubate both tubes at 42°C for 50 seconds.

Incubate both tubes on ice for 2 minutes.

Add 250ul of LB broth to each tube and incubate at room temperature for 10 minutes.

Flick each tube gently. Add 100ul of this transformation mix to the appropriate plates.

Results:

+DNA: LB/amp--> 1 colony, Glow: uncertain

+DNA: LB/amp/ara--> 9 colonies, Glow: yes

-DNA: LB/amp--> 7 colonies, GLow: no

-DNA: LB--> many colonies, Glow: no